AN ABSTRACT OF THE THESIS OF
David Talbot Jordan for the degree of Doctor of Philosophy
in Horticulture presented on August 15. 1989.
Title: Ammonium in Grapevines: Seasonal Levels in Tissue
and Xylem Extracts, and a Tendril Model System.
Abstract approved:
1
r
Patrick J/i/Breen
Recent interest in ammonium concentration in grape
(Vitis vinifera L.) cluster tissue has resulted from
established relationships between two cluster disorders
and ammonium concentration in rachis tissue.
Little is
known about the dynamics of ammonium concentration and
ammonium assimilation in rachis tissue.
Inflorescence necrosis caused large crop losses in
some Willamette Valley, Pinot noir, 1988.
Cultivars and
clones had different susceptibility, and vineyards had
different incidence of the disorder.
Ammonium
concentrations in rachis were positively correlated with
the severity of the disorder.
Free ammonium concentrations were determined in
rachis and berry tissue, and xylem extracts from shoots of
field grown Cabernet Sauvignon vines.
Half the vines were
covered with 50% shade cloth.
Ammonium concentrations
were highest at or before bloom followed by a decline to
about veraison with low levels through to harvest.
The
decline in xylem and rachis ammonium concentrations
preceded that in berries by about 20 days.
Shade caused a
three fold increase in xylem ammonium concentration near
bloom and a two fold increase in the concentration, postveraison, in the rachis.
Berries from shaded vines had
seven fold higher ammonium concentration at veraison than
berries from exposed vines, but early and harvest
concentrations were similar.
Detached tendrils were a useful model system to study
ammonium toxicity and assimilation in tissue closely
related to that of clusters.
Tendrils lost fresh weight
and later became necrotic when continuously supplied 330
mM ammonium sulfate via the cut end.
Concentration of
free ammonium in treated tendrils greatly increased
compared to tendrils in distilled water.
This increase
was halved when oc-ketoglutarate or glutamate was added to
the ammonium solution.
This response was attributed to
stimulated ammonium assimilation as reflected by increased
free amino acid, especially glutamate and ^-aminobutyrate,
concentration in the tissue.
Methionine sulfoximine (MSX)
did not prevent the decrease in free ammonium caused by
o^-ketoglutarate.
Sucrose or glucose added to the
ammonium solution provided little beneficial effect.
Ammonium in Grapevines:
Seasonal Levels in Tissue and Xylem Extracts,
and a Tendril Model System
by
David T. Jordan
A
THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed August 15, 1989
Commencement June 1990
APPROVED:
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Proxessor of Hortioujxure in charge of major
Head of Depaitinent of Horticulture
Dean of Gradi^^e Schoo^
Date thesis presented August 15. 1989
Typed by DTJ.
ACKNOWLEDGMENTS
As a kiwi that found his wings and made his new nest
in Oregon, this has been a very fulfilling, exciting, and
educational time of my life.
The success of my stay and
study is, in most part, due to the many fine people that I
had the pleasure to interact with.
Special thanks go to my advisor, Pat Breen, whose
scholarly advice and concern were inspiration and
invaluable.
Pat's philosophy of science and methodical
approach to research were some of my most professionally
valuable experiences.
Remarkably, we found that we had
something in common — the same propensity to laugh
(loudly) at the same jokes and humorous situations.
These
qualities made our meetings educational, memorable and
enjoyable.
In my viticulture world two people need special
mention.
Porter Lombard is a tireless source of
information and personal care.
Porter is one of the most
generous (materially and of his time) people that I have
met.
His guidance through the wilds of Oregon (and this
may mean on the road with him driving !) were greatly
valued.
I also thank Steve Price for the many and lengthy
discussions we had about my research.
These were very
rewarding and often resulted in interpretations and
experimental approaches found in this thesis.
His
suggestion to use tendrils as a model is acknowledged.
Thanks also to the remaining members of my committee:
Tim Righetti, Neil Christensen and Ron Wrolstad.
Their
time and assistance are gratefully acknowledged.
The faculty, staff and fellow students of the
Horticulture Department have been wonderful colleagues and
friends.
Special thanks to two groups.
First our
research group of Gui Wen, Kirk, Jim, Adly and Steve
formed a very effective forum for scientific discussions.
I greatly valued our times together, professionally and
socially.
group".
The other group is the "first floor coffee
I enjoyed the chance to solve the ills of the
world with them.
The Oregon Wine Advisory Board provided financial
support for the late rachis necrosis research, a disorder
that rarely affects their industry.
I thank the New Zealand Government for their
financial support via a National Research Advisory Council
scholarship.
The security of this support meant that I
was able to complete this study and work more effectively.
Lastly, I thank my friends and family back in New
Zealand.
My colleague and friend Richard Smart was my
first Viticulture instructor and his advice on my study
meant that I came to Oregon.
To my family, I thank them
for their patience and support while we have been
separated by a vast ocean.
TABLE OF CONTENTS
Page
CHAPTER 1.
INTRODUCTION
CHAPTER 2.
LITERATURE REVIEW
Introduction
Plant / Soil Interface
Transport within the Plant
Xylem : General
Xylem : Grapevine
Phloem : General
Transport Driving Force
Ammonia Assimilation
Enzymes
Assimilation Regulation
Ammonia Toxicity
Elevated Tissue Ammonia: Sources
Ammonia Detoxification
Grape Disorders
Inflorescence Necrosis
Late Rachis Necrosis
5
5
6
8
8
9
12
13
14
15
22
24
27
28
30
30
31
CHAPTER 3.
INFLORESCENCE NECROSIS: ONE CAUSE
OF POOR FRUIT SET IN GRAPES
35
Introduction
Description
Symptoms
Other Fruit Set Disorders
Distribution 1988
Research on Possible Causes
Summary
References
35
35
36
38
40
44
50
51
SEASONAL XYLEM AND CLUSTER TISSUE
AMMONIUM LEVELS OF CABERNET SAUVIGNON
GRAPEVINES
52
Abstract
Introduction
Materials and Methods
Results and Discussion
Summary
References
52
54
57
60
80
82
CHAPTER 4.
1
Pacre
CHAPTER 5.
CHAPTER 6.
TENDRIL MODEL SYSTEM TO INVESTIGATE
AMMONIUM TOXICITY AND ASSIMILATION
IN GRAPEVINE REPRODUCTIVE TISSUE . .
Abstract
Introduction
Materials and Methods
Results and Discussion
Summary
References
.
86
86
88
91
96
113
114
EPILOGUE
117
Introduction
Ammonium Concentration in Grape Tissue
Ammonium Concentration Determination
Seasonal Trend
Tissue Development
Disorders, is Ammonium Involved ?
Ammonium Toxicity
Tendril Model System
Ammonium Toxicity
Assimilation
Ammonium Accummulation
Conclusion
117
117
117
118
122
122
125
126
126
127
130
131
BIBLIOGRAPHY
132
APPENDICES
141
LIST OF FIGURES
Figure
Page
2-1.
Ammonia assimilation pathways and
nitrogen redistribution in plants.
17
2-2.
GS/GOGAT assimilation cycle.
18
3-1.
Pinot noir clusters at shatter affected
by INec.
37
3-2.
Pinot noir cluster near harvest severely
affected by INec.
39
3-3.
Pinot noir cluster at shatter with
poor set.
41
3-4.
INec severity in four Pinot noir
vineyards in the Willamette Valley
surveyed in 1988.
42
3-5.
INec severity in five clones of Pinot
noir in a clonal evaluation trial.
45
3-6.
Relationship between INec severity and
ammonium concentration in the rachis
tissue.
47
4-1.
Ammonium concentration of rachis and
berries in exposed vines in 1987 and
1988.
61
4-2.
Dry weight of rachis and berries in
1988.
66
4-3.
Total ammonium content of berries from
exposed and shaded vines in 1987.
67
4-4.
Total ammonium content of rachis from
1987 and 1988.
68
4-5.
Ammonium concentration in xylem exudates
from vacuum extracted shoots.
71
4-6.
Ammonium concentration of rachis and
berry tissue from exposed and shaded
vines in 1987.
76
Figure
Page
5-1.
Percent fresh weight change,
concentration of free ammonium, and
total nitrogen of tendrils over the
55 hr experiment treated with
distilled water, ammonium sulfate,
ammonium sulfate plusoc-ketoglutarate,
and ^<-ketoglutarate.
97
5-2.
Concentration of total free amino acid,
glutamate and glutamine amino acids in
tendrils treated with the same solutions
listed in Fig. 5-1.
101
5-3.
Free ^-aminobutyric acid concentration
in tendrils treated with the solutions
listed in Fig. 5-1.
106
5-4.
Free ammonium concentration in
tendrils treated with glutamate or
oc-ketoglutarate added to ammonium
sulfate.
107
5-5.
Fresh weight change and free ammonium
concentration of tendrils treated with
sucrose or glucose added to ammonium
sulfate, or used separately.
112
A-l.
Ammonium concentration in tendrils
treated with a range of ammonium
solution concentrations.
141
A-2.
Ammonium concentration in tendrils
treated with distilled water or
330 mM ammonium sulfate under
different oxygen concentrations.
142
A-3.
Fresh weight change of tendrils treated
with distilled water or 330 mM ammonium
sufate under different oxygen
concentrations.
143
A-4.
Rachis dry weight trend over 1988 from
the field sampling (Chapt. 4).
144
A-5.
Berry dry weight trend over 1988 from
the field sampling (Chapt. 4).
145
A-6.
Total ammonium content in berry tissue,
1988, from the field sampling (Chapt. 4).
146
Figure
Page
A-7.
Mean berry weight of Chenin blanc
clusters from vines in Summerland,
British Columbia, that had a history
of late rachis necrosis or freedom
from the disorder.
147
A-8.
Percent dry weight of Chenin blanc
rachis from vines in Summerland,
British Columbia, that had a history
of late rachis necrosis or freedom
from the disorder.
148
A-9.
Ammonium concentration of Chenin blanc
rachis from vines in Summerland,
British Columbia, that had a history
of late rachis necrosis or freedom
from the disorder.
149
A-10.
Total nitrogen in Chenin blanc rachis
from vines in Summerland, British
Columbia, that had a history of late
rachis necrosis or freedom from the
disorder.
150
LIST OF TABLES
Table
Page
2-1.
Comparison of nitrogen fraction
concentration reported for grape and
kiwifruit xylem exudates.
5-1.
Free ammonium concentration in tendrils
treated with distilled water, or ammonium
solutions with or without added
o<-ketoglutarate or oc-ketoglutarate plus
methionine sulfoximine.
10
110
AMMONIUM IN GRAPEVINES:
SEASONAL LEVELS IN TISSUE AND XYLEM EXTRACTS,
AND A TENDRIL MODEL SYSTEM
CHAPTER 1
Introduction
Grape is the fruit crop with the largest world
production (Westwood, 1978).
it has an ancient history.
Along with this distinction
Fossil leaves and seeds of grape
have been discovered in deposits from the Tertiary period of
geologic time (Winkler et al., 1974).
Not only does the
vine have a long history, its culture also extends back to
early times.
Winkler et al. (1974) stated that grape
culture began in Asian Minor and that records of
cultivation date back to 2440 BC.
is not a recent science.
Also, wine production
Plinys, along with others,
described many types of wine in their early written
records (Winkler et al., 1974).
The history and importance of the grape crop also
mean that it is one of the most researched fruit crops
grown.
Even with the considerable research effort that
has occurred over many years, there still remain many
aspects of the vine and its production that are not known.
Two disorders that affect the cluster are good examples.
Both inflorescence necrosis (syn., early bunch stem
necrosis, Jackson and Coombe, 19883^) and late rachis
necrosis (syns., shanking, stiellahme, waterberry,
dessechement de la rafle, disseccamento del rachide, bunch
stem die-back, pedicel necrosis, stalk necrosis, pedicel
girdling, Christensen and Boggero, 1985; Silva et al.,
1986) are disorders that have symptoms of necrotic pedicel
and peduncle tissue and which the cause(s) is not known.
Both can cause considerable yield loss and late rachis
necrosis causes the loss of quality in wine and table
grapes.
Inflorescence necrosis is a recently described
disorder that has received little research attention.
However, over at least the last century there has been
considerable research effort directed at the determination
of the cause of late rachis necrosis.
Still, the cause
remains unknown.
Although there are many suggested causes for late
rachis necrosis, recently the most popular hypothesis is
that some form of nitrogen or, more specifically, a toxic
ammonium concentration in the rachis is responsible
(Christensen and Boggero, 1985; Silva et al., 1986;
Jordan, 1985; Jordan et al., 1988a,b; Perez pers. comm.,
1987).
High concentrations of ammonium have also been
identified in rachis tissue affected by inflorescence
necrosis (Jordan et al., 1989).
Consequently, nitrogen
metabolism and vine ammonium are receiving research
attention.
Our knowledge of these aspects of vine
physiology is far from complete.
Increased information about nitrogen and ammonium
physiology will not only benefit our endeavors to discover
the cause(s) of the two disorders.
Potentially, it will
be valuable for ethyl carbamate (urethane) studies.
Ethyl
carbamate is a carcinogen that is found in small ammounts
in wine (Ough et al., 1988a).
The wine industry wants to
eliminate or limit the levels of this compound in their
products to maintain sales.
Recently, Ough and coworkers
(Ough et al., 1988a, b)have shown that arginine is a
critical precursor for ethyl carbamate formation.
Stress
increases arginine levels in avocado (Nevin and Lovatt,
1987), citrus and Poncirus (Rabe and Lovatt, 1984, 1986),
and squash (Rabe and Lovatt, 1986).
Accompanied with
these increased arginine levels are increased ammonium
levels.
The link between ammonium and arginine in grape
is not well defined.
Increased information about ammonium
levels and its metabolism in grape may be beneficial to
develop procedures to reduce ethyl carbamate levels in
wine via reduced arginine in the berries.
The objectives of the research in this thesis were:
1) characterize the ammonium concentration in rachis and
berry tissues throughout the season and relate these to
the development stages when infloresence necrosis and late
rachis necrosis occur;
2) establish if the ammonium concentration in xylem
extracts could have an important influence on the
concentration of ammonium in rachis and berry tissue;
3) develop a model system using grape tissue that could be
used to study ammonium toxicity and assimilation in grape.
This thesis describes the dynamic nature of ammonium
concentration in grape tissue and how detached tendrils
proved to be a valuable model system to study how tissue
closely related to reproductive tissue reacts to different
ammonium environments.
CHAPTER 2
LITERATURE REVIEW
Introduction
Nitrogen assimilation in autotrophic plant growth
represents a major metabolic flux.
Ammonia is the primary
inorganic nitrogen form involved in the synthesis and
catabolism of organic nitrogen.
Although ammonia is
critical for plant growth and development it is
potentially very toxic.
Consequently, the plant operates
on a fine line between what are adequate and toxic ammonia
levels.
Ammonia assimilation is the only way plants can
reduce elevated ammonia levels.
Unlike many other
molecules or ions, ammonia is difficult to compartmentalize because it is very membrane mobile.
Consequently, plants are unable to use compartmentalization as a protection strategy against elevated
ammonia.
This strategy is used with other harmful
materials where the vacuole serves to isolate them from
the cytoplasm with movement restricted by the tonoplast
(Salisbury and Ross, 1985).
This review describes the role of ammonia in plants
and, where available, in grape.
Highlighted is ammonia
assimilation and how this operates as a "detoxification"
mechanism to protect against toxic ammonia levels.
Also
described are ammonia toxicity symptoms and our lack of
precise knowledge about why or how ammonia is toxic.
The
review concludes with evidence for, and speculation of,
the potential involvement of ammonia toxicity in two grape
cluster disorders.
For convenience, throughout this review "ammonia"
includes both the molecule NH3 and ammonium, the cation
NH4+.
No attempt is made to distinguish between the
molecule and the ion.
Note, aqueous ammonia and ammonium
are in chemical equilibrium within tissue solutions —
H20 + NH3(aq) ^ NH4+ + 0H[ammonium hydrolysis constant (K) is 5.52 * 10-10 ,
Hageman, 1984].
Plant / Soil Interface
Most plants are capable of nitrate and ammonia uptake
from the root environment (Haynes and Goh, 1978).
However, nitrate is considered the major form because it
is more available in most soils than ammonia which is
rapidly converted to nitrate (nitrification) by
microorganisms (Hageman, 1984).
Root temperature and soil pH greatly affect ammonia
and nitrate availability and uptake in the root
environment (Hageman, 1984).
For example, Lycklama (1963)
found ammonia absorption was highly temperature dependent
with a 270C optimum for ryegrass in nutrient solution pH
4.0 to 6.5.
However, absorption was independent of
temperature with pH between 6.5 to 8.5.
Lycklama (1963)
suggested that this pH/temperature interaction reflected
ammonia being taken up as NH4OH between pH 6.5 to 8.5
rather than as NH4+ or NH3 at pH 4.0 to 6.5.
Little nitrification occurs at low pH or low soil
temperature (Buckman and Brady, 1969).
Increased ammonia
levels are available for plant uptake under these root
environments.
Calcifuge (acid-loving) plants are adapted
to take advantage of high soil ammonia at low pH (Haynes
and Goh, 1978).
These plants utilize ammonia in
preference to nitrate.
Ammonia uptake by roots, although not clearly
understood, is suggested to be passive whereas nitrate
uptake is an active process (Mengel and Kirkby, 1982;
Marschner, 1986).
Mengel and Kirkby (1982) reviewed
possible ammonia uptake mechanisms.
They proposed
that NH4+ may be deprotonated at the root cell
plasma membrane.
Consequently, NH3 may pass through the
membrane with the release of one H+ per ammonia.
This
proton release and that associated with a membrane bound
exchange carrier (ie., ammonia/proton cotransport)
explains the decreased pH that occurs with ammonia uptake.
Transport Within the Plant
Xylem: General
Although nitrogen solutes frequently are the major
component of dry matter in xylem extracts, and second only
to carbohydrate in the phloem (Pate, 1980, 1983), often
little of the total nitrogen is transported as nitrate or
ammonia.
Most nitrate is reduced by nitrate and nitrite
reductases, enzyme complexes that catalyze the reduction
of nitrate to nitrite and then to ammonia, before being
incorporated into organic forms.
However in some plants,
e.g., cocklebur (Xanthium strumarium) and cotton
(Gossypium), nitrate accounts for up to 95% of the xylem
nitrogen transported from the root (Pate, 1983).
Plants
of this type, often herbaceous species (Mengel and Kirkby,
1982), have little nitrate reductase activity in the root
but high activity in leaves.
Xylem nitrogen in plant species with active root
nitrate reductase is almost all in an organic form.
Generally, root nitrate reductase is high in woody species
(Mengel and Kirkby, 1982).
Pate (1983) listed a range of
nitrogen compounds found in xylem extracts.
These include
amides (glutamine, asparagine, and substituted amides),
amino acids, and alkaloids.
Usually only one or two
compounds predominate and they have a low carbon nitrogen ratio.
Xylem: Grapevine
Grapevine xylem composition is not clearly defined.
A large range in nitrogen solute concentrations are
reported in root pressure exudates (Andersen and Brodbeck;
1989a,b; Roubelakis-Angelakis and Kliewer, 1979; Marangoni
et al., 1986).
Sampling time, species, cultivar, plant
nutrition, environment and sampling site on the plant
(Pate, 1980; Ferguson, 1980), and possibly artifacts due
to sample preparation, could explain the large
concentration range found.
Table 2.1 lists the broad xylem nitrogen constituents
for comparison between four reports on grape and that for
kiwifruit (Actinidia deliciosa var. deliciosa), another
vine.
Prominent in this comparison are the abnormally
high concentrations Marangoni et al. (1986) reported.
Even at their sample time with low concentration, each
fraction was greater than all other reported grape levels.
It seems that Marangoni et al. (1986) experienced errors
or sample preparation artifacts.
Other than the
abnormally high concentrations, there are other indicators
of procedural errors.
For example, ammonia nitrogen in
10
Table 2.1
Comparison of nitrogen fraction concentration
(mM - nitrogen basis) reported for grape and kiwifruit
xylem exudates.
Ammonia
Nitrogen
Nitrate
Nitrogen
Amides
and free
Amino
Acids
4.6
1.1
0.1
3.4
[l]*a
9.7
0.6
0.9
8.3
[l]b
6.0
0.4
0.1
5.6
[2]a
12.7
2.1
0.2
10.6
[2]b
1.6
0.1
0.05
1.4
[3]a
5.9
0.3
0.6
5.0
[3]b
28.8
7.3
1.2
20. 3Y
[4]a
276.0
108.0
47.7
121.0Y
[4]b
36.1
1.4
1.5
27.4
[5]a
50.0
1.5
1.8
31.2
[5]b
Total
Soluble
Nitrogen
Source
Grape
Kiwifruit
* Adapted from [1]
[2]
[3]
[4]
[5]
Andersen and Brodbeck (1989a)
Andersen and Brodbeck (1989b)
Roubelakis-Angelakis and Kliewer (1979)
Marangoni et al. (1986)
Clark et al. (1986)
a or b indicate low and high total nitrogen samples from
each source.
y amino/amide fraction estimated as remainder from total
nitrogen less ammonia and nitrate nitrogen
fractions.
11
sample "b" was about 40 % of the total soluble nitrogen.
This is very high compared to the 3 - 20 %, with less than
10 % common, in other reports.
Roubelakis-Angelakis and Kliewer (1979) generally
found low concentrations.
They noted a big difference
between years but this was based on only one sample per
year.
Potentially, sample time differences relative to
vine physiological development could explain the year to
year difference.
Although Andersen and Brodbeck (1989a,b)
samples had intermediate concentrations, note they sampled
Vitis rotundifolia rather than Vs. vinifera used by
Marangoni et al. (1986) and Roubelakis-Angelakis and
Kliewer (1979).
Consequently, it is difficult to
establish what are "normal" nitrogen solute concentrations
in xylem extracts from Vi. vinifera.
Nitrogen solute concentration in xylem extracts
from kiwifruit vines is generally higher (ignoring
Marangoni et al., 1986) than that in grape (Table 2.1).
Clark et al. (1986) highlighted the large concentration
variation between sampled kiwifruit vines.
Unfortunately,
it seems that xylem extract determinations are inherently
variable and, at best, can only provide broad composition
information.
Nitrate nitrogen is about 2-15 % of the total soluble
12
nitrogen in grape xylem extracts (Table 2.1).
This
indicates that grape have significant root nitrate
reductase activity.
Nitrate reductase activity is present
in grape leaves (Perez and Kliewer, 1982; R.E. Smart,
pers. comm., 1985) but it has not been compared to root
nitrate activity.
Pate (1973) compared the relative
proportion of nitrate nitrogen to total nitrogen in many
plant xylem extracts.
The relative proportion of about
2-15% total nitrogen as nitrate in grape is similar to
plants where most nitrate reduction occurs in the roots.
Plants with most nitrate reduction in leaves or shoots
have greater than 50% nitrate nitrogen in xylem extracts.
For example, sunflower xylem extracts have about 50 %
nitrate nitrogen with 10 mM nitrate concentration
(Marschner, 1986).
This concentration is 10-100 times
that often found in grape xylem (Table 2.1).
Again, this
indicates that grape must have high nitrate reductase
activity in roots.
Phloem: General
Phloem exudates usually contain 10-20 times the xylem
concentration of nitrogen solutes (Pate, 1983).
Although
nitrate concentration maybe high in the xylem it is often
absent or much lower in phloem.
Pate (1980) highlighted the importance of phloem for
13
nitrogen supply to fruit.
In lupin (Lupinus albus), Pate
(1980) estimated that 89% fruit nitrogen is supplied by
the phloem.
The importance of phloem transport for ammonia is not
clear.
Reports are inconsistent about the relative
ammonia concentration in xylem and phloem extracts.
For
example. Hocking (1980) found tree tobacco (Nicotiana
glauca) had almost five times the ammonia concentration in
phloem compared to xylem extracts.
In contrast,
Richardson and Baker (1982) reported equal or lower
concentrations in phloem compared to xylem of cucurbits.
It has proved difficult, or impossible, to obtain
phloem extracts in many plants.
Nitrogen solute
composition of grapevine phloem has not been reported.
Transport Driving Force
Solute transport in xylem is predominantly by mass
flow (Marschner, 1986; Mengel and Kirkby, 1982).
However,
interactions occur between xylem solutes and cell walls of
vessels and xylem parenchyma.
Marschner (1986) described
these interactions as "exchange adsorption of polyvalent
cations and reabsorption of mineral elements and the
release (secretion) of organic compounds by surrounding
living cells (xylem parenchyma and phloem)".
Release or
secretion mechanisms can influence nitrogen solute
14
concentration along the xylem length (Pate, 1983;
Marschner, 1986).
For example. Pate et al. (1964) found
xylem nitrate concentration decreases along the xylem
length.
However, while nitrate decreases, organic
nitrogen, especially glutamine, increases.
Xylem-to-phloem transfers of nitrogen within a stem
or shoot can also affect the translocated nitrogen
concentration (Simpson, 1986).
Amino acids and amides are
the major nitrogen compounds involved in these exchanges.
Simpson (1986) believes that the exchanges occur via
transfer cells which are located along xylem and phloem.
Although mass flow is the main driving force in xylem
transport, transpiration can have different effects on
nitrogen solute distribution within the plant (Marschner,
1986).
Marschner (1986) gave the example of
15
N
distribution in beans fed labeled nitrate and ammonia.
Distribution of
15
N from nitrate closely followed
transpiration, whereas from ammonia it was independent of
transpiration.
This could indicate the greater importance
of phloem transport for ammonia and its organic nitrogen
products than xylem transport.
Ammonia Assimilation
Ammonia is the critical inorganic nitrogen form for
15
organic nitrogen synthesis and catabolism in plants.
Although nitrate is often the major inorganic form taken
up by plants, it is reduced in the plant to ammonia via
nitrite in root plastids or leaf chloroplasts by nitrate
and nitrite reductases (Joy, 1988).
Assimilation rapidly
follows with often little ammonia accumulation.
Enzymes
Before about 1973, the key first step to ammonia
assimilation was considered to be catalyzed by glutamate
dehydrogenase (Joy, 1988).
This enzyme catalyzes the
synthesis of glutamate from OC-ketoglutarate and ammonia
(Eqn. 1).
It is widely distributed in plants.
However,
after 1973 glutamine synthetase was found to have at least
a ten fold higher affinity for ammonia than glutamate
dehydrogenase.
Glutamine synthetase is located in
chloroplasts and root plastids, where most assimilation
occurs, which also indicates this enzyme could be the
primary assimilation pathway.
Glutamine synthetase
catalyzes glutamine synthesis from glutamate, ammonia, and
ATP (Eqn. 2).
Equation 1
NH3 + Oc-ketoglutarate + NAD(P)H
"■*■ L-glutamate +
NAD(P)+
Equation 2
L-glutamate + NH3 + ATP
^ glutamine + ADP + P^
16
The detection of glutamate synthase (GOGAT ,
glutamine amide:
2-oxoglutarate amino transferase,
oxidase) further supported the primary assimilation role
of glutamine synthetase.
GOGAT catalyzes the amide group
transfer from glutamine to o^-ketoglutarate for glutamate
synthesis (Eqn. 3).
Glutamate is more readily utilized in
amino acid synthesis than glutamine (Fig. 2.1).
Glutamine
synthetase and glutamate synthase form a complex often
called "GS-GOGAT pathway" or "glutamate synthase cycle"
(Fig. 2.2).
This complex is often considered the primary
assimilation pathway (Joy, 1988).
Equation 3
Glutamine + oc-ketoglutarate + reduced ferredoxin
[or NAD(P)+H3
2 L-glutamate + oxidised ferredoxin
[or NAD(P)+]
GOGAT has been renamed by the Enzyme Commission of the
IUB.
The systematic name of:
enzyme (Eqn. 3) is L-glutamate:
1) the ferredoxin-dependent
ferredoxin oxidoreductase
(transaminating);
2) the pyridinenucleotide-dependent enzyme (Eqn. 3) is L-glutamate:
NADP+ oxidoreductase (transaminating).
17
Lysine
^
Aspartate ^^^
Threonine
Homosenne
rine
Methionine
»
(Homoserine)
*
■
Alanine
Glycine
^f-Serine
■Cysteine
-Glutamate'
Ammonia-^-Glutamine
Asparagine
Proline
Tryptophan
:itruline
OrnithineHistidine
Carbamoylphosphate
Arginine
Gama-amino
butyric
Nucleic Acids
'—Nucleotides
Figure 2.1.
X Ureides
Ammonia assimilation pathways and nitrogen
redistribution in plants.
(1988) .
Adapted from Joy
18
GLUTAMINE
ADP + PH
Reductant
O^-Ketoglutarate
2 GLUTAMATE
Figure 2.2.
GS/GOGAT assimilation cycle.
Givan (1979).
Adapted from
19
Although GS/GOGAT is still considered the primary
ammonia assimilation path, Oaks (1987) and others (e.g.,
Tischner, 1987) provided supporting evidence for an
important role of glutamate dehydrogenase.
Glutamine
synthesized by glutamine synthetase from ammonia in the
root can serve as a nitrogen donor for the synthesis of
other amino acids (Fig. 2.1) or it can be exported to the
upper part of the plant.
With amino acid synthesis
glutamate is regenerated, via transamination, so it can
serve as substrate for further glutamine synthesis.
However, when glutamine is exported there is no glutamate
regeneration.
Oaks (1986) suggested that glutamate
dehydrogenase provides the alternative mechanism for
glutamate synthesis.
However, she overlooked the
potential glutamate supply from the shoot via the phloem.
Glutamate is exported in the phloem, so it could supply
the root (Pate, 1980).
Other evidence for a major role of glutamate
dehydrogenase is based on experiments that used
inhibitors and
15
N or
C, or a combination of the three.
Oaks (1980) listed experiments that not only showed the
importance of the GS/GOGAT assimilation path but a
concurrent role for glutamate dehydrogenase.
Methionine sulfoximine (MSX, an irreversible inhibitor
of glutamine synthetase), aminooxyacetate (AOA, a
20
transaminase and glycine decarboxylation inhibitor), and
azaserine (glutamate synthase inhibitor) are the common
inhibitors used in ammonia assimilation experiments.
Unfortunately, there are no specific inhibitors of other
ammonia assimilation pathways — glutamate dehydrogenase,
asparagine synthetase or carbamylphosphate synthetase
(Oaks, 1986).
Also, Sieciechowicz et al. (1989)
found that MSX was not a specific glutamine synthetase
inhibitor.
In their experiments with pea leaves, MSX
reduced ammonia release from other pathways, e.g.,
asparaginase.
Consequently, inhibitor studies can be
difficult to interpret.
Experiments that use labeled substrates plus
inhibitors have provided more complete information than
using inhibitors or labeled substrate alone (Oaks, 1986).
Ammonia is the common form
N is supplied and
C is
supplied as an organic or amino acid.
Differences in ammonia affinity (Km) between
assimilation enzymes have been used to identify their
relative importance in ammonia assimilation.
For example,
glutamine synthetase has a much higher affinity (0.001 to
0.002 mM Km) than glutamate dehydrogenase (10 to 80 mM Km)
(Stewart et al., 1980).
Consequently, glutamine
synthetase was considered the primary assimilation path
rather than glutamate dehydrogenase.
21
Recent enzyme studies have identified isozymes that
have different ammonia affinities.
Some early
interpretations of the relative importance of enzymes
based on Km values have been revised.
For example,
Tischner (1987) described several reports where ammonia
induced the formation of glutamate dehydrogenase isozymes
that had much reduced Km values (e.g., 76 mM reduced down
to 3 mM).
But these Km values are still several fold
higher than those for glutamine synthetase.
Ammonia assimilation mutants have been used to
further establish the relative importance of assimilation
pathways.
Joy (1987) identified such mutants in
Arabidopsis. barley and pea.
Photorespiration is a major
source of aiomonia in leaves (Miflin and Lea, 1980; Walker
et al., 1984).
When mutants lacking chloroplast glutamine
synthetase are grown in air plus light (i.e., conditions
that promote photorespiration) they rapidly accumulate
ammonia.
Mutants deficient of ferredoxin dependent GOGAT
accumulate glutamine and ammonia under the same
conditions.
These experiments do not support the
potential role of glutamate dehydrogenase because,
although present in normal levels in the mutants, it did
not assimilate significant amounts of the accumulated
photorespiratory ammonia (Joy, 1988; Srivastava and Singh,
1987).
22
Ammonia assimilation enzymes in grape have received
little research attention.
Glutamine synthetase
(Roubelakis-Angelakis and Kliewer, 1983b) and glutamate
dehydrogenase (Roubelakis-Angelakis Kliewer, 1983a)
activity have been detected in grape leaf and root
extracts.
These enzymes are also present in berry
extracts (Ghisi et al., 1984).
However, Roubelakis-
Angelakis and Kliewer (1983b) were unable to detect GOGAT
activity in grape roots.
This could reflect problems with
enzyme extraction rather than the absence of enzyme.
However, if GOGAT activity is low in grape tissue, other
ammonia assimilation pathways, e.g., glutamate
dehydrogenase, could be more important in grape than other
plants.
Assimilation Regulation
Enzymes associated with ammonia assimilation are
regulated by many mechanisms.
Tischner (1987) listed the
following factors that regulate glutamine synthetase :
1] Nitrogen supply to the cells;
2] Divalent cation availability;
3] Feedback inhibition by end products of glutamine
metabolism;
4] Covalent modification by adenylation/deadenylation;
23
5] Energy charge.
Feedback regulation and ATP availability (linked with
photosynthesis activity) are believed to be the most
important regulatory mechanisms (Tischner, 1987; Valpuesta
et al., 1987).
GOGAT activity is regulated by mechanisms similar to
those that regulate glutamine synthetase.
For example,
aspartate accumulation causes feedback control of GOGAT
(Valpuesta et al., 1987).
Carbon substrate supply is critical for ammonia
assimilation (Givan, 1979). ©<-Ketoglutarate is the
primary carbon substrate for glutamate synthesis
(glutamate dehydrogenase and GOGAT, Eqn 2 and 3).
Consequently, ammonia assimilation is restricted by an
inadequate supply of oc-ketoglutarate.
Givan (1979) stated
that carbon substrate supply is determined by the level of
available reserve carbohydrate and the rate it is
degraded.
Carbohydrate catabolism increases during active
ammonia assimilation (Givan 1979).
Direct supply of
©<-ketoglutarate depends on the activity of the
tricarboxylic acid (TCA) cycle.
Hence, availability of
precursors for the TCA cycle is critical to the supply of
carbon substrates.
24
Ammonia Toxicity
Ammonia toxicity in higher plants has been shown
repeatedly.
However, the exact biochemical cause and
toxic concentrations are not well defined (Given, 1979).
Many workers consider NH3/acf\ to be the toxic component of
aqueous ammonical nitrogen (Hageman, 1984).
Ammonia can
readily penetrate membranes to increase intracellular
and NH4+ concentrations (Hageman, 1984).
NH3
As stated
earlier, this ease of membrane mobility prevents ammonia
from being compartmentalized by the plant as is found with
other nitrogen forms, e.g., nitrate in cell vacuoles
(Salisbury and Ross, 1985).
Serious physiological and morphological disorders are
caused by toxic ammonia levels.
1) Chlorosis.
These include :
Chlorophyll synthesis can be inhibited
(Sauer et al., 1987) and chloroplast structure
changed (Puritch and Barker, 1967).
2) Reduced COj fixation (Hageman, 1984; Magalhaes and
Wilcox, 1984).
3) Uncoupled photophosporylation (Hageman, 1984; Magalhaes
and Wilcox, 1984; Sauer et al., 1987).
4) Inhibited photodependent activation of ATP synthetase
(Sauer et al., 1987).
5) Inhibited NADP+ reduction (Sauer et al., 1987;
Magalhaes and Wilcox, 1984).
25
6) Blocked starch synthesis (Magalhaes and Wilcox, 1984).
7) Reduced carboxylase activity (Magalhaes and Wilcox,
1984).
8) Depleted organic acid levels (Hageman, 1984).
9) Inhibited cell division (Sauer et al., 1987).
10) Deficient K+ and Ca++.
Plants grown in ammonia
solutions are often found to be deficient in
these nutrients and this is suggested to be the
reason for ammonia toxicity (Magalhaes and
Wilcox, 1984; Goyal et al., 1982).
11) Excess protons generated during ammonia assimilation,
if not eliminated from the cytoplasm, can result
a physiologically toxic intracellular pH
environment (Raven, 1987; Salsac et al., 1987;
Hageman, 1984).
Reduced growth, chlorosis, and/or necrosis are common
ammonia toxicity symptoms.
For example, toxic ammonia
levels in avocadoes causes leaf necrosis (Nevin and
Lovatt, 1987) and in radish interveinal chlorosis is
observed before marginal necrosis develops (Goyal et al.,
1982).
Reduced growth of plants only supplied ammonia in
comparison to nitrate or nitrate plus ammonia supplied
plants is mainly due to retarded root development (Lewis
et al., 1987; Lips et al., 1987; Lindt and Feller, 1987).
26
Restricted root growth results from the consumption of
carbon substrates imported from the shoot for ammonia
assimilation rather than the substrates being available
for root growth.
Lindt and Feller (1987) determined
carbon fluxes and a carbon budget for cucumber plants
grown in solutions containing nitrate or ammonia as the
nitrogen source.
They found that roots on nitrate
consumed about 50 % of the carbon supplied from the shoot
for biomass production whereas roots on ammonia consumed
only 20 %.
The difference resulted from a two fold
increase in carbon recycled to the shoot via the xylem (as
assimilated nitrogen compounds) by plants on ammonia
compared to those on nitrate.
Plants have different tolerances to elevated ammonia
levels.
Hageman (1984) reviewed examples of different
tolerances among species.
He suggested that differences
could even exist within a species.
Evidence of these
tolerances is gained when "normal" tissue ammonia levels
are compared.
For example, levels in radish shoots are
about 120 ug g-1 (Goyal et al., 1982) whereas tomato
shoots have about 2300 ug g-1 (Magalhaes and Wilcox,
1984) .
Goyal et al. (1982) showed that ammonia toxicity
symptoms occur in radish when ammonia is elevated to about
2000 ug g-1.
However, at this concentration tomatoes do
not express ammonia toxicity.
Grape cluster tissue seems
27
similar to tomato in that it has high "normal" ammonia
levels (Christensen and Boggero, 1985; Silva et al.,
1986).
Elevated tissue ammonia: Sources
Tissue ammonia levels are dynamic pools influenced by
imput and export components.
Ammonium input or supply to
a given tissue can occur directly by xylem or phloem
translocation and penetration of the plasmalemma.
Also,
many catabolic processes can generate ammonia within the
cell (Durzan and Stewart, 1983).
Elevated xylem ammonia levels can reflect root zones
with high availability of ammonia.
Cold soils or high
ammonia fertilizers increase both soil ammonia levels and
its uptake (Hageman, 1984).
Photorespiration is the major source of ammonia in
leaves of many plants (Miflin and Lea, 1980; Walker et
al., 1984).
This is unlikely to be a major source of
ammonia in grape rachis or berry tissue which have low
photosynthetic capacity.
Durzan and Stewart (1983) listed 30 reactions that
release ammonia in plant tissue.
Consequently, there are
many possible metabolic pathways for ammonia supply.
These supply components can be a greater ammonia source
28
than direct xylem supply (Miflin and Lea, 1980).
Elevated
ammonia could result if one or more of these metabolic
pathways has increased activity and the assimilation
capacity is exceeded.
Stress increases free ammonia levels in plant tissue
(Rabe and Lovatt, 1986; Nevin and Lovatt, 1987; Srivastava
and Singh, 1987).
Temperature, water, salt, infection and
pollution (Srivastava and Singh, 1987), nutrient deficiency
(Rabe and Lovatt, 1986), and low light (Smart et al.,
1988) stresses increase tissue ammonium levels.
There are
many possible explanations for this stress response.
Stress could affect carbon skeleton availability needed
for ammonium assimilation, e.g., ©<-ketoglutarate supply is
reduced when the tricarboxylic acid cycle activity is
restricted by stress.
Deamination is higher in stressed
than nonstressed plants (Srivastava and Singh, 1987), so
stressed plants have increased ammonia released by this
process.
Also, Srivastava and Singh (1987) list examples
where stress affects ammonia assimilation and, hence,
tissue ammonia levels.
Ammonia Detoxification
Assimilation is the main defense mechanism that
plants have to prevent ammonia accumulation (Givan, 1979).
Ammonia can also be volatilized from leaves but often this
29
does not constitute a large loss (Marschner, 1986; Hocking
et al.,1984).
Givan (1979) reviewed assimilation pathways involved
in ammonia detoxification.
He considered that there were
three important assimilation pathways — GS/GOGAT (Eqn. 2
and 3), glutamate dehydrogenase (Eqn. 1) and asparagine
synthetase (Eqn. 4) - for the removal of free ammonium.
In the earlier ammonia assimilation section the relative
importance of the first two pathways was discussed.
Givan
(1979) also based the relative role on ammonia affinity.
GS/GOGAT had the highest affinity so was considered the
primary detoxification mechanism.
Glutamate dehydrogenase
and asparagine synthetase generally have 3 to 4 orders of
magnitude lower affinity (although that appears to depend
on which isozymes are considered) than glutamine
synthetase, so Givan (1979) considered these pathways were
only important at high ammonia levels.
Equation 4
Aspartate + glutamine + ATP
asparagine + AMP + PP^ +
L-glutamate
Elevated ammonia levels result in an initial increase
of amides, particularly glutamine (Givan, 1979).
indicates the important role of GS/GOGAT.
This
However, Lovatt
and coworkers (Rabe and Lovatt, 1984, 1986; Nevin and
Lovatt, 1987) found arginine and ammonia concentrations
30
increased in stressed plants.
This increased accumulation
of arginine paralleled an increase in de novo arginine
biosynthesis.
Consequently this, and possibly other
assimilation pathways, could be involved in ammonia
detoxification.
Grape Disorders
Inflorescence necrosis (INec) and late rachis
necrosis (LRN) are physiological disorders that cause
necrotic rachis tissue but, as their names imply, occur at
different phenological stages.
INec (syn. early bunch
stem necrosis, Jackson and Coombe, 1988a,b) affects grape
inflorescences at or around flowering.
Whereas LRN (syn.
shanking, New Zealand; stiellahme, Germany, Switzerland;
waterberry, USA; palo negro, Chile; dessechement de la
rafle, France; Christensen and Boggero, 1985) affects the
rachis just after the onset of berry softening (veraison)
through to harvest.
Inflorescence Necrosis
Symptoms of INec are necrotic pedicels and flowers or
small berries.
Slightly affected inflorescences only have
a few necrotic pedicels.
and often abscise.
Affected flowers do not develop
With increased severity more pedicels
are affected and the necrosis can extend into peduncle
31
tissue.
There has been very little research on this disorder.
This reflects that it has often gone unnoticed and been
confused with "normal" fruit set problems.
However, after
its recent description (Jackson and Coombe, 1988a,b) it is
now recognized as an important cause of poor fruit set.
High rachis ammonia levels are associated with INec
affected tissue (Jordan et al., 1989).
Also, ammonia
applied as ammonium to inflorescences a week before bloom
induces typical ERN symptoms (Jackson and Coombe,
1988a,b).
Late Rachis Necrosis
INec and LRN have similar symptoms.
LRN in slightly
affected clusters shows as necrotic girdles, often about
2-3 mm wide, on a few pedicels.
Severely affected
clusters have completely necrotic pedicels and the
peduncle can have necrotic regions.
Berries attached to
affected tissue do not develop normal composition and
finally wither with the associated vascular disfunction.
Consequently, yield and, more importantly, fruit quality
are greatly reduced when vines are moderately to severely
affected.
The LRN disorder in Europe has been described
anatomically (Brendel et al., 1983).
First stomata and
32
the epidermis and hyperdermis cells that surround the
stomata in affected pedicels become necrotic.
Initially,
the necrosis spreads into the cortical tissue (collenchyma
and parenchyma) of the pedicel and, later, into the
peduncle.
Finally, phloem cells become necrotic.
In contrast to INec, LRN has been extensively studied
internationally.
Many causes have been suggested,
including water, nutrient, and hormone imbalances, soils
high or low in organic matter, low temperature at bloom,
over- and under-cropping (Christensen and Boggero, 1985;
Theiler and Muller 1986).
Pathogens have never been
consistently isolated from affected tissue.
Nutrient imbalances as the possible cause have
received the greatest research attention.
European
research found low calcium and magnesium are associated
with LRN (e.g., Feucht et al., 1975; Cocucci et al.,
1988).
However, these associations were not confirmed in
the USA (Christensen and Boggero, 1985) or New Zealand
(Jordan, 1984).
Recently, LRN research in the USA, Chile and New
Zealand has concentrated on the role of nitrogen.
Christensen and Boggero (1985) and Silva et al. (1986)
showed petiole and rachis nitrogen and ammonia levels are
higher in affected than unaffected vines.
However, tissue
33
sampling was always after symptoms had developed.
It is
not possible to determine if these associations are cause
or effect.
In New Zealand, a survey of table grape vineyards
showed those affected with LRN had higher soil biomassnitrogen, ammonia, calcium, phosphate, pH and lower
potassium than soils in unaffected vineyards (Haystead et
al., 1988).
These results did not indicate that nitrogen
is exclusively correlated with LRN incidence.
Gysi (1984)
found that high nitrogen fertilizer applications increased
LRN compared to lower or nil rates.
Also, unpublished
results by Haystead and coworkers using
N showed that
vine nitrogen metabolism is different in affected than
unaffected vines.
Combined, these results reinforce the
role of nitrogen in LRN.
Similar symptoms to LRN are induced in unaffected
grape tissue by applying exogenous ammonium solutions
(Jordan, 1985; Jordan et al., 1988a,b).
a common symptom of ammonia toxicity.
Also, necrosis is
These results
strengthen the association between LRN and ammonia but are
not definitive.
Although there has been extensive LRN research that
has spanned a century, we do not know the cause and
physiology associated with symptom development.
The
strongest lead is with nitrogen and its forms within the
34
vine.
However, our knowledge of vine nitrogen metabolism
is far from complete.
35
CHAPTER 3
INFLORESCENCE NECROSIS : ONE CAUSE OF POOR FRUIT SET
IN GRAPES
Introduction
Inflorescence necrosis (INec) is a disorder that can
severely affect fruit set in grapes.
In 1988, this
disorder was widespread in the Willamette Valley, Oregon
and other West Coast regions of the United States.
In
some vineyards which were severely affected, crop losses
of up to 50 % were attributed to the poor set caused by
INec.
INec was again observed in 1989, which suggests
that this disorder occurs most years but in the past it
has not been distinguished from other causes of poor set.
INec has only been described recently; little
information is known about its cause or physiology.
In
this article INec is described along with the preliminary
research results that indicate clonal and cultivar
differences in susceptibility and that high ammonium
concentrations are found in affected tissue.
Description
This disorder was recently described by David Jackson
and Brian Coombe in New Zealand and Australia respectively
36
(Jackson and Coombe, 1988a,b).
It is not a new problem.
It now seems that INec has occurred for many years but was
overlooked, or loosely described as "poor set" or
attributed to Botrytis infection.
INec has now been
recorded in many world grape growing regions.
Jackson and Coombe (1988a,b) have used the name
"Early Bunch Stem Necrosis" for this disorder.
However,
field observations have identified that pedicels, flowers
and small berries, as well as the cluster peduncle (bunch
stem), are affected.
Consequently, the term inflorescence
was considered better than bunch stem because it
encompasses all affected tissue.
Also, it clearly
indicates when the disorder occurs.
Symptoms
INec only affects clusters.
Slightly affected
clusters have a few necrotic flowers and pedicels (flower
or berry stalks).
Figure 3.1 shows necrotic flowers plus
pedicels in a moderately affected Pinot noir cluster.
With increased severity, affected clusters have many
affected pedicels and the necrosis can extend into the
peduncle which results in necrotic sections.
In extreme
situations, complete clusters are necrotic from extensive
peduncle damage.
Our symptom description is broader than that
37
Fig. 3.1.
INec.
Pinot noir clusters at shatter affected by
Note necrotic flowers in the cluster.
38
published by Jackson and Coombe (1988afb) who emphasized
peduncle tissue.
We consider individual necrotic flowers
and pedicels are also symptoms of INec.
Close inspection
of many so called "healthy" clusters identified that they
also had some necrotic flowers and pedicels.
Although
these clusters had adequate set (about 45 to 60 % set is
often considered adequate), a slight increase in severity
of INec would have reduced set.
INec can appear before bloom, at bloom and/or as late
as early fruit set.
Although the disorder occurs near
bloom, necrotic tissue and barren sections of clusters are
still visible at harvest (Fig. 3.2).
Fruit set is reduced because affected flowers or
young berries do not develop.
or remain on the cluster.
They can either fall from
Figure 3.2 shows a Pinot noir
cluster with large, barren sections where INec affected
berries have fallen. If many clusters are severely
affected as in figure 3.2, dramatic yield reductions can
be caused by INec.
Other Fruit Set Disorders
"Coulure" is a French term used to describe reduced
fruit set in grape where flowers fail to develop into
berries.
INec appears to be a disorder that could be
grouped under this broad category.
39
Fig. 3.2.
Pinot noir cluster near harvest severely
affected by INec.
Note necrotic sections of peduncle
and how few berries developed.
40
"Filage" and "millerandage" are other fruit set
disorders (Winkler et al., 1974) which differ from INec.
Filage is a flower disorder where initiated flowers do not
develop.
Affected clusters have tendril like sections
without flowers.
Millerandage (often called "hen and
chickens") is due to poor seed set.
Seedless or low
seeded berries do not fully develop.
INec is not simply due to poor pollination or
fertilization.
Non-pollinated flowers or unfertilized
berries remain or fall when green or yellow unlike the
necrotic berries in INec affected clusters.
Necrotic
flowers and pedicels distinguish INec from other fruit set
problems.
Figure 3.3a shows a Pinot noir cluster with
poor set.
Both INec affected flowers/berries and green
berries fell at shatter from this cluster (Fig. 3.3b).
Distribution 1988
INec occurred throughout New Zealand, Australia,
Pacific Northwest and California vineyards in the 1988
season.
We surveyed North Willamette Valley vineyards in
mid-summer, 1988, about three weeks after bloom when fruit
set was being determined, and visually scored INec
severity and incidence.
Figure 3.4 shows the severity
ranged from about 23 to 46 % over four of the surveyed
vineyards.
Severely affected 'Pinot noir* vineyards had
41
Fig. 3.3.
Pinot noir cluster at shatter with poor set.
a) Cluster after removal from the vine;
b) After cluster tapped on to hand.
INec affected
flowers and berries are necrotic (at tip of pencil)
whereas other berries are green.
42
UCD2A
2
2
VINEYARD
Fig. 3.4.
INec severity (visual score of percent necrotic
flowers) in four Pinot noir vineyards in the
Willamette Valley surveyed in 1988.
The clones
(UCD 4 = Pommard and UCD 2A = Wadenswil) in each
vineyard are listed on the top of the bars.
in vineyard 4 was unknown.
The clone
43
crop loss greater than 50 % and cluster weights were about
50 % lower than the previous year.
All vineyards surveyed
had some level of INec.
Within some vineyards there were large differences in
INec and the distribution was not easily explained.
One
vineyard had an area with very severe INec which caused
total crop loss.
An old dairy yard had been in this area
before the vineyard was developed.
In this example, a
carry-over from the prior use of the land may have
influenced INec incidence.
Also, the disorder tended to
occur in vineyards with severe spring boron deficiency,
but we suspect that these were separate phenomena.
Vineyards with excessive growth were more likely to be
affected.
Although INec was formally recorded for the first
time in 1988, industry members now think that it was not
the first time that it had occurred in Oregon.
Similar
symptoms were seen in 1981 and 1983 when there were large
or total crop losses in Willamette Valley Early Muscat and
Muscat Ottonel.
In other years INec has been seen but
with few commercial consequences.
Normal yield and
cluster sizes occur when fruit set is in the range 45 to
60 %.
Consequently, some loss from INec is unlikely to
reduce yield.
Grape cultivars vary in INec susceptibility.
In
44
1988,
Pinot noir in the Willamette Valley was greatly
affected, much more than most other cultivars.
Gewurz-
traminer and some muscats seem more susceptible cultivars.
Even cultivars that traditionally have good set can be
affected, e.g., Riesling.
Clones of some cultivars appeared to vary in INec
susceptibility.
In the 1988 vineyard survey (Fig. 3.4)
two vineyards had two clones of Pinot noir.
Poitunard (UCD
4) had greater severity of INec than the Wadenswil (UCD
2A) clone.
Clonal differences in INec susceptibility were
further highlighted when clones of Pinot noir in a clonal
evaluation trial were scored.
Colmar (538) was the most
severely affected with 72 % necrotic flowers (Fig. 3.5).
The least severely affected clone was, again, Wadenswil
with about 25 % severity.
Research on Possible Causes
This disorder has received little research attention.
This reflects how it has generally gone unnoticed or not
distinguished from other fruit set problems.
cause is unknown.
The specific
Apparently, pathogens are not involved
since Jackson and Coombe (1988a,b) were unable to isolate
any from affected tissue.
Research conducted in New Zealand and Australia
45
PINOT NOIR CLONE
Fig. 3.5.
INec severity (visual score of percent necrotic
flowers) in five clones of Pinot noir in a clonal
evaluation trial.
46
during the last two seasons has primarily described the
symptoms and the response to various nutrient and vine
treatments.
Last season in Oregon preliminary
investigations were made in to the role of nitrogen in
this disorder.
However, greater research effort is in
progress in both Oregon and California this season (1989).
INec seems to be similar to late rachis necrosis
(shanking, waterberry, stiellahme) a disorder that affects
the clusters near harvest.
Both can be induced by similar
methods (e.g., heavy shade), and develop necrotic regions
in affected clusters.
We do not have a complete
understanding of this late cluster disorder, but
information gained from research with it has been used to
develop INec research programs and hypotheses.
Present theories for a possible cause center on
nutritional imbalances, particularly elevated nitrogen or
ammonium levels.
In 1988 high ammonium levels were
associated with affected tissue.
Riesling clusters with a
range of INec severity were sampled about two weeks after
bloom.
Clusters with high (e.g., above 2.0 mg NH4+g
dw)
concentrations of ammonium in the rachis were more
severely affected than clusters with low concentrations
(Fig. 3.6). Above this concentration, none of the clusters
had slight severity of INec.
However, there was
variability in the severity at low ammonium concentrations
47
100
a:
UJ
o
o
a:
o
2
3
4
AMMONIUM CONCENTRATION
(mg NH4+ g~1 dw)
Fig. 3.6.
Relationship between INec severity (visual
score of percent necrotic flowers) and aitunoniuin
concentration in the rachis tissue.
48
in the rachis.
This could reflect problems associated
with visual scores rather precise flower counts.
INec incidence is increased by dense shade.
Lombard
(unpublished) found shaded (76 % shade) vines had 31 %
INec incidence compared to 2 % in nonshaded vines.
Again,
high ammonium concentrations were associated with the
disorder.
Concentration of ammonium in rachis tissue was
three fold higher in shaded vines compared to the
concentration in rachis from exposed nonshaded vines.
These associations of high ammonium concentrations in
affected tissue suggest that toxic ammonium levels in the
tissue could be the cause of INec.
Ammonium is
potentially very toxic to plants when levels are elevated.
Often necrosis is a symptom of ammonium toxicity, e.g.,
leaf necrosis in avocado (Nevin and Lovatt, 1987).
However, the possibility that elevated ammonium levels are
not the cause but an effect of the disorder cannot be
discounted.
Increased INec when vines are shaded could suggest a
role of photosynthetically produced assimilates.
Plant
tissue requires active photosynthesis to generate
carbohydrates and other carbon substrates.
These
substrates are critical for ammonium assimilation and
prevent ammonium accumulation to toxic levels in the
49
tissue.
If ammonium assimilation is limited by carbon
substrate supply, ammonium could accumulate and cause the
necrosis now called INec.
This hypothesis is being tested
in our laboratory.
Shade can be considered a stress.
Other stresses,
e.g., nutrient deficiency (Jackson, 1988), and severe leaf
removal, also increase INec incidence and severity.
The
limited experience with INec prevents associations being
made with other stresses.
Cool wet seasons, especially spring conditions, seem
to correlate with greater INec incidence than warm
seasons.
The month prior to bloom 1988 was one of the
wettest and coolest periods on record in the Willamette
Valley (Lombard et al., 1988).
The reason(s) for the
climatic effect on INec are not known.
If nitrogen or
ammonium are involved, perhaps cold spring temperatures
decrease the ability of the plant to assimilate ammonium
or increase uptake of these nutrients, although this last
point seems unlikely.
Also, the competition
between shoot tip and cluster sinks for carbon substrates
needed in ammonium assimilation may change under different
environments.
Different assimilation activities could
result in different concentrations of ammonium in the
tissue.
No remedy or prevention is available because the
50
cause of INec is not clearly understood.
If nitrogen is
involved, methods to reduce nitrogen, particularly
ammonium, in the clusters should be considered.
Good
shoot exposure and less, but not deficient, vine nitrogen
would help.
Summary
In summary, INec is a disorder that reduces fruit set
in grapes.
Affected cluster tissue is necrotic and
affected sections of the cluster can abscise, cluster
tissue and subsequently prevents berry development.
In
some 'Pinot noir' vineyards in the Willamette Valley,
severe INec greatly reduced fruit set and final yield.
specific cause is unknown but a relationship exists with
elevated ammonium levels and INec affected tissue.
A
51
References
Jackson, D. 1988. Poor fruit set in vines - the
significance of early bunch-stem necrosis.
Industry J. Aug:42-43.
Wine
Jackson, D.I. and B.G. Cooinbe. 1988a. Early bunchstem
necrosis in grapes - a cause of poor fruit set. Vitis
27:57-61.
Jackson, D.I. and B.G. Cooinbe. 1988b. Early bunchstem
necrosis - a cause of poor set, p. 72-75. In: R.E.
Smart, R.J. Thorton, R.J. Rodriguez, and J.E. Young
(eds). Proceedings of the Second International
Symposium for Cool Climate Viticulture and Oenology,
Auckland, New Zealand. New Zealand Soc. Vitic.
Oenol., Auckland.
Lombard, P.B., A.N. Miller, M. Westwood, K. Redmond, M.T.
AliNiazee, K. Powers, L. Smith, J. Maul, T. Darnell,
and K. Sherer. 1988. Phenology report, July 19, 1989.
Phenology report, Oregon State University, Corvallis,
Oregon, 13:1-2.
Nevin, J.M. and C.J. Lovatt. 1987. Demonstration of
ammonia accumulation and toxicity in avocado leaves
during water-deficit
stress. Yearbook, South African
Avocado Growers1 Assn. 10:51-54.
Winkler, A.J., J.A. Cook, W.M. Kliewer, and L.A. Lider.
1974. General viticulture. University of California
Press, Berkeley
52
CHAPTER 4
SEASONAL XYLEM AND CLUSTER TISSUE AMMONIUM LEVELS OF
CABERNET SAUVIGNON GRAPEVINES
Abstract
Free ammonium concentrations were determined in
rachis and berry tissue, and shoot xylem extracts from
six-year-old, field grown 'Cabernet Sauvignon* (Vitis
vinifera L.) vines sampled at phenological stages in 1987
and 1988.
Half the vines in 1987 were covered with 50 %
shade cloth.
Xylem contents were vacuum extracted from
cut shoots.
Concentrations early in the season for both
years were about 2.1 and 3.0 mg NH4+ g-1 dw (dry weight),
and at harvest about 0.10 and 0.05 mg NH4+ g-1 dw for the
rachis and berry tissue respectively.
Ammonium
concentration in the xylem declined from about
3.5 ug NH4+ ml-1 near bloom to 0.4 ug NH4+ ml-1 at
harvest.
The pre-veraison decline in xylem and rachis
ammonium concentrations preceded the decline in berry
ammonium concentration by about 20 days.
Shade caused a
three fold increase in xylem ammonium concentration near
bloom with no effect after this stage.
Ammonium levels in
rachis near bloom were not affected by shade, but by
harvest the rachis from shaded vines had about two times
53
higher levels.
There was a different trend in ammonium
levels of berries.
Berries from shaded vines had seven
fold higher ammonium concentration around veraison than
berries from exposed vines, but early and harvest
concentrations were similar.
The difference between
berries, and perhaps rachis, from shaded and exposed vines
was explained by delayed development of berries on shaded
vines.
54
introduction
Ammonium
concentration in cluster tissue has recently
received increased research attention.
This is because
elevated ammonium levels are found in cluster tissue
affected by two rachis disorders.
Jordan et al. (1989)
showed a positive relationship between inflorescence
necrosis (syn., early bunch stem necrosis, Jackson and
Coombe, 1988a,b) severity and rachis ammonium levels.
A
similar relationship exists with late rachis necrosis
(syns., shanking, stiellahme, waterberry, dessechement de
la rafle, disseccamento del rachide, bunch stem die-back,
pedicel necrosis, stalk necrosis, pedicel girdling)
(Christensen and Boggero, 1985; Silva et al.,1986).
Suggested from these relationships is that high
concentrations of ammonium are toxic to rachis tissue and
cause the necrotic symptoms.
Ammonium (NH3 is often
considered the toxic component of aqueous ammonical
nitrogen, Hageman, 1984) toxicity in higher plants has
been shown repeatedly with necrosis the common symptom.
For example, toxic ammonium levels in avocados causes leaf
necrosis (Nevin and Lovatt, 1987).
The association between grape disorders and tissue
ammonium were based on analyses of tissue sampled after
the symptoms had developed.
The dynamics of grape tissue
ammonium levels are not known.
It is possible that the
55
relationships just reflect an effect of the disorders.
Both inflorescence necrosis and late rachis necrosis
increase if vines are shaded.
Shade increased the
incidence of inflorescence necrosis incidence by 15 fold
in potted Cabernet Sauvignon vines (Jordan et al., 1989)
and incidence of late rachis necrosis by six fold in field
grown Thompson seedless (Perez, pers. comm., 1987).
In
both studies tissue from shaded vines had three fold
higher ammonium concentration than tissue from exposed
vines.
Toxic ammonium levels may result from more than one
factor.
Tissue ammonium is a dynamic pool influenced by
input and export components.
supply (input) ammonium.
The xylem can directly
Ammonium moves by mass flow in
the xylem and apoplast, and penetrates the plasmalemma
(Hageman, 1984).
Grapevine xylem sap contains ammonium
(Roubelakis-Angelakis and Kliewer, 1979; Marangoni et al.,
1986).
However, ammonium concentration in the xylem
throughout the season, and how this may influence cluster
disorders, are not known.
This paper reports sampling of field-grown Cabernet
Sauvignon vines to test the hypotheses : 1) Maximum
ammonium concentrations in rachis occur at stages when
inflorescence necrosis and late rachis necrosis develop;
2) Shade causes increased ammonium concentration in rachis
56
tissue; and 3) Ammonium concentrations in clusters reflect
xylem ammonium concentration.
1) For convenience, throughout this paper "ammonium"
includes both the aqueous molecule NH3 and ammonium, the
cation NH4 .
Note, measured ammonium is the combined pool
of ammonia and ammonium.
57
Materials and Methods
Plant Material and Experimental Design
Vitis vinifera L. cv. Cabernet Sauvignon vines in a
Willamette Valley, Oregon, commercial vineyard were
sampled in 1987 and 1988.
The vines were planted in 1981
and trained on a standard vertical trellis.
The soil is a
silty clay loam (Bellpine).
In 1987 six vine pairs were selected for similar
growth and size in one vineyard block.
From each pair,
one randomly selected vine was shaded with 50 % shade
cloth from 18 days before first bloom and throughout the
season.
Single, fruitful shoot samples were collected
randomly on nine mornings from each vine in 1987.
The
sample times were (date and days after first bloom): Prebloom (7 June, -3), Bloom (15 June, 5), Post-bloom (22
June, 12), Between bloom and veraison (28 July, 48), Preveraison (15 August, 66), Veraison (27 August, 78),
Immediately post-veraison (13 September, 95), Between
veraison and harvest (24 September, 106), and Harvest (15
October, 127).
No shade treatment was used in 1988.
Eight random
vines were sampled from the same block used in 1987.
58
Shoots were sampled early morning at five times (date and
days after first bloom): Bloom (10 July, 0), Between bloom
and veraison (6 August, 27), Veraison (4 September, 56),
Between veraison and harvest (17 September, 69), Harvest
(29 October, 111).
Leaves were stripped from the sampled shoots before
transport back to the laboratory.
Basal clusters from
each shoot were frozen at -18 C for later processing.
Xylem Exudate Extraction
Exudate was extracted under suction (Bollard, 1953).
In 1987 insufficient exudate volume was obtained from the
first two sample times.
Consequently, exudate composition
results are only presented for the post-bloom through to
harvest samples.
per shoot.
Exudate volume ranged from 0.2 to 7.2 ml
The freshly collected exudate was centrifuged
(750 g) for 10 min and then the supernatent diluted to
10 ml (the volume required for analysis).
The exudate
solution was promptly frozen at -18 C.
The last three exudate samples in 1988 were too
dilute for accurate analysis.
Consequently they, and
subsamples from the first two samples, were concentrated
at 60 C in a vacuum oven.
This procedure increased the
ammonium concentration in the exudate solution.
It seems
that ammonium was released from amino acids and other
59
ammonium containing molecules in the exudate.
Consequently, pre-concentration values only from the first
two samples are presented.
To confirm bloom concentration
further samples were taken 21 June 1989.
Non-diluted
exudates of four shoots from single vines were combined
and replicated eight times.
Ammonium Analyses
A procedure developed by Carlson (pers. comm., 1987)
was used to extract free ammonium in the tissue.
Rachis
and berry tissue samples were ground after being freeze
dried in 1987 and forced air dried at 63 C in 1988.
A
O.lg dried sample was extracted in 10 ml 2 % (v:v) acetic
acid.
Extracts in 16 mm test tubes were vigorously shaken
for 1 hr then let stand for 30 min at room temperature
before being filtered through an in-tube, serum filter
(Plasma/Serum separator, Karlan Chem. Corp., Torrence,
California).
The filtrate was stored at -18 C until
analyzed.
Ammonium concentrations of tissue extracts and xylem
exudates were determined on an ammonium analyzer developed
by Carlson (1978) or its commercial form (Wescan Ammonium
Analyzer Model 360, Alltech Assoc., Inc./ Wescan
Instruments, San Jose, California).
60
Results and Discussion
Rachis Ammonium Concentration
Maximum ammonium concentration (2.0 mg NH4
g
x
dw)
in rachis was at bloom and declined from bloom to veraison
(Fig. 4.1) in 1988 and exposed vines in 1987.
was more pronounced in 1988 than 1987.
The decline
However in both
years, ammonium concentration immediately before veraison
through to harvest was low (about 0.2 mg NH4
little change over this period.
g-1 dw) with
From veraison through to
harvest the ammonium concentration was about three times
higher in 1987 than 1988.
This may reflect the slower
development of the clusters in 1987 compared to 1988.
Note, bloom to veraison was about 22 days longer in 1987
than 1988.
Neither inflorescence necrosis or late necrosis were
observed in these cluster samples.
However, in 1988 Pinot
noir in the Willamette Valley was severely affected by
inflorescence necrosis (Jordan et al., 1989).
This
disorder occurs around bloom but, if ammonium is involved,
it may not just reflect bloom levels but also high
ammonium concentrations in pre-bloom tissue.
Bloom, or
pre-bloom was when ammonium concentration was naturally
high in rachis tissue so, potentially, this is a time that
any extra ammonium could result in toxic levels and cause
inflorescence necrosis.
61
RACHIS
z
o
I ' 2-
o 1987
• 1988
Ir
L
A
±
o
+
o <«-
V
4
V
i
•*»
—i—i—i—i—H
z
r
■»
0
T
s
?1
h
1
1
i -i—r
0
i—
BERRIES
o 1987
• 1988
yT 2
o+
I"
2
-O-w-
0-
H
0
Fig. 4.1.
1
1 1 1 1 1 1 1 1 1 1 1—
20
40
60
80
100
120
DAYS AFTER BLOOM
Ammonium concentration of rachis and berries in
exposed vines in 1987 (open circles) and in 1988
(filled circles).
arrows.
Veraison is indicated by the
Error bars are standard errors of the means.
With low variation, the error bars are covered by the
symbol.
62
Christensen and Boggero (1985) and Perez (pers.
comm., 1987) found rachis tissue affected by late rachis
necrosis, a post-veraison disorder, typically has two to
three fold higher ammonium concentration than unaffected
tissue.
In the present study, a three fold increase in
ammonium concentration of rachis tissue at post-veraison
or harvest would have resulted in a concentration less
than that at bloom.
If an increase of this magnitude can
cause necrosis late in the season it suggests :
1] Sensitivity of rachis tissue to ammonium decreased over
the season; 2] Tissue analyses average the amount of
ammonium over the total tissue sample, perhaps ammonium is
not uniformly distributed throughout the tissue and
localized concentrations could be high in critical cells,
e.g., at or around the stomata or lenticel cells where
symptoms occur first (Brendel et al., 1983);
3] If
neither 1] or 2] occur, the conclusion is that ammonium is
unlikely to be toxic late in the season and not
responsible for the late necrosis disorder.
Future
research should investigate the dynamics of ammonium
sensitivity and use procedures for micro-determination of
ammonium within the tissue.
The difference in ammonium concentration of rachis
between the two seasons may have reflected the different
climate patterns of the two years.
Spring of 1988 was
cool and wet which delayed bloom by about 2.5 weeks
63
compared to 1987 (Lombard et al., 1988).
However,
clusters developed quicker in 1988 than 1987.
This is
seen in the 22 day shorter bloom to veraison period in
1988 than 1987.
Free ammonium in tissue is affected by
the supply of carbon substrate for ammonium assimilation
(Givan, 1979).
Active shoot tips and clusters are sinks
that compete for how these substrates are partitioned.
Hence, if season affects the competitive advantage of one
or other sink, or the production of carbon substrate, the
ammonium concentration could be affected.
Rachis compared to berry tissue ammonium concentration
Ammonium concentration in rachis and berry tissue in
both seasons showed the same seasonal trends (Fig. 4.1).
Although the trend was the same, the ammonium
concentration in berries was more than twice that in the
rachis (about 3.0 compared to 1.2 mg NH4+ g-1 dw in 1987
and 3.1 compared to 0.5 mg NH4+ g-1 dw in 1988) at the
first berry sample.
Also, the decline in pre-veraison
concentration of ammonium was much greater in berries than
in rachis.
This resulted in similar (about
0.11 mg NH4+ g-1 dw) post-veraison concentrations for both
tissues.
Higher concentration of ammonium in berries than the
rachis indicates that berries receive more, assimilate
64
less or produce more ammonium than the rachis.
The rapid
decline of ammonium concentration in berries coincides
with the period of rapid berry development and increased
carbohydrate supply.
As stated earlier, ammonium
assimilation is dependent on the supply of carbon
substrates.
The decline could indicate enhanced
assimilation that resulted from increased carbon substrate
availability.
Also, the decline could reflect restricted
xylem supply of ammonium because the decline occurred when
xylem disfunction has been found in pedicels (Lange and
Thorpe, 1987; During et al., 1987; Findlay et al., 1987).
Another possibility to explain the trend in ammonium
concentration is that metabolic processes that release
ammonium within the berry also decline with berry
development.
Durzan and Stewart (1983) listed the many
catabolic processes that generate ammonium within cells.
Also, they provided examples in other plants where
nitrogen metabolism changed with development stage.
Phenylaline ammonia-lyase (PAL) is considered to be an
important enzyme for the release of ammonium (Durzan and
Stewart, 1983).
In berries, PAL activity increases at
veraison, associated with anthocyanin accumulation
(Hrazdina et al., 1984).
At this time ammonium
concentration is low in berries which suggests that PAL
metabolism does not greatly affect ammonium concentration
and that metabolic supply within the berry may not be a
65
major source of ammonium.
The trend of high ammonium concentration early in the
season and low concentration after veraison cannot be
explained by a dilution effect from gained dry weight of
rachis and berries.
Figure 4.2 shows there was little
change in rachis weight in 1987 (rachis from 1988 had the
same dry weights as the rachis from exposed vines in 1987,
Appendix 4) from bloom until about 50 days after bloom.
This was the period that ammonium concentration declined
in rachis tissue (Fig. 4.1).
Also, the rate that berry
dry weight changed (Fig. 4.2) did not correspond with the
change in ammonium concentration of the berries (Fig.
4.1).
For example, dry weight of berries from exposed
vines in 1987 (berries from 1988 had the same dry weights
as berries from exposed vines in 1987, Appendix 5) had an
almost constant increase over the season whereas ammonium
concentration rapidly declined just prior to veraison and
showed little change before or after this.
Ammonium amount vs concentration
The general lack of dilution effect from dry weight
changes in the tissue was also evident when the amounts of
ammonium in the berries (Fig. 4.3) and rachis (Fig. 4.4)
were calculated.
The amount of ammonium in the berries in
both years increased up to veraison (about 9.0 mg NH4 )
66
2.5
RACHIS
o EXPOSED
• SHADED
2.0-
C3
LOT
K
a
1.0 +
0.50.0
15
§
W
H
9-
>en
6-
a
1
h
H
1
1
1
1
1
1
h
BERRIES
©EXPOSED
•SHADED
12+
o
1
H
1
1 1 1 1 1 1 1 1 1 1 h
40
20
60
80
100
120
DAYS AFTER BLOOM
Fig. 4.2.
Dry weight of rachis and berries in 1988.
Plots with open circles are of tissue from exposed and
with open circles are of tissue from exposed vines
cloth) vines.
Veraison is indicated by the arrows.
Error bars are standard errors of the means.
With low
variation the error bars are covered by the symbol.
67
60
80
100
DAYS AFTER BLOOM
Fig. 4.3.
Total ammonium content of berries from exposed
(open circles) and shaded vines (filled circles) in
1987.
Veraison is indicated by the arrow.
are standard errors of the means.
Error bars
With low variation
the error bars are covered by the symbol.
68
1.5
1987
o EXPOSED
♦ SHADED
1.2-
0.0
1.5
-i
1
1
1
1
1
1
1
1
1
h
1988
1.20+- 0.9
o
H
1
h
100
120
DAYS AFTER BLOOM
Fig. 4.4.
1988.
Total ammonium content of rachis from 1987 and
The plot with open circles in 1987 are of
rachis from exposed vines and with filled circles from
shaded vines.
Veraison is indicated by the arrows.
Error bars are standard errors of the means.
With low
variation the error bars are covered by the symbol.
69
and then rapidly declined (Fig. 4.3).
This was similar to
the trend in ammonium concentration (Fig. 4.1) but the
pre-veraison increase was not observed.
This difference
up to veraison is an example of when an increase in berry
dry weight may have decreased the ammonium concentration.
Total ammonium in rachis tissue (Fig. 4.4) did not
follow the same trend as ammonium concentration (Fig. 4.1)
or dry weight of the rachis (Fig. 4.2).
Total ammonium in
rachis tissue had two maximums in 1987.
The first
occurred between bloom and veraison and the second just
after veraison (both about 0.7 mg NH4+).
Between these
maximums, total ammonium decreased to about 0.1 mg NH4 .
Although there were fewer samples in 1988 than in 1987,
total ammonium in rachis from 1988 also appeared to follow
a similar trend to that in 1987 (Fig. 4.4).
However, the
timing of the last maximum and the mid season minimum
occurred at earlier development stages in 1988 compared to
1987.
For example, the last maximum occurred at veraison
in 1988 whereas it was 17 days after veraison in 1987.
These differences are difficult to explain.
Ammonium
content in tissue is a dynamic pool that is influenced by
input (supply) and output (export) factors.
From the
following discussion of ammonium in xylem extracts, it
seems that differences in direct supply of ammonium could
not explain the differences in ammonium content of rachis
tissue throughout the season.
Perhaps the differences in
70
ammonium content reflected changes in ammonium
assimilation, an output factor.
Xylem extract ammonium concentration
The ammonium concentration in xylem extracts was high
early in the season but low thereafter (Fig. 4.5).
First
extracts at (1988 and 1989) or soon after (exposed 1987)
bloom had ammonium concentration of about 3 to
4 ug NH4+ ml-1.
For the rest of the season the
concentration was 1 ug NH4+ ml-1, or less.
A similar
seasonal trend is found with total nitrogen in xylem
extracts from apple shoots (Cooper et al., 1972).
Previously, ammonium concentration of grape xylem
extracts have been determined with samples expressed by
root pressure.
reported.
A wide range in ammonium concentration is
Marangoni et al. (1986) found about
1900 ug NH4+ ml-1 28 days after budbreak whereas others
report concentrations of 7 to 40 ug NH4+ ml
(Andersen
and Brodbeck, 1989 a,b) and 1.8 to 5.4 ug NH4+ ml"1
(Roubelakis-Angelakis and Kliewer, 1979).
Root pressure
sampling is limited to a period around budbreak.
Xylem
extracts from times later in the season have not been
reported.
Figure 4.5 shows that the early season ammonium
concentrations of 2 to 13 ug NH4+ ml-1 were within the
ranges reported by Andersen and Brodbeck (1989a,b) and
71
20
T
"1
1987
o EXPOSED
• SHADED
^ 1988
^ 1989
16-T
&
\-
?
TJ
12 -V
1
o 1
•y
o +
o '«*• a
V
\
UJ
z
X
z
o
2
J?
4
T
L \
s
1 ^^v
■^
<
0
1
h
1
1
1
20
40
1
1
60
1
1
1
80
1
1
100
1
120
DAYS AFTER BLOOM
Fig. 4.5.
Ammonium concentration in xylem exudates from
vacuum extracted shoots.
Full season sampling 1987
(circles) and limited early season samples in 1988
(open triangles) and 1989 (filled triangles).
The
plot with open circles are from exposed vines and with
filled circles from shaded vines in 1987.
indicated by the arrow.
errors of the means.
Veraison is
Error bars are standard
With low variation the error
bars are covered by the symbol.
72
Roubelakis-Angelakis and Kliewer (1979).
Also, the
concentrations were less than the 27 ug NH4+ ml-1 in
extracts expressed by root pressure from kiwifruit,
another vine (Clark et al., 1986).
High concentration of ammonium in xylem extracts
early in the season could reflect : 1) high root uptake ;
2) deamination of stored amino acids; 3) low ammonium
assimilation in the roots.
Cold soil temperature at
budbreak through bloom is predicted to increase ammonium
supply from the soil (Hageman, 1984) and, hence, the
potential for increased uptake and translocation of
ammonium.
Andersen and Brodbeck (1989a) showed this with
potted grapevines.
They found that cold (4 to 8 C) root
temperatures increased xylem extract concentration of
ammonium by 50 % compared to normal temperatures (22 to
28 C) .
Ammonium concentration in the rachis tissue (Fig.
4.1) followed a similar seasonal trend to that in the
xylem extracts (Fig. 4.5) but the xylem concentration
declined about 20 days earlier.
The high ammonium
concentration in rachis at bloom could reflect the high
concentration of ammonium supplied by the xylem.
However,
to establish this a measure of xylem flow rate is
required.
determined.
Xylem flow rates to the clusters were not
Blanke and Leyhe (1987) found cluster
73
transpiration per berry increases up to about veraison and
thereafter shows little change.
An increase in
transpiration should have elevated the ammonium supply to
the cluster up to veraison.
However, because free
ammonium is a dynamic pool, a change in supply does not
necessarily result in a change in the pool size because
output factors (e.g., assimilation) could change at the
same time.
Even with this concept of ammonium
concentration as a dynamic pool, the increase in total
ammonium in berries up to veraison (Fig. 4.3) could
reflect a change in xylem supply with increased
transpiration.
Recent xylem function and anatomy studies have shown
that xylem supply to the berry stops or is severely
restricted at about veraison (Lange and Thorpe, 1988;
During et al., 1987; Findlay et al., 1987).
If ammonium
in grape is predominantly xylem mobile its supply would be
restricted by this xylem disfunction.
Ammonium is
generally considered to be xylem mobile (Richardson et
al., 1982; Pate, 1976) although Hocking (1980) found more
ammonium in the phloem than xylem of tree tobacco
(Nicotiniana qlauca).
Also, the supply of ammonium in
leaves is considered to be independent of transpiration
(Marschner, 1985) which is interpreted to indicate that
ammonium is translocated in the phloem and not the xylem.
However, the results from this study could have been
74
influenced by changes in ammonium concentration in the
xylem rather than differential translocation within the
vascular system.
Pate (1980) described research with
lupin fruit which suggested that 89 % of the nitrogen in
the fruit is supplied by the phloem.
Most of this
nitrogen is supplied as amino acids which when deaminated
would release ammonium in the tissue.
Hence, the phloem
rather than the xylem maybe more is important for the
supply of ammonium in grape, its nitrogen composition
should be determined because it is not known.
Vacuum extraction to determine xylem composition has
some associated problems.
During early season, small
volumes of extract were obtained from the grape shoots in
this study.
Also, extracted fluid may be contaminated by
solution that originated from non-vessel tissue or nonfunctional xylem vessels (Hardy and Possingham, 1969;
Ferguson, 1980).
Comparisons between extracts obtained by
vacuum and root pressure extracts show different
composition (Hardy and Possingham, 1969; Ferguson, 1980;
Canny and McCully, 1988).
Also, vacuum extracts have been
criticized because they represent the average composition
from along the shoot.
Often a large concentration
gradient exists for individual constituents (Cooper et
al., 1972; Pate et al., 1964; Simpson, 1986).
What is
unknown is how the ammonium concentration in the shoot
75
extracts compared with the xylem supply received by the
cluster.
Ideally, xylem extracts should have been
collected from the cluster, however, attempts to do this
were unsuccessful.
Shaded compared to exposed vines
Artificially shaded vines had higher ammonium
concentration in xylem extracts early in the season (Fig.
4.5), in berries at around veraison (Fig. 4.6), and in
rachis from veraison through to harvest (Fig. 4.6)
compared to extracts and tissue from exposed vines.
Shade
caused a three fold increase in xylem ammonium
concentration from about 4.5 to 13.0 NH4+ ug ml
only at
12 days after bloom, the first xylem extract date.
Ammonium concentration for the remainder of the season was
similar for both shaded and exposed vines.
This response
suggests at least two possible mechanisms.
First, shade
is unlikely to have increased uptake of nitrogen by the
roots.
Conradie (1986) showed that early in the season
the demand for nitrogen in potted vines was greater than
root supply.
This shortfall is supplied from remobilized
nitrogen reserves stored within the vine.
Perhaps shade
increased this remobilization and the release of ammonium.
The second suggestion is based on the expectation
that shade reduced carbon substrate, energy and reduction
76
RACHIS
o EXPOSED
• SHADED
z
o
■
■o
^
UJ
2 -
|
1
*■
^w
^
^^
I^
X,
§1
2
■X
•
1——1
o
1
1
1
I
o
o
h-—i—^—1—-{
1
1
1—
BERRY
o EXPOSED
4
i'^
UJ
"
|
en
0+ 2
o -<*-
0H
0
1
h
H
20
40
1
1
60
1
1
80
1
1
100
1
h
120
DAYS AFTER BLOOM
Fig. 4.6.
Ammonium concentration of rachis and berry
tissue from exposed (open circles) and shaded (filled
circles) vines in 1987.
arrows.
Veraison is indicated by the
Error bars are standard errors of the means.
With low variation the error bars are covered by the
symbol.
77
potential and so limited ammonium assimilation.
elevated ammonium concentrations would result.
Thus,
At bloom,
carbohydrate reserves in the vine are depleted to the
lowest levels in the season (Winkler et al., 1974).
Consequently, at bloom the vine is dependent on currently
assimilated carbon substrate rather than that remobilized
from carbon reserves.
This stage is when shade is likely
to have maximum effect on carbon substrate supply for
ammonium assimilation.
Shaded vines are likely to be in a low energy state,
so may use free amino acids as source of energy.
Amino
acids can be metabolized to provide intermediates of the
tricarboxylic acid cycle for ATP synthesis and ultimately
feature in gluconeogenesis.
This metabolism of amino
acids would be predicted to result in an increased
concentration of ammonium in the tissue and xylem.
The
difference between shaded and exposed vines would be less
late in the season when plant development has slowed,
i.e., less demand for energy supply via amino acid
metabolism.
Although ammonium concentration in xylem extracts
near bloom was affected by shade, this difference did not
show in ammonium concentration of rachis or berry tissue
(Fig. 4.6).
This suggests that early season tissue levels
are not greatly influenced by the supply of ammonium from
78
the xylem.
Ammonium concentration of berries from shaded vines
had a similar seasonal trend but delayed by about 20 days
compared to berries from exposed vines (Fig. 4.6). This
response appeared to reflect delayed development of
berries on shaded vines.
This delayed development was
seen at 95 days after bloom when only about 15 % of the
berries were colored on shaded vines compared to 75 % on
exposed vines.
The effect of delayed development on ammonium
concentration in berries from shaded vines was also found
in total ammonium content (Fig. 4.3).
However, berries
from shaded vines had a lower maximum ammonium content
than berries from exposed vines.
This difference
reflected the difference in dry weight between the two
treatments (Fig. 4.2).
However, the difference in dry
weight did not affect ammonium content late in the season.
Consequently, changes or differences in dry weight cannot
fully explain the dynamics of ammonium concentration.
Ammonium assimilation appeared to have had considerable
effect on ammonium levels in berries.
Lombard (pers. comm., 1988) found artificial shade
(76% shade) increased ammonium concentration of the rachis
between bloom and veraison by about three fold from 1.2 to
4.0 mg NH4+ g-1 dw in potted Cabernet Sauvignon vines
79
grown in a greenhouse.
Fig. 4.6 shows that rachis from
shaded vines at a similar stage had only about 25% higher
ammonium concentration than rachis from exposed vines.
The difference between the two experiments could reflect
the deference (75 compared to 50 %) in degree of shade.
Delayed development seen in berries could also
explain the difference in ammonia concentration of rachis
from shaded and exposed vines (Fig. 4.6).
However, rachis
from shaded vines late in the season had about 0.25 mg
NH4+ g
higher ammonium levels than rachis from exposed
vines, whereas the levels in berries were similar.
This
difference between rachis and berries suggests the two
tissues had different ammonium assimilation capacity.
Over the season, this difference in assimilation is
indicated by the way berry ammonium was reduced by about
3.0 mg NH4+ g-1, whereas rachis ammonium decreased by
about 1.3 mg NH4+ g-1.
Alternatively, the late season
difference between berries and rachis could reflect lower
ammonium supply to berries compared to the rachis.
If
xylem supply to berries is restricted post-veraison,
berries would receive little ammonium compared to the
rachis.
These possibilities need to be tested.
Ideally,
the dynamics of the tissue ammonium have to be determined.
80
Summary
Rachis, berry and xylem ammonium concentrations were
highest during early season.
Ammonium concentration
declined first in the xylem extracts, then the rachis and
finally in the berries.
These declines occurred at or
before veraison to result in low ammonium concentrations
post-veraison through to harvest.
The decrease in
ammonium concentration could not be simply explained by
dilution from increases in tissue dry weight.
Shaded
vines had higher concentration of ammonium in xylem
extracts early in the season, in rachis from veraison
through to harvest, and in berries near veraison.
Although neither disorder was observed, at bloom, the
development stage that inflorescence necrosis occurs, was
when rachis tissue had the highest ammonium concentration
of any time throughout the season.
From veraison through
to harvest, the development stage when late rachis
necrosis occurs, was when the rachis had the lowest
seasonal ammonium.
Hence, the hypothesis that elevated
ammonium levels in rachis tissue occur at stages when the
two disorders occur was only supported for infloresence
necrosis and not late rachis necrosis.
The final hypothesis that cluster ammonium levels
reflect the ammonium concentration in the xylem was not
confirmed.
Although the ammonium concentration in xylem
81
extracts was high early in the season, the decline
preceded that in rachis and berry tissue.
Free ammonium
in the tissue appeared to be largely independent of the
ammonium concentration in the xylem.
Acknowledgments
We thank Dr. Frank Baynes for allowing us to sample
his Woodhall Vineyard and Dr. Robert Carlson, University of
California, Davis for the use of his facilities to
complete the 1987 ammonium analyses.
The Oregon Wine
Advisory Board provided financial support for the research
and the National Research Advisory Council, New Zealand, a
study scholarship for DTJ.
82
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temperature preconditioning on flux and chemical
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Andersen, P.C. and B.V. Brodbeck. 1989b. Diurnal and
temporal changes in the chemical profile of xylem
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Bollard, E.G. 1953. The use of tracheal sap in the study
of apple-tree nutrition. J. Exp. Bot. 4:363-368.
Blanke, M.M. and A. Leyhe. 1987. Stomatal activity of the
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Brendel, G., F. Stellwaag-Kittler, and R. Theiler. 1983.
Die patho-physiologischen kriterien der stiellahme.
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Canny, M.J. and M.E. McCully. 1988. The xylem sap of maize
roots: its collection, composition and formation.
Aust. J. Plant Physiol. 15:557-566.
Carlson, R.M. 1978. Automated separation and
conductimetric determination of ammonia and dissolved
carbon dioxide. Anal. Chem. 50:1528—1531.
Christensen, L.P. and J.D. Boggero. 1985. A study of
mineral nutrition relationships of waterberry in
Thompson Seedless. Amer. J. Enol. Vitic. 36:57-64.
Clark, C.J., P.T. Holland, and G.S. Smith. 1986. Chemical
composition of bleeding xylem sap from kiwifruit
vines. Ann. Bot. 58:353-362.
Conradie, W.J. 1986. Utilisation of nitrogen by the grapevine as affected by time of application and soil
type. S. Afr. J. Enol. Vitic. 7:76-83.
Cooper, D.R., D.G. Hill-Cottingham, and M.J. Shorthill.
1972. Gradients in the nitrogenous constituents of
sap extracted from apple shoots of different ages. J.
Exp. Bot. 23:247-254.
83
During, H., A. Lange, and F. Oggionni. 1987. Patterns of
water flow in Riesling berries in relation to
developmental changes in their xylem morphology.
Vitis 26:123-131.
Durzan, D.J. and F.C. Stewart. 1983. Nitrogen metabolism,
p. 55-265. In: F.C. Stewart and F.G.S. Bidwell (eds).
Plant physiology, a treatise. Vol. Ill: Nitrogen
metabolism. Academic Press, New York.
Ferguson, A.R. 1980. Xylem sap from Actinidia chinensis :
Apparent differences in sap composition arising from
the method of collection. Ann. Bot. 46:791-801.
Findlay, N., K.J. Oliver, N., Nii, and B.G. Coombe. 1987.
Solute accumulation by grape pericarp cells. IV.
Perfusion of pericarp apoplast via the pedicel and
evidence for xylem malfunction in ripening berries.
J. Exp. Bot. 38:668-679.
Hageman, R.H. 1984. Ammonium versus nitrate nutrition of
higher plants, p. 67-85. In: R.D. Hauk (ed). Nitrogen
in Crop Production. ASA-CSSA-SSSA, Madison.
Hardy, P.J. and J.V. Possingham. 1969. Studies on
translocation of metabolites in xylem of grapevine
shoots. J. Exp. Bot. 20:325-335.
Haystead, A., R. Leigh, D. Jordan, and R. Smart. 1988.
Soil fertility and shanking in table grapes, p. 8082. In: R.E. Smart, R.J. Thorton, R.J. Rodriguez, and
J.E. Young (eds). Proceedings of the Second
International Symposium for Cool Climate Viticulture
and Oenology, Auckland, New Zealand. New Zealand Soc.
Vitic. Oenol., Auckland.
Hocking, P.J. 1980. The composition of phloem exudate and
xylem sap from tree tobacco (Nicotiana glauca Grah.).
Ann. Bot. 45:633-643.
Hrazdina, G., G.F. Parsons, and L.R. Mattick. 1984.
Physiological and biochemical events during
development and maturation of grape berries. Amer.
J. Enol. Vitic, 35:220-227.
Jackson, D.I. and B.G. Coombe. 1988a. Early bunchstem
necrosis in grapes - a cause of poor fruit set. Vitis
27:57-61.
84
Jackson, D.I. and B.G. Coombe. 1988b. Early bunchstem
necrosis - a cause of poor set, p. 27-75. In: R.E.
Smart, R.J. Thorton, R.J. Rodriguez, and J.E. Young
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Auckland, New Zealand. New Zealand Soc. Vitic.
Oenol., Auckland.
Jordan, D.T., P.B. Lombard, and S. Price 1989.
Inflorescence necrosis in grapes : description and
preliminary research. Amer. Soc. Enol. Vit. Annual
Meeting, Anaheim, P. 2. (Abst.)
Lange, A. and M. Thorpe. 1988. Why do grape berries
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Rodriguez, and J.E. Young (eds). Proceedings of the
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AliNiazee, K. Powers, L. Smith, J. Maul, T. Darnell,
and K. Sherer. 1988. Phenology report, July 19, 1989.
Phenology report, Oregon State University, Corvallis,
Oregon, 13:1-2.
Marschner, H. 1986. Mineral nutrition of higher plants.
Academic Press, London.
Marangoni, B., C. Vitagliano, and E. Peterlunger. 1986.
The effect of defoliation on the compostion of xylem
sap from Cabernet franc grapevines. Amer. J. Enol.
Vitic. 37:259-262.
Nevin, J.M. and C.J. Lovatt. 1987. Demonstration of
ammonia accumulation and toxicity in avocado leaves
during water-deficit stress. Yearbook, South African
Avocado Growers' Assn. 10:51-54.
Pate, J.S. 1976. Nutrients and metabolites of fluids
recovered from xylem and phloem: Significance in
relation to long distance transport in plants, p.
253-345. In: l.F. Wardlaw and J.B. Passioura (eds).
Transport and transfer processes in plants. Academic
Press, Canberra.
Pate, J.S. 1980. Transport and partitioning of nitrogenous
solutes. Ann. Rev. Plant Physiol. 31:313-340.
85
Pate, J.S., W. Wallace, and J. van Die. (1964). Petiole
bleeding sap in the examination of the circulation of
nitrogenous substances in plants. Nature (London)
204:1073-1074.
Richardson, P.T., D.A. Baker, and L.C. Ho. 1982. The
chemical composition of cucurbit vascular exudates.
J. Exp. Bot. 33:1239-1247.
Roubelakis-Angelakis, K.A. and W.M. Kliewer. 1979. The
composition of bleeding sap from Thompson Seedless
grapevines as affected by nitrogen fertilization.
Amer. J. Enol. Vitic. 30:14-18.
Silva, H., G. Gil, and J. Rodriguez. 1986. Desecacion del
escobajo de la vid, palo negro. Un excesso de
nitrogeno amoniacal ? Rev. Fruticola 7:52-54.
Simpson, R.J. 1986. Translocation and metabolism of
nitrogen: whole plant aspects, p. 71-96. In: H.
Lambers, J.J. Neeteson, and I. Stulen (eds).
Fundamental, ecological and agricultural aspects of
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86
CHAPTER 5
TENDRIL MODEL SYSTEM TO INVESTIGATE AMMONIUM TOXICITY
AND ASSIMILATION IN GRAPEVINE REPRODUCTIVE TISSUE
Abstract
Detached tendrils are a useful model system to study
ammonium toxicity and assimilation in tissue closely
related to that of clusters.
Cabernet Sauvignon (Vitis
vinifera L.) tendrils from greenhouse grown, potted vines
lost turgidity and fresh weight, and later became necrotic
when continuously supplied 330 mM ammonium sulfate via the
cut end.
Concentration of free ammonium in treated
tendrils increased about 12 mg NH4+ g-1 dw compared to
tendrils in distilled water.
about 4 to 6 mg NH4+ g
This increase was halved (to
) when 330 mM oc-ketoglutarate or
glutamate was added to the ammonium solution.
This
response was attributed to stimulated ammonium
assimilation.
Added ©C-ketoglutarate increased free amino
acid concentration in the tissue by about
1 mg NH4
g
dw. Glutamate and o-aminobutyric acid
concentrations increased five and two fold respectively in
tendrils treated with 330 mM ammonium sulfate plus 330 mM
^-ketoglutarate compared to tendrils in ammonium alone.
©C-Ketoglutarate alone increased glutamate concentration
eight fold and o_aminobutyric acid concentration five fold
87
compared to the concentrations of tendrils in distilled
water.
The patterns in amino acid changes with each
treatment suggest that one or more ammonium assimilation
pathways were operating.
Glutamate dehydrogenase,
glutamine synthetase and glutamate synthase (GOGAT) could
all have been active.
Methionine sulfoximine (MSX), a
glutamine synthetase inhibitor, did not prevent the
decrease in free ammonium caused by oc-ketoglutarate.
This suggested that the assimilation path catalyzed by
glutamate dehydrogenase was involved in the ability of
oc-ketoglutarate to stimulate assimilation.
Sucrose or
glucose (165 and 330 mM) added to 330 mM ammonium solution
provided little beneficial effect.
Sucrose (330 mM), but
not glucose, reduced free ammonium concentration about
4 mg NH4+ g-1 dw compared to the concentration in tendrils
treated with ammonium alone.
88
Introduction
Ammonium1 and ammonium assimilation in grape vines
have recently received increased research attention
because high ammonium levels have been found in tissue
affected by two rachis disorders.
Jordan et al. (1989)
showed a positive relationship between the severity of
inflorescence necrosis (syn., early bunch stem necrosis,
Jackson and Coombe, 1988a,b), a disorder that affects the
rachis near bloom, and rachis ammonium concentration.
A
similar relationship exists with late rachis necrosis
(syns., shanking, stiellahme, waterberry, dessechement de
la rafle, disseccamento del rachide, bunch stem die-back,
pedicel girdling, stalk necrosis) (Christensen and
Boggero, 1985; Silva et al., 1986; Perez pers. comm.,
1987).
Suggested from these relationships is that high
concentrations of ammonium are toxic to the rachis tissue
and causes the necrotic symptoms.
Ammonium assimilation is the only way plants can
prevent ammonium accumulation (Givan, 1979).
Glutamine
synthetase (GS, catalyzes the synthesis of glutamine from
glutamate and ammonium) and glutamate synthase (GOGAT,
glutamine amide: 2-oxoglutarate amino transferase,
oxidase, catalyzes the synthesis of two glutamate from
glutamine and oc-ketoglutarate) as an enzyme complex
(GS/GOGAT) is often considered the primary assimilation
89
pathway for anunonium (Joy, 1988).
However, the relative
importance of other assimilatory pathways is being
reassessed.
Oaks (1987) considers that glutamate
dehydrogenase (GDH, catalyzes the synthesis of glutamate
from oc-ketoglutarate and ammonium) is involved in the
primary assimilation of ammonium.
Very little is known
about ammonium assimilation in grapevines.
GS
(Roubelakis-Angelakis and Kliewer, 1983b) and GDH
(Roubelakis-Angelakis and Kliewer, 1983a) activity have
been detected in grape leaf and root extracts.
These
enzymes are also present in berry extracts (Ghisi et al.,
1984).
However, GOGAT activity has not been reported in
any grape tissue and was not detected in root extracts
(Roubelakis-Angelakis and Kliewer, 1983b).
Ammonium
assimilation in rachis tissue has not been studied.
Ammonium assimilation and toxicity studies in higher
plants have been on the whole plant or with various tissue
model systems.
Examples of model systems are: detached
leaves (Walker et al., 1984), leaf discs (Platt et al.,
1977), callus cultures (Dwivedi et al., 1984), cell
suspension cultures (Morillo and Sanchez de Jimenez,
1984), and isolated chloroplasts (Woo et al., 1987;
Puritch and Barker, 1967).
With these model systems the
tissue can be exposed to ammonium and substrates of
ammonium assimilation.
Also, assimilation inhibitors,
e.g., methionine sulfoximine (MSX) a GS inhibitor, are
90
used to help identify the primary assimilation pathways.
Perennial fruit crops, like grape, are often difficult to
work with because of seasonal availability of fruit
tissue.
A model system for grape rachis studies that does
not have the same seasonal limitations was sought.
The
present study evaluates tendrils as a potential model
system to study tissue reaction to ammonium.
Tendrils are
the most closely related tissue to that of clusters as
they both have a common origin within the bud (Winkler et
al., 1974).
From preliminary studies, detached tendrils
and rachis have similar symptoms (fresh weight loss and
tissue necrosis) when their cut ends are exposed to
ammonium solutions.
Ammonium assimilation in tendrils
treated with high ammonium concentrations is enhanced by
the supply of oc-ketoglutarate, a critical substrate for
ammonium assimilation.
This response was repeated when
glutamate was used rather than oc-ketoglutarate but sucrose
and glucose did not substitute.
The benefit from oc-keto-
glutarate and glutamate suggested that ammonium
assimilation in grape reproductive tissue is limited by
carbon substrate supply.
1) For convenience, throughout this paper "ammonium"
includes both the aqueous molecule NH3 and ammonium, the
cation NH4+.
Note, measured ammonium is the combined pool
of tissue ammonia and ammonium.
91
Materials and Methods
Four experiments are reported here.
Materials and
methods common to all the experiments are described first
and then followed by specific information for each
experiment.
Plant Material
Potted Vitis vinifera L. cv. Cabernet Sauvignon were
grown in a greenhouse with supplemental light to maintain
a 16 hr daylength.
Turface (a calcined montmorillonite
clay. International Minerals and Chemical Corporation,
Mundelein, Illinois) media was used and the plants
received liquid fertilizer applied regularly.
The
fertilizer was modified from Smith et al. (1980) with 10 g
20-10-20 (N,P,K; Peters , W.R. Grace and Co., Allentown,
Pennsylvania) I-1, 5 g calcium nitrate 1
, 2 g magnesium
sulfate l-1, and 0.4 g commercial micro-nutrient mix
(Peters, 15.0 % sulfur, 1.45 % boron, 3.2 % copper, 7.5 %
iron, 8.15 % manganese, 0.046 % molybdenum, and 4.5 %
zinc) I"1.
Plant age varied from the equivalent of the
first through to the third season of growth.
Tendrils were cut from the shoot at nodes opposite
the most recently expanded leaf and, at most, three
tendril bearing nodes below this node.
Collected tendrils
were promptly transferred to the laboratory and about a
92
10 mm basal section removed as they were recut immediately
before being placed in about 8 ml of treatment solution.
Solutions were contained in 12 ml vials with a watertight, rubber cap that had a small hole for the tendril.
In all except the first experiment, a single tendril
per vial was used as a replicate.
Tendrils plus treatment
vials were kept at room temperature under laboratory light
throughout each experiment.
Analyses and measurements
Change in tendril fresh weight over the experimental
period was the difference between the weight at setup and
that at end of the experiment.
This weight change
(negative if weight loss or positive if weight gain) was
expressed as a percentage of the setup weight to
compensate for differences in initial tendril size.
For ammonium determinations tendrils were forced air
dried at 63 C.
To limit tissue loss, the dried tendrils
were hand ground in small paper envelopes.
A procedure
developed by Carlson (pers. comm., 1987) was used to
extract the free ammonium.
About 0.05 to 0.10 g of dried
tissue was extracted in 10 ml 2 % (v:v) acetic acid.
Extracts in 16 mm test tubes were vigorously shaken for
1 hr then let stand for 30 min at room temperature before
being filtered through an in-tube serum filter
93
(Plasma/Serum separator, Karlan Chem. Corp., Torrence,
California).
analyzed.
The filtrate was stored at -18 C until
Ammonium concentration was determined on an
ammonium analyzer (Wescan Ammonium Analyzer Model 3 60,
Wescan, California).
Experiment Designs and Treatments
oc-Ketoqlutarate.
Four treatment solutions were used:
distilled water (control); 330 mM ammonium sulfate (NH4+);
NH4+ plus 330 mM oc-ketoglutarate; and 330 mM ©C-ketoglutarate.
The ammonium sulfate concentration was
selected after preliminary experiments showed this
concentration to be toxic within 48 hr after the
experiment was setup.
All solutions were buffered to pH
6.0 with 2-(N-morpholino)-ethanesulfonic acid (MES).
The
experimental period was 55 hr.
In this experiment there were three replicates with
seven tendrils per replicate.
They were divided into
three or four per vial.
At harvest, the combined tendril tissue of each
replicate was cut into small sections and divided into two
samples.
One sample was dried for ammonium (see above
description) and total nitrogen analysis.
Total nitrogen
in the tissue was determined using a micro-Kjeldahl digest
on a 0.1 subsample of the dried tissue.
The ammonium
94
concentration of the digest was analyzed on the same
analyzer used for tissue ammonium analyses.
The other fresh sample (1.0 g) was prepared for amino
acid analyses.
First, this sample was promptly
homogenized in 5 ml of 75 % (v:v) ethanol with three
further 5 ml volumes used as washes.
After
centrifugation, the supernatent was evaporated to dryness
in a rotary evaporator at 10 C.
The sample was then
resuspended in lithium citrate buffer.
Amino acid
content and concentration of the sample were
determined in a Beckman 121 MB Amino Acid Analyser
equipped with a sulfanated, polystyrene bead, cation
exchange column (AA 10).
Glutamate compared to oc-ketoglutarate.
The six treatment
solutions in this experiment were : distilled water
(control); 33 0 mM ammonium sulfate (NH4+); NH4+ plus
170 mM oc-ketoglutarate; NH4+ plus 340 mM ©C-ketoglutarate;
NH4+ plus 170 mM glutamate; and NH4+ plus 340 mM
glutamate.
The pH of all solutions in this, and
subsequent, experiments was adjusted to 6.0 with sodium
hydroxide.
replicates.
Four tendrils in separate vials were the
The experimental period was 72 hr.
Methionine sulfoximide (MSX).
The four treatment
solutions used in this experiment were: distilled water
95
(control); 330 mM ammonium sulfate (NH4+); NH4+ plus
340 mM<x-ketoglutarate; and NH4+ plus 340 mM
Oc-ketoglutarate plus 10 mM
(MSX).
separate vials were the replicates.
Six tendrils in
The experimental
period was 51 hr.
Sugars.
Six treatments were used in this experiment :
distilled water (control); 330 mM ammonium sulfate (NH4+);
NH4+ plus 165 mM sucrose; NH4+ plus 330 mM sucrose; NH4+
plus 167 mM glucose; and NH4+ plus 330 mM glucose.
tendrils in separate vials were the replicates.
experimental period was 48 hr.
The
Four
96
Results and Discussion
Model System
Detached tendrils remained green and turgid for at
least 3 days when their cut ends were kept in distilled
water.
Ammonium toxicity symptoms develop in tendrils
placed in 330 mM (6 g NH4
1
) ammonium sulfate.
These
tendrils wilted, evident by the drooping and non-erect
nature, and the tips became necrotic (brown/black) which
can extend back to the solution level.
The necrosis was
similar to that in detached clusters treated with ammonium
solutions (Jordan, 1985; Jordan et al., 1988) and similar
to the necrosis seen in the field associated with late
rachis necrosis.
These similarities indicated the
suitability of tendrils as a model system to study
ammonium toxicity and assimilation in cluster or closely
related tissue.
Other advantages that make tendrils a useful model
system are tissue availability and simple tissue unit.
Cluster tissue studies are restricted by the seasonal
availability of clusters.
Tendrils can be produced with
no seasonal restriction from greenhouse grown plants.
Also, up to 10 tendrils per shoot make tendrils more
attractive to work with than clusters because at best two,
and usually only one, clusters are available per shoot.
Another advantage of tendrils is that they are a less
97
o
8
o
fc -10
I ~15
C
-20
•^O
Fig. 5.1.
NHn4
4
HH.
nnj
+
KGUU
KOUU
Percent fresh weight change (A), concentration of
free ammonium (B), and total nitrogen (C) of tendrils
over the total 55 hr experiment treated with distilled
water (dl^O) , 330 mM ammonium sulfate (NH4) , 330 mM
ammonium sulfate plus 340 mM oc-ketoglutarate (NH4 +
KGLU), and 340 mM <x-ketoglutarate (KGLU).
are standard error of the means.
Error bars
Means with the same
letter are not significantly different (LSD, p < 0.05).
98
complex tissue unit compared to clusters with berry,
pedicel and various levels of branched peduncle tissues.
The change in fresh weight of the tendril over the
experiment proved to be a useful index of ammonium
toxicity.
Tendrils in water often gained or lost little
weight whereas in ammonium the tendrils lost up to 10-20 %
of their weight (Fig. 5.1A).
Other indices of ammonium
toxicity were evaluated during the development of the
model system (Jordan, unpublished).
For example,
increased ethylene generation or respiration was not
associated with ammonium toxicity stress.
Consequently,
percent change in fresh weight of tendrils was used in all
subsequent experiments.
Ammonium toxicity
In the 55 hr period of first experiment with oc-ketoglutarate, 33 0 mM ammonium sulfate caused a 19 % loss of
tendril fresh weight compared to less than 1% loss for
tendrils in distilled water (Fig. 5.1A).
The addition of
OQ-ketoglutarate to the ammonium solution prevented the
weight loss caused by ammonium (Fig. 5.1A).
Tendrils in
ammonium plus oc-ketoglutarate and cs-ketoglutarate alone
had the same weight loss as those in distilled water.
These weight loss responses were seen in symptoms.
Tendrils in ammonium lost turgidity and started to droop
after 24 hr, whereas tendrils in treatments with oc-keto-
99
glutarate appeared as turgid as those in distilled water.
At the end of the experiment the tendrils in ammonium were
severely wilted and had necrotic regions.
However,
tendrils in ammonium plus os-ketoglutarate were slightly
wilted whereas tendrils in distilled water andoc-ketoglutarate alone appeared turgid.
The pattern in fresh weight loss generally reflected
free ammonium concentrations in the tendrils.
Tendrils in
ammonium solution had nine fold higher ammonium
concentration than those in distilled water (14.0 compared
to 1.5 mg NH4+ g-1 dw; Fig. 5.IB).
The addition of
cc-ketoglutarate to the ammonium solution reduced free
ammonium by nearly 50 %.
Tendrils in an oc-ketoglutarate
solution alone had the same ammonium concentration as
those in distilled water (Fig. 5.IB).
Although concentration of free ammonium in tendrils
was different between ammonium and ammonium plus cs.-ketoglutarate treatments, figure 5.1C shows there was no
difference (p < 0.05) in total nitrogen (expressed on an
ammonium concentration base).
Total nitrogen in tendrils
treated with these two solutions was about
3 3 mg NH4+ g-1 dw.
This concentration was about twice
that in tendrils in either distilled water or oQ-ketoglutarate alone.
100
The 16 mg NH4
g ■L increase m total nitrogen of
tendrils in ammonium could be explained by the increase in
free ammonium in the tissue.
Although the increase in
free ammonium was about 12 mg NH4+ g-1, the variation in
both the ammonium and total nitrogen concentrations of
tendrils in distilled water and ammonium could explain the
4 mg NH4+ g"1 shortfall.
However, the 12 mg NH4+ g-1
increase in total nitrogen of tendrils in ammonium plus
oc-ketoglutarate cannot be explained by the 6 mg NH4+ g
increased free ammonium concentration, even if the
variation is considered.
The apparent excess in total
nitrogen with this treatment reflected the amount of
ammonium assimilated amino acids and protein (it is
unlikely that the ammonium was metabolized to increase the
nitrate concentration).
Although only significant at
p < 0.1, tendrils in ammonium plus oc-ketoglutarate had
1 mg NH4+ g-1 dw higher amino acid concentration than the
tendrils in the other treatments, all of which had the
same amino acid concentrations (Fig. 5.2A).
Protein
content was not measured but was assumed to be estimated
from the difference between total nitrogen and the sum of
the free ammonium and amino acid.
Hence, about 4 mg of
the increase in total nitrogen of the tendrils treated
with ammonium plus oc-ketoglutarate was due to increased
protein synthesis.
101
Fig. 5.2.
Concentration of total free amino acid,
glutamate and glutamine amino acids in tendrils
treated with the same solutions listed in Fig. 5.1.
Error bars are standard error of the means.
Means
with the same lower case letter are not significantly
different (LSD) at p < 0.05, those with the same upper
case letter are not significantly different at p<0.10.
102
The reduced ammonium concentration and apparent
increase in concentration of free amino acid when os-ketoglutarate was added to the ammonium solution indicated
that this tricarboxylic acid stimulated ammonium
assimilation.
Similar responses to exogenously supplied
oc-ketoglutarate have been found with other higher plants
(Matsumoto et al., 1971).
Although amino acid analyses are inherently variable,
significant differences were found in three primary amino
acid concentrations of tendrils in the four treatment
solutions.
Concentrations of other amino acids were not
significantly (p < 0.05) different.
Figure 5.2B shows
that the glutamate concentration of tendrils in ammonium
plus oc-ketoglutarate and oc-ketoglutarate alone increased 5
and 8 fold respectively compared to the concentrations of
tendrils in distilled water or ammonium.
Little
difference in glutamine concentrations were found (Fig.
5.2C).
However, there was 1.1 mg NH4+ g
higher
glutamine concentration in tendrils treated with ammonium
plus oc-ketoglutarate than those treated with ^c-ketoglutarate alone.
Note, glutamine represented up to about
50 % of the total free amino acids in tendrils, whereas
glutamate accounted for only 2 to 5 %.
Both glutamate and glutamine are primary amino acids
103
formed during ammonium assimilation (Givan, 1979; Joy,
1988).
The shifts in glutamate and glutamine
concentrations with added oc-ketoglutarate, with or without
ammonium, are difficult to explain on an assimilation path
basis without concurrent labeling and enzyme activity
studies.
However, if glutamate dehydrogenase (which
catalyzes the synthesis of glutamate from oc-ketoglutarate
and ammonium) was involved, glutamate concentration would
be expected to increase without an associated change in
glutamine concentration.
This is what was observed (Fig.
5.2B and 5.2C) which indicates that glutamate
dehydrogenase could have been active.
However, Mohanty
and Fletcher (1980) found that exogenously supplied
ammonium increased both glutamate dehydrogenase and GOGAT,
and reduced glutamine synthetase activity in nonphotosynthetic suspension cultures of rose.
Consequently, it
is difficult to make an interpretation about the relative
roles of these enzymes.
Givan (1979) considered the assimilation path
catalyzed by glutamate dehydrogenase was only involved in
ammonium assimilation at high ammonium concentrations and
that glutamine synthase and GOGAT were the primary
assimilatory enzymes.
However, the relative roles of
these enzymes is being reassessed.
Oaks (1986) provided
evidence that glutamate dehydrogenase is a major synthetic
enzyme in ammonium assimilation.
104
The lowered glutamine concentration in tendrils
treated with oc-ketoglutarate alone and the indication that
this amino acid was high in tendrils treated with ammonium
plus oc-ketoglutarate (Fig. 5.2C), suggests that both
glutamine synthetase and GOGAT could also be operating.
This is particularly evident when the glutamine and
glutamate concentrations in tendrils treated with oc-ketoglutarate alone are compared to concentrations treated
with ammonium plus oc-ketoglutarate or distilled water.
When treated with oc-ketoglutarate alone the glutamate
concentration increased with an apparent reduction in
glutamine concentration.
This response could be explained
if the treatment had stimulated GOGAT activity.
When
ammonium is added to oc-ketoglutarate, the smaller increase
in glutamate and higher glutamine concentrations compared
to OR-ketoglutarate alone indicated that glutamine
synthetase activity was stimulated or increased.
The high ammonium concentration used in the treatment
solutions caused high tissue ammonium concentration
(Fig. 5.IB) so it is possible that ammonium assimilation
pathways that only operate at high ammonium concentration
would have been expressed.
This could explain the
difference between the glutamate and glutamine
concentration patterns seen with the two oc-ketoglutarate
treatments.
At normal tissue concentrations of ammonium.
105
oc-ketoglutarate appeared to stimulate GOGAT.
Whereas
when excess ammonium was supplied, the increase in
concentration of glutamate indicated that«Q-ketoglutarate
stimulated glutamate dehydrogenase and/or glutamine
synthetase.
Gamma-aminobutyric acid concentration was about 0.5
(six fold) and 0.3 mg NH4+ g-1 (two fold) higher in
tendrils treated with ©c-ketoglutarate, compared to
tendrils in distilled water and ammonium respectively
(Fig. 5.3).
Q
-Aminobutyric acid is synthesized from
glutamate (Joy, 1988).
Consequently, treatments that
increased glutamate concentration would be expected to
stimulate increased ^-aminobutyric acid synthesis.
was generally true.
This
The reason is not known for the
increase of ^-aminobutyric acid rather than other amino
acids formed from glutamate.
Perhaps the other amino
acids were incorporated in to protein.
Kishinami and
Ojima (1980), working with cultured rice cells, also found
that ammonium treatment increased concentration of ^aminobutyric acid.
Wallace et al. (1984) found rapid
accumulation of {(-aminobutyric was triggered by low
temperature, darkness, and mechanical damage.
the reasons for these increases is not known.
However,
106
dH20
Fig. 5.3.
NH,
NH4
+
KGLU
KGLU
Free ft-aminobutyric acid concentration in
tendrils treated with the solutions listed in Fig.
5.1.
Error bars are standard error of the means.
Means with the same letter are not significantly
different (LSD, p < 0.05
107
£.\J -
z
w
"^
o
o
2
3
2
2
2
<
o KLIOGLUTARATE + NH4
• GLUTAMATE + NH4
a
<
dw)
TRATI
2
O
r
^^^^^
^^
^^^1-r
^
1 12- +
^i 8- ?
4-
T^
T ^^^
^^^^
1
^^^
^<v>\, 1
^ <s ^
»
:
NNl,
s
^ dH20
0-
1
0
1
1——1
100
1
200
1
1
1
300
KETOGLUTARATE or GLUTAMATE CONCENTRATION
(mM)
Fig. 5.4.
Free ammonium concentration in tendrils
treated with glutamate (filled circles) or Qt-ketoglutarate (open circles) added to 330 mM ammonium
sulfate.
Distilled water treatment labeled dl^O.
Error bars are standard error of the means.
400
108
Glutamate compared to oc-ketoglutarate
When added to ammonium solutions, glutamate was as
effective as or-keto-glutarate at reducing the increase in
free ammonium of tendril tissue (Fig. 5.4) .
Each 170 mM
addition of glutamate or oc-ketoglutarate reduced the
ammonium concentration in the tendrils by about
4.5 mg NH4+ g
dw compared to the concentration in
tendrils treated with ammonium alone.
At the highest
concentration (340 mM) of glutamate or oc-ketoglutarate the
free ammonium concentration was about 7 mg NH4+ g
This was 4 mg NH4+ g
dw.
higher than the concentration of
tendrils in distilled water.
The same benefit from glutamate as oc-ketoglutarate
suggests that the tendril tissue has more than one
assimilation path to reduce elevated ammonium
concentration.
Glutamate is a substrate for glutamine
synthesis catalyzed by glutamine synthetase.
This
reaction consumes ammonium and would explain the benefit
of glutamate addition.
In contrast, oc-ketoglutarate
serves as the substrate for two assimilation paths.
The
path catalyzed by glutamate dehydrogenase directly
consumes ammonium, whereas the GOGAT path synthesizes
glutamate to serve as substrate for ammonium assimilation.
Note, the amino subunit for the synthesis of glutamate via
GOGAT is the transamination product from glutamine.
109
Ammonium is not a substrate in this step.
Methionine sulfoximine effect
The addition of 10 mM MSX did not prevent the
reduction in ammonium concentration of tendrils treated
with or-ketoglutarate compared to those treated with
ammonium alone.
7.5 mg NH4+ g
ammonium plus
Table 5.1 shows that there was a
reduction in free ammonium of tendrils in
-ketoglutarate with or without MSX.
This
inhibitor is generally considered to be an irreversible
inhibitor of glutamine synthetase (Oaks, 1988), although
Sieciechowicz et al. (1989) found MSX also reduced release
of ammonium from metabolic pathways in pea leaves.
The
failure of MSX to prevent oc-ketoglutarate from lowering
the ammonium concentration in tendrils supplied with
ammonium suggests that the assimilation stimulated by
oc-ketoglutarate did not involve glutamine synthetase.
Another assimilation pathway, most likely that catalyzed
by glutamate dehydrogenase, must have operated.
This is
supported by the change in amino acid concentrations as
stated earlier.
Sugars treatments
Sucrose and glucose were not as beneficial as oc-ketoglutarate or glutamate at reducing ammonium induced fresh
110
Table 5.1.
Free ammonium concentration in tendrils
treated with distilled water, or ammonium solutions (NH4)
with or without added cc-ketoglutarate or oo-ketoglutarate
plus methionine sulfoximine (MSX).
Solution
Ammonium Concentration
(mg NH4+ g-1 dw)
Distilled water
Ammonium sulfate (NH4)
330 mM
1.3 b
11.2 a
NH4 plus 330 mM
oc-ketoglutarate
3.3 b
NH4 plus 330 mM
cc-ketoglutarate plus
10 mM MSX
3.8 b
* Means with the same letter are not significant (LSD, p <
0.05)
Ill
weight loss or the increase in tissue ammonium
concentration (Fig. 5.5).
At 150 mM, glucose increased
the loss of fresh weight to -8.0 % compared to -0.5 % of
tendrils treated with ammonium alone.
This trend in
weight loss mirrored the increase in concentration of
ammonium in tendrils treated with ammonium plus glucose
solutions (Fig. 5.5).
An addition of 150 mM glucose to
the ammonium solution increased the ammonium concentration
in tendrils from 11.2 to 13.4 mg NH4+ g-1 dw.
Sucrose
added to the ammonium solution did not affect fresh
weight.
However, there was a slight linear decrease
(about 2.2 mg NH4+ g
for each 165 mM sucrose added) in
free ammonium in tendrils treated with sucrose (Fig. 5.5).
Givan (1979) cited research of Prianischnikow (1922) that
showed that glucose added to ammonium increased amide
synthesis and reduced the accumulation of ammonium.
Srivastava and Singh (1987) stated that both sucrose and
glucose inhibit glutamate dehydrogenase activity.
However, they believe the exact mechanism for the action
of these sugars is complex and difficult to identify.
grape tendrils, sucrose, rather than glucose, provided
substrate and/or stimulated an aspect of ammonium
assimilation.
In
112
10
*
ui
A
o
z
$ dHjO
u
i
o
ED
=£
GLUCOSE 5
SUCROSE *
5
I
L
^v
SUCROSE +
-5-
t
a -io
-15
15
o
NHA/'
1
1
1
1—
GLUCOSE + NH4
1
1
1
GLUCOSE + NFL
12-
^ *
UJ T
96 g dH20
GLUCOSE •
3-
SUCROSE *
0
0
50
100
150
200
250
300
350
SUGAR CONCENTRATION (mM)
Fig. 5.5.
Fresh weight change (A) and free ammonium (B)
concentration of tendrils treated with sucrose (open
triangles) or glucose (open circles) added to 330 mM
ammonium sulfate, or used separately (sucrose - filled
triangle and glucose - filled circle).
water treatment labeled dl^O.
error of the means.
Distilled
Error bars are standard
113
Summary
Detached tendrils proved to be a useful model system
to study ammonium toxicity and ammonium assimilation in
tissue closely related to reproductive tissue.
The loss
in turgidity, expressed as loss in fresh weight, and
increased concentration of free ammonium in tendrils
treated with ammonium can be reduced or prevented if
oc-ketoglutarate or glutamate are added in combination
with ammonium.
This indicates that ammonium assimilation
in tendrils, and presumably in cluster tissue, is limited
by carbon substrate supply and not the lack of enzyme
capacity.
MSX did not prevent the reduction in ammonium
concentration caused by oc-ketoglutarate in tendrils
treated with ammonium.
This suggested that glutamate
dehydrogenase is involved in the oc-ketoglutarate
response.
Further research is required to determine which
ammonium assimilation pathway is most important in tissue
with high ammonium concentration.
Acknowledgments
We thank Dr. S. Gurrusiddaiah at the Bioanalytical
Center, Washington State University, Pullman, Washington
for the amino acid analyses.
The Oregon Wine Advisory
Board provided financial support for this research.
was a recipient of a New Zealand, National Research
Advisory Council scholarship.
DTJ
114
References
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mineral nutrition relationships of waterberry in
Thompson Seedless. Amer. J. Enol. Vitic. 36:57-64.
Dwivedi, U.N., B.M. Khan, S.K. Rawal, and A.F.
Mascarenhas. 1984. Biochemical aspects of shoot
differentiation in sugarcane callus: I. Nitrogen
assimilating enzymes. J. Plant Physiol. 117:7-15.
Ghisi, R., B. lannini, and C. Passera. 1984. Changes in
the activities of enzymes involved in nitrogen and
sulphur assimilation during leaf and berry
development of Vitis vinifera. Vitis 23:257-267.
Givan, C.V. 1979. The metabolic detoxification of ammonia
in tissues of higher plants. Phytochem. 18:375-382.
Jackson, D.I. and B.G. Coombe. 1988a. Early bunchstem
necrosis in grapes - a cause of poor fruit set. Vitis
27:57-61.
Jackson, D.I. and B.G. Coombe. 1988b. Early bunchstem
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Smart, R.J. Thorton, R.J. Rodriguez, and J.E. Young
(eds). Proceedings of the Second International
Symposium for Cool Climate Viticulture and Oenology,
Auckland, New Zealand. New Zealand Soc. Vitic.
Oenol., Auckland.
Jordan, D.T. 1985. Narrowing the research focus. Southern
Horticulture Grapegrower and Winemaker 3:53-55.
Jordan, D.T., P. Breen, and S. Price. 1988. Pedicel
necrosis caused by ammonium toxicity in detached
grape clusters. HortScience 23:374 (Abst.).
Jordan, D.T., P.B. Lombard, and S. Price. 1989.
Inflorescence necrosis in grapes : description and
preliminary research. Amer. Soc. Enol. Vit. Annual
Meeting, Anaheim, P. 1. (Abst.)
Joy, K.W. 1988. Ammonia, glutamine, and asparagine: a
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to cultured rice cells. Plant Cell Physiol. 21:581589.
115
Matsumoto, H., N. Wakiuchi, and E. Takahashi. 1971.
Changes of some mitochondrial enzyme activities of
cucumber leaves during ammonium toxicity. Physiol.
Plant., 25:353-357.
Mohanty, B. and J.S. Fletcher. 1980. Ammonium influence
on nitrogen assimilating enzymes and protein
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rose. Physiol. Plant., 48:453-459.
Murillo, E. and E. Sanchez de Jimenez. 1984. Alternative
pathways for ammonium assimilation in Bouvardia
ternifolia cell suspension cultures. J. Plant
Physiol. 117:57-68.
Oaks, A. 1986. Biochemical aspects of nitrogen metabolism
in a whole plant context, p. 133-151. In: H.
Lambers, J.J. Neeteson, and I. Stulen (eds).
Fundamental, ecological and agricultural aspects of
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Dordrecht.
Platt, S.G., Z. Plaut, and J.A. Bassham. 1977. Ammonia
regulation of the carbon metabolism in
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Puritch, G.S. and A.V. Barker. 1967. Structure and
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Roubelakis-Angelakis, K.A. and W.M. Kliewer. 1983a.
Ammonia assimilation in Vitis vinifera L. I.
Isolation and properties of leaf and root glutamate
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Roubelakis-Angelakis, K.A. and W.M. Kliewer. 1983b.
Ammonia assimilation in Vitis vinifera L. II. Leaf
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116
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117
CHAPTER 6
EPILOGUE
Introduction
As with most research, the research reported in this
thesis has generated more questions than it answered.
Also, some interpretations of the results could not be
made because critical pieces of information were not
available.
In this epilogue I take the opportunity to indicate
the direction of future research based on what was learnt,
and to state some of the questions raised.
First, aspects
related to in vivo ammonium concentration in tissue are
discussed.
Finally, experiments are described that will
further develop the tendril model system to increase our
knowledge of ammonium toxicity and assimilation in grape,
and enable improved interpretation of the early results
with this model system.
Ammonium Concentration in Grape Tissue
Ammonium concentration determination
All tissue analyses reported in this thesis were of a
sample of the whole tissue, i.e., cell types, or zones.
118
were not separated, so only an average ammonium
concentration was established.
This approach has the
inherent disadvantage that it does not inform us about the
range of concentrations between different cells.
This
range could be large and differ between different tissue.
When considering inflorescence necrosis and late rachis
necrosis it will be important to develop micro-analysis
techniques to determine ammonium concentrations in
different cells.
In particular, the cells surrounding the
lenticels and stomata in the rachis should be separated
from the rest of the tissue.
This seems a daunting task.
Approaches are being developed that can scan tissue at the
cellular level to determine nutrient content.
Perhaps a
similar system will be available to scan for the ammonium
content.
Seasonal trend
Chapter 3 described for the first time the trend in
ammonium concentration in rachis and berry tissue and in
xylem extracts but did not answer the question — why does
the concentration change throughout the season ?
Also,
why does different tissue have similar seasonal trends in
ammonium concentration although they occur at different
times ?
119
I predict that there is a complex answer to the first
question about the reason(s) for the seasonal changes in
ammonium concentration.
Free ammonium in the tissue is a
dynamic pool with many input and output factors.
Consequently, future research should concentrate on the
identification, and quantification (if possible), of the
major factors that effect the free ammonium pool and how
these change over the season.
The attempt to determine
the ammonium concentration in the xylem extracts was a
first, and somewhat timid, step towards quantifying one of
the supply factors.
However, this needs to be extended to
the collection of xylem extracts at the cluster and to the
determination of xylem flow rates.
Vacuum extraction
techniques could be developed to get xylem extracts from
the cluster.
Also, an element that is exclusively xylem
mobile could be used to estimate xylem flow rates to the
cluster.
In the past calcium has been used but, more
recently, silica is being used.
With both elements, the
rate of accumulation in the tissue and the concentration
in the xylem are used to determine xylem flow rate over
defined periods.
The emphasis in the thesis has been ammonium in
grapevines so, rather than just considering the general
xylem flow to the cluster, the flow of ammonium (or
nitrogen) would be preferred.
A possible approach to
estimate this flow would involve the use of
N.
120
Initially this work would have to be done on potted vines.
During the first season plants would be fed labeled or nonlabeled fertilizer.
The plants receiving the label would
be used the second season to determine the contribution of
reserve nitrogen stored within the stem and roots.
These
reserves would be labeled from the treatment the previous
year.
The other half of the vines would be fed labeled
fertilizer in the second season.
This would directly
determine the supply of nitrogen from current uptake.
With critical sample periods the relative contribution of
reserve and current uptake sources could be established.
Combined with this study the nitrogen fractions (ammonium,
nitrate, amide and amino) in xylem extracts should be
separated to determine in what form the label arrives at
the cluster from the different sources.
Further labeling studies have been considered.
Direct feeding of labeled ammonium to the attached cluster
at different developmental stages would provide a method
to determine if the cluster tissue changes in its ability
to assimilate ammonium.
It is possible to use a wick
arrangement to draw treatment solutions in to the cluster.
After treatment the cluster tissue would be harvested and
processed to again determine in which fraction(s) the
label is located.
Care has to be taken that the uptake is
quantified if the analysis is to be quantitative.
121
The above proposals were suggested to address some of
the "input" factor questions.
However, they could also
help with some of the "output" factors.
The fractionation
studies allow for an estimate of the role ammonium
assimilation plays in reducing the free ammonium levels in
the tissue.
An estimate of the seasonal change in
assimilation would also be obtained.
Ammonium assimilation may be limited by the supply of
carbon substrate.
I predict that these substrates are not
supplied at a constant rate to the cluster throughout its
development.
The beneficial effect of oc-ketoglutarate in
the model system suggests that the supply of this, and
other substrates, could greatly influence the ability of
the tissue to assimilate ammonium.
A link between the
model system and the field would be made if the carbon
substrates concentrations are also determined throughout
the season as was done for ammonium.
The possibility that ammonium is exported from the
cluster should not be forgotten.
Perhaps ammonium or
assimilated ammonium in the form of amino acids are
exported at different rates during the season.
This
output factor appears difficult to identify and quantify
unless a total nitrogen budget for the cluster is
developed where, at least, all input factors are
122
quantified.
The difference between input (deposits) and
the tissue content (savings) is the quantity exported
(expenditure).
Tissue development
Tissue development appeared to greatly influence the
ammonium concentration in the tissue.
Young
(physiologically) tissue had higher concentration than old
tissue.
Consequently, it appeared that shade delayed the
ammonium concentration trends because it delayed the
development of the tissue.
A comparison of tissue from
the shaded and exposed vines when the difference in
ammonium concentration was greatest could help determine
what ammonium metabolism changes occurred.
Disorders, is ammonium involved ?
Although there are many reports on the relationship
between high ammonium concentration and tissue affected
with inflorescence necrosis and late rachis necrosis, we
do not know if high (toxic) ammonium is the cause or an
effect.
Careful research is needed to test the hypothesis
that toxic ammonium concentrations cause the disorders.
It is of interest to note that both inflorescence
necrosis and late rachis necrosis are limited to the
rachis tissue (although we wonder if the necrosis and
123
abscission seen in tendrils on field grown plants may
also be related to these disorders).
Adjacent stems,
petioles, leaf lamina, and berries are not affected.
This tissue discrimination is puzzling.
rachis different ?
Why is the
Also if ammonium is involved, the
berries have higher ammonium concentrations than the
rachis.
Why are the berries less sensitive to
ammonium than the rachis ?
However, as stated
earlier, these concentration differences are based on
the average ammonium concentration within the whole
tissue.
It is possible that ammonium concentrations
differ between cells and that whole tissue analyses
do not indicate what the concentrations are in
critical (sensitive) cells.
Although the sensitivity of our present analyses is
questioned, they may be giving us a real indication of
tissue differences in ammonium concentrations.
Perhaps
the differences between tissue types reflect different
ammonium assimilation capacity.
this theory would be valuable.
Sampling tissue to test
First glutamate
dehydrogenase, glutamine synthetase and GOGAT should be
assayed (see later discussion about assimilation in the
tendril model system).
Experiments are needed that influence the ammonium
concentration in rachis tissue and establish if the
124
disorders are induced when the ammonium concentration is
artificially elevated.
Possible treatments that could be
used to increase ammonium concentration are:
1) direct
application of ammonium via either the wick system
described above or with application of biuret free urea
(this last treatment has been successfully used by Lovat
et al. (1988) to increase leaf ammonium concentration);
2) the wick approach could be used to apply inhibitors of
ammonium assimilation;
3) increase soil ammonium to
encourage ammonium uptake;
4) increase photorespiration
with lowered carbon dioxide concentration.
At sites that repeatedly have the disorders,
treatments that reduce ammonium in the rachis should be
evaluated.
If these treatments are successful in the
reduction of ammonium concentration, does the incidence
and severity of the disorders change ?
Potential
treatments to reduce the ammonium concentration in the
rachis:
1) root pruning to reduce uptake;
2) shoot
girdling to improve carbon substrate for ammonium
assimilation;
3) heavy nitrate application to the soil to
increase nitrate rather than ammonium uptake;
4) decrease
photorespiration with increased carbon dioxide.
Concurrent with the experiments that directly
attempt to increase or decrease ammonium concentration,
should be experiments with treatments that are known to
125
increase the disorders.
With each treatment the
ammonium concentration in the rachis should be
monitored to establish that elevated ammonium is
consistently associated with treatments that induce the
disorders.
Examples of treatments that induce the
disorders are: 1) high vigor rootstocks;
berry removal from the cluster;
pruning;
2) early
3) severe summer
4) heavy shade (artificial with cloth or
natural from dense leaves);
5) susceptible cultivars.
A single direct approach to establish if ammonium is
the cause or an effect is not possible.
Consequently, an
approach with multiple experiments is necessary that tests
the hypothesis from different directions.
Ammonium toxicity
Although ammonium toxicity is well recognized we do
not know exactly how ammonium is toxic.
This is a major
limitation to any study that suspects that toxic ammonium
concentrations are the cause of a disorder
disfunction.
or tissue
Research to improve our knowledge about the
mechanism of ammonium toxicity would be valuable.
Initially, this probably will be investigated at the
cellular (e.g., with protoplast or cell cultures) or
subcellar level.
considered.
Ultimately, the whole tissue should be
126
Other aspects of ammonium toxicity (if it exists) in
rachis tissue that are not known are the critical
concentration that is toxic and if cell sensitivity to
ammonium is constant throughout the season.
aspects are directly linked.
These two
For example, if the cells
become more sensitive with time the critical concentration
will decrease over the same period.
To study this we need
to know how cells are sensitive to ammonium and have the
ability to determine ammonium concentration at the
cellular or subcellular level.
Tendril Model System
Ammonium toxicity
The mechanism of ammonium toxicity can be further
investigated at the whole tissue level with the tendrils
in this model system.
Applications of high ammonium
concentrations are toxic to the tendrils.
Although the
wilting and necrosis occur, we do not know the physiology
of the development of these symptoms.
I suspect that the
wilting is initiated by membrane perturbation which
results in water loss, i.e., turgor is lost before there
is water loss.
Other factors that also affect cell turgor
and water loss can occur which make the index of fresh
weight change used in the model experiment not unique to
ammonium toxicity.
However, until we know more about the
127
mechanism of the toxicity we are not in a position to
develop a specific index.
Weight change has the
advantages that it is simple to measure and is nondestructive.
Preliminary experiments used a range of ammonium
concentrations as treatments to establish a concentration
that produced toxic symptoms within a period that the
control tissue remained "healthy".
Appendix 1 shows the
response between ammonium concentration within the tissue
and a wide range of treatment concentrations.
This work
could be extended to establish critical concentrations of
ammonium for symptom development.
Assimilation
The amino acid analyses discussed in the paper on the
tendril model system indicated that changes in ammonium
assimilation occurred and explained the responses seen
with oc-ketoglutarate and glutamate treatments.
These
indirect measures of assimilation should be confirmed with
direct enzyme assay studies.
Initially, the activities of
glutamate dehydrogenase, glutamine synthetase and GOGAT
should be determined.
As stated in the literature review,
GOGAT activity has not been detected in grape.
I believe
that this just reflects problems with enzyme extraction
rather than the absence of the enzyme.
To confirm that
128
GOGAT exists in grape tissue it may be possible to probe
for the enzyme using polyclonal antibodies developed for
this enzyme.
Anderson et al. (1989) used these antibodies
in a study of alfalfa root nodules.
Enzyme extraction procedures and the interpretation
of their in vitro activities are not simple.
Grape tissue
contains high concentrations of phenolic substances that
can greatly interfere with the extracted enzyme.
Also, it
is difficult to determine how directly related are the in
vitro and in vivo activities.
Further experiments with inhibitors of ammonium
assimilation enzymes could provide more information about
the responses seen with oc-ketoglutarate and glutamate.
For example, what is the effect of MSX when glutamate is
added.
If glutamine synthetase is stimulated by glutamate
then MSX should prevent the reduction in ammonium
concentration measured when glutamate is used.
Also, it
would be informative to do an amino acid analysis of
tissue treated with oc-ketoglutarate with or without MSX.
I suspect that when MSX is used that the glutamate
concentration is much higher and there is no change in
glutamine concentration compared to when MSX is not
added, i.e., glutamate dehydrogenase activity is the sole
assimilation pathway when MSX is used whereas glutamine
129
synthetase is normally active with oc-ketoglutarate when
no inhibitor is added.
Other inhibitors are available that target other
enzymes.
For example, Srivastava and Singh (1987) stated
the 5,5idithiobis(2-nitrobenzoate) (DTNB) can completely
inhibit glutamate dehydrogenase.
Interestingly, these
authors also describe that 0-mercaptoethanol stimulates
glutamate dehydrogenase in leaves of Urtica.
Perhaps, a
treatment with 8-mercaptoethanol should be considered.
A further refinement of the model system would be to
use
15
N labeled ammonium.
It is possible to get an
indication of the assimilation pathway from the location
of the label.
For examples: 1) the nitrogen in the amide
group of glutamine and the amino group of glutamate should
be in equilibrium if the GS/GOGAT is the predominate
pathway (Goodwin and Mercer, 1983);
2) if glutamate
dehydrogenase catalyzes the primary pathway, the label
from ammonium should be first incorporated into the amino
group of glutamate and then in to the amide group of
glutamine (Durzan and Stewart, 1983) .
Although these
labeling studies are used they are not without
problems.
Oaks (1986) maintained that these studies
are limited by the sensitivity of the assay methods.
She provided an
14
analogy with the incorporation of
C02 by leaves on C4 plants.
In these plants.
130
malate or aspartate, early products in the
assimilation of CO2, mask the incorporation of label
into phosphoglycerate.
This was initially interpreted to
mean that ribulose-l,5-bisphosphate carboxylase was
absent.
Consequently, often an approach with multiple
treatments is taken where labeled substrate and inhibitors
are used.
However, interpretation of the results can
still be difficult.
Ammonium accumulation
Necrosis and wilting of the tendrils in the model
system when treated with ammonium is only seen in the
tissue above the solution level.
appears healthy.
Tissue in the solution
This phenomena is interesting.
Evaporation from the surface of the tissue or an aerobic
environment seem necessary for symptom development.
I
tested the effect of oxygen concentration on the fresh
weight change and ammonium concentration of tendrils
(Appendices 2 and 3).
At very low oxygen concentration
there seemed to be an effect on the ammonium concentration
and fresh weight change.
large.
However, the effects were not
Evaporation could be the key factor for the above
solution response.
Perhaps ammonium is transported to the
surface of the tendril with the evaporation
(transpiration) of tissue water.
With water loss ammonium
would accumulate at the surface.
Note, late rachis
131
necrosis symptoms are first seen in cells that surround
the stomata and lenticels — where most transpiration
occurs and the most likely zone for the accumulation of
substances that do not evaporate with the water.
In a
preliminary experiment, lanolin was applied to the surface
of the tendril to limit transpiration.
These tendrils did
not develop symptoms of ammonium toxicity, although they
did take up solution.
Further experiments are necessary
to develop the theory that evaporation from the tissue
surface is critical for the development of toxic ammonium
concentrations.
Conclusion
Much basic and applied research is needed before we
have a good knowledge of ammonium toxicity and
assimilation in grape tissue.
This and more specific
research will be used to determine the role of elevated
ammonium in tissue affected by the disorders inflorescence
necrosis and late rachis necrosis.
132
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APPENDICES
141
2
UJ
o
^.
o
o
1—
1
CP
+'4-
—5
CT
E
12
3
4
5
SOLUTION CONCENTRATION
(mg NH4+ ml-1)
Appendix Fig. 1.
Ammonium concentration in tendrils
treated with a range of ammonium sulfate solution
concentrations (mg NH^"1" ml-1) .
dard errors of the means.
Error bars are stan-
With low variation, the
error bars are covered by the symbol.
142
4
Appendix Fig. 2.
8
12
16
20
OXYGEN CONCENTRATION (%)
Ammonium concentration in tendrils
treated with distilled water (open circles) or 330 mM
ammonium sulfate (filled circles) under different
oxygen concentrations.
errors of the means.
Error bars are standard
With low variation, the error
bars are covered by the symbol.
143
Ld
CD
z
<
o
hX
o
Ld
X
LU
5
10
15
20
OXYGEN CONCENTRATION (%)
Appendix Fig. 3.
30
Fresh weight change of tendrils
treated with distilled water (open circles) or 330 mM
ammonium
sulfate (filled circles) under different
oxygen concentrations.
errors of the means.
covered by the symbol.
Error bars are standard
With low variation, the bar is
144
.£.vJ -
2.0- X
o
5=
X
1.5-
X "^
1.0-
o
0.5-
0.0- —i—i—i—i—i—i—i—i—i—i—i—i—i—
0
20
40
60
80
100
120
DAYS AFTER BLOOM
Appendix Fig. 4.
Rachis dry weight trend over 1988
from the field sampling (Chapt. 3).
standard errors of the mean.
Error bars are
With low variation,
error bars are covered by the symbol.
145
IE
O
UJ
Q
>Ld
CD
60
80
100
DAYS AFTER BLOOM
Appendix Fig. 5.
Berry dry weight trend over 1988 from
the field sampling (Chapt. 3).
ard errors of the mean.
Error bars are stand-
With low variation, error
bars are covered by the symbol.
146
60
80
100
DAYS AFTER BLOOM
Appendix Fig. 6.
Total ammonium content in berry tis-
sue, 1988, from the field sampling (Chapt. 3).
bars are standard errors of the mean.
Error
With low vari-
ation, error bars are covered by the symbol.
147
2.0
o
o UNAFFECTED
•--•• AFFECTED
E
o
1.5
|3
1-0 +
m
6
0.5
+
0.0
0
Appendix Fig. 7.
+
+
+
+
10
20
30
40
50
DAYS AFTER FIRST SAMPLE
60
Mean berry weight of Chenin blanc
clusters from vines in Summerland, British Columbia,
that had a history of late rachis necrosis
("affected", filled circles) or freedom from the
disorder ("unaffected, open circles).
First sample
(11 August, 1988) was 22 days prior to veraison.
Error bars are standard errors of the mean.
70
148
25
23-
o
O
o UNAFFECILD
T
♦--- AFFECILD
s
21--
'>-—-^
^^^^ \.
s^
S
/
-O
\
\
S^
s^
\
/yT
UJ
O
UJ
19-
T
\
\
I'
.^s^
][
\
\
_-^<!'
1715
1
0
Appendix Fig. 8.
1
1
1
1
—\
10
20
30
40
50
DAYS AFTER FIRST SAMPLE
1
60
Percent dry weight of Chenin blanc
rachis from vines in Summerland, British Columbia,
that had a history of late rachis necrosis
("affected", filled circles) or freedom from the
disorder ("unaffected, open circles).
First sample
(11 August, 1988) was 22 days prior to veraison.
Error bars are standard errors of the mean.
70
149
Z
p
800-
o
o UNAhhtCTED
- - - AFFECTED
/
^T 600z ^
/
o+
o ^
5
z> ^
400-
2
200-
s
/
/
/
{
/
T
/
/
i /
"T*''
^~^o
<
0-
1
0
Appendix Fig. 9.
1
1
1
[—i—i—
10
20
30
40
50
DAYS AFTER FIRST SAMPLE
60
70
Ammonium concentration of Chenin blanc
rachis from vines in Summerland, British Columbia,
that had a history of late rachis necrosis
("affected", filled circles) or freedom from the
disorder ("unaffected, open circles).
First sample
(11 August, 1988) was 22 days prior to veraison.
Error bars are standard errors of the mean.
With low
variation, error bars are covered by the symbol.
150
30
25
^
Ul
o
o
Dl
f—
2
_J
^
O
H-
%
13
T—
1
20--
cn
+
15
X
z
o>
10-
o o UNAFFECTED
- - - AFFECTED
E
50
+
+
10
20
30
440
+
50
60
70
DAYS AFTER FIRST SAMPLE
Appendix Fig. io.
Total nitrogen in Chenin blanc rachis from
vines in Summerland, British Columbia, that had a
history of late rachis necrosis ("affected", filled
circles) or freedom from the disorder ("unaffected,
open circles).
First sample (11 August, 1988) was 22
days prior to veraison.
errors of the mean.
Error bars are standard