Fruit Ripening – A Developmental Event Driven by Hydration/ Dehydration
Chaim Frenkel
Rutgers – the State University of New Jersey
Department Plant Biology and Pathology
New Brunswick, NJ 08901 USA
(848) 932-6236
frenkel@aesop.rutgers.edu
Fruit ripening, a form of senescence in plants,
is a genetically programmed event manifesting
abundant transcriptional and translational activity.
Expressed sequence tag (EST) dataset in ripening tomato fruit
assembled to different functional categories
Fei Z et al (2004) The Plant Journal 40:47–59
Based on a ripening pattern fruit are classified into:
1. Climacteric fruit, displaying Climacteric rise
in respiration and attended pattern in ethylene
evolution during the ripening process.
2. Non climacteric fruit, which manifest continuous
down drift in respiration and ethylene evolution.
Ethylene
Ethylene production and action stimulate ripening
in climacteric fruit but appears not to be important
in the ripening of non climacteric fruit.
Climacteric and non climacteric respiration in ripening fruit
Banana- a climacteric fruit
Strawberry- non climacteric fruit
Autonomous stress might trigger the onset of fruit ripening
It is not clear, however, what might be a cellular or metabolic cue(s) that stimulate the ripening
transcriptional program, ethylene action not withstanding.
Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied by
and is driven by autonomous stress. Stress might be a major metabolic shift that, apparently,
trigger the expression of ripening related genes and formation of corresponding gene products.
Defense/stress response
Distribution of gene products
by different categories in ripening
apricot fruit
Distribution of ESTs in ripening tomato
by different functional categories
But what type of stress?
Chemical Stress?
An early study revealed that fruit ripening is
accompanied by the accumulation of and is
apparently driven by production of reactive
oxygen species (ROS).
However, accumulation and activity of ROS
might jeopardize the ripening-dependent
transcriptional and translational programs.
The molecular basis for ROS-driven fruit
ripening wait clarification, but Stay tuned:
oxidative stress might instigate water stress,
as shown later.
ethylene
H2O2
lipid
hydroperoxide
Ethylene evolution is relation to H2O2 and lipid hydroperoxide
accumulation in ripening tomato fruit (Frenkel and Eskin 1977)
Hydration stress might be an alternative stress form,
which initiate and drive the fruit ripening process.
To examine this conjecture, we carried out measurement of the water status
in ripening fruit and, furthermore, to asses whether hydration changes is a causation
in the onset of fruit ripening .
We used azeotropic (toluene) distillation
to extract and measure the water content
in ripening climacteric and non climacteric fruit.
fr
Toluene
Fruit tissue sample
(around 25 g)
Water, an azeotrop, is removed from the fruit tissue sample by distillation with toluene.
The moisture is collected in a water trap and its volume is measured.
A.O.C.S official protocol 1989
Fetzer WR (1951) Anal Chem 23:1062-1069
Changes in tissue moisture content in
ripening climacteric fruit.
Arrows indicate the Breaking (insipient color)
ripening stage.
A schematic chart showing stages in fruit background
color during the ripening process
Green
White
Breaking Pink
Red
Changes in tissue moisture content in
ripening non climacteric fruit.
Arrows indicate the Breaking (insipient color)
ripening stage.
35
68
Banana
starch
66
30
25
water
peel
62
60
20
water
pulp
15
g starch/100 g pulp
Moisture Content (%)
64
58
10
56
5
54
60
70
80
90
100
Days After Bunch Emergence
Changes in moisture content in the pulp and peel of developing banana fruit.
The data (from Offem and Thomas [1993]Food Res Intn26: 187) was presented in table form and was
plotted and presented in graph form. Similar results (not shown) were observed in plantain fruit.
Other studies, in a different context noted occurrence of water deficit before
the onset of ripening in both climacteric and non climacteric fruit.
The present and other studies suggest that decline in tissue water content,
preceding the onset of fruit ripening, might be a universal phenomenon.
Ethylene might induce water deficit
Ethylene was applied to potato tuber, an ethylene-responsive tissue,
to examine whether the phytohormone might also lead to a decline
in water content.
The results suggest that ethylene-induced water deficit might account,
in part, for ethylene action in fruit ripening.
The results, showing comparable changes in water content and
total weight, suggest expulsion onto or uptake of moisture from the
ambient atmosphere in ethylene-treated potato tuber.
Ethylene-induced changes in potato tuber:
A. water content
B. Total weight
Ripening-associated water deficit: Coincidental to or a cause of fruit ripening?
It is not clear whether the ripening-related hydration/ dehydration is merely coincidental to
or, alternatively, ,might have a regulatory role in the onset of ripening.
We examined this question with the use of an ethylene-mutant tomato*, which does not
produce ethylene but can be ripen by applied ethylene.
We asked: Would artificially-induced water deficit lead to the initiation of ripening,
even in the absence of ethylene ?
*
antisense to 1-aminocyclopropane carboxylate synthase, an intermediate in ethylene biosynthesis
Oeller PW et al (1991) Science 254:437–9.
60 hours at 12° C
-------------- Cold Stress ----------------- I -----------------Stress Acclimation -----------Changes in water content in ethylene-mutant tomato fruit, induced by cold stress and acclimation
Fruit was held for 60 h at 12° C and next at 23° C for stress acclimation for up to 98 h. Tissue water content was
measured periodically.
Effect of ethylene and comparable effect of cold stress and acclimation on ripening
of ethylene-mutant tomato fruit.
Ethylene-treated tomatoes were held at 23◦C and ventilate continuously for 5 days
with air containing 10 μL ethylene/L air.
Cold-treated fruit were held at 12◦ C for 60 h and next for 5 days at 23◦ C.
Ripening of ethylene-mutant tomato induced by cold stress and acclimation
Effect of cold stress and acclimation on ripening, in fruit held at 12° C for 60 h and next at 23° C for
up to 8 d for stress acclimation.
What is the cellular and metabolic origin of ripening-related water deficit?
Cyclic changes in tissue water content might reflect changes in the hydration state
of two major water compartments in plant cells:
a. the central vacuole and
b. the apoplast.
Apoplast: Matter located between the plasma membrane of
neighboring cells and harboring the cell wall
Central vacuole
Cyclic changes in vacuolar water content would necessitate comparable changes in the state of water:
Increase in water content would be predicated on solute uptake,
to decrease the chemical potential (increase the osmotic pressure).
Decrease in water content would result from solute expulsion,
to increase the chemical potential (decrease the osmotic pressure).
This is not a likely scenario, particularly in
detached tissues, off-the-vine ripened tomato
or ethylene-treated potato tuber, for example.
Ethylene-treated
potato tuber
Cell wall might mediate cyclic changed in fruit water content.
Cell walls might have an efficacy to mediate changes in the state of water content, based on
changes in the physical confinement of the solvent, brought about by cell wall re-modeling:
cell wall loosening, which increases the osmotic pressure leads to increased hydration.
Cell wall tightening, resulting in lowering the osmotic pressure lead to water expulsion and
dehydration.
Cell wall swelling has been used to measure degree of cell wall hydration.
Swelling of cell wall material (CWM) isolated
from kiwifruit at different stages of ripening.
CWM was stained with toluidine blue and
allowed to settle.
Redgwell RJ et al (1997) Planta 203:162-173
Changes in tissue moisture content and comparable changes
in swelling efficacy of cell walls:
R2 = 0.9563
(A) ripening tomato fruit
(B) ripening strawberry fruit
(C) in ethylene-treated potato tuber
A high correlation coefficient (R2) values between
tissue water content and swelling proficiency of cell walls,
suggest that these variables are strongly interrelated.
R2 = 0.6812
R2 = 0.9626
The results suggest, furthermore, that changes in cell wall
swelling might reflect and actually account for corresponding
changes in tissue water content.
Low chemical potential
High chemical potential
Peroxidase, Oxidase
H2O2, O2
Relaxed
High osmotic pressure
Tightened
Low osmotic pressure
A scheme showing the hydration state of relaxed and tightened plant cell wall
Cell wall in a relaxed state, draw and hold water because molecular and segmental mobility of the wall
constituents lowers the chemical potential (increase the osmotic pressure) of water molecules. ( ).
Cell wall tightening, resulting from oxidative cross-linking and consequent restriction on molecular mobility,
leads to an increase in the wall water potential (decrease in osmotic pressure ( ).
High-chemical potential water escapes the system resulting in cell wall dehydration.
H2O2, O2
A scheme showing cell wall-bound phenyl propanoid (ferulic acid)
monomer undergoing oxidative coupling to a wall-bound dimer.
The scheme suggests that cell wall remodeling is predicated on onset of oxidative metabolism.
Oxidant-induced cell wall remodeling and consequent changes in wall and tissue hydration state
might be a cellular and metabolic context for regulation of fruit ripening by oxidative metabolism.
Cell wall constituents are denoted by A through G:
A; β-(1-4) linked xylan backbone B; Xylose-arabinose linkage C; 5-O-feruloyl lignin
D; 5-O-diferuloyl group (5-5linked dimer) E; 5-O-diferuloyl group(8-5dimer)
F; 3-O-acetyl group G; arabinose-lignin
Mathew S (2004) Critical Reviews in Biotechnology 24:59–83
Changes in the relative abundance of cell wall-bound
and cross-linked phenolic acid residues measured in
on-the-vine ripened tomato fruit at various stages of
ripening including (A) Green, (B) Breaker, (C) Pink,
and (D) Red.
The results show a progressive increase in the abundance of
mostly ferulic acid dimers as fruit become ripe.
These results suggest that progressive cross-linking and
tightening of cell wall structure might be a molecular basis
for water expulsion from cell walls and corresponding
decrease in tissue water content.
Some of the dimers were tentatively identified:
Molecular mass
504
560
518
Molecular Mass
Compound
β–β, coniferyl alcohol/coniferyl alcohol
8-8 ferulate/ferulate
8-β, ferulate/coniferyl alcohol
Low chemical potential
High chemical potential
Peroxidase, Oxidase
H2O2, O2
Relaxed
High osmotic pressure
Tightened
Low osmotic pressure
A scheme showing the hydration state of relaxed and tightened plant cell wall
A similar reasoning might account to dehydration associated with human aging.
Age-related changes
in body water in humans
20 years
65-80 years
Decrease in
total body water
17%
Decrease in
extra-cellular water
40%
Shi S, Klotz U (2011) Current Drug Metabolism 12:603
FEMALE
MALE
24
intra cellular water
extra cellular water
24
22
20
18
Plot 1
intra cellular water
Average Body Water Content (kg)
Average Body Water Content (kg)
26
extra cellular water
22
20
18
16
14
16
70
72
74
76
Age (years)
78
80
70
72
74
76
78
80
Age (years)
Change in intra and extra cellular water in aging male and female populations
Age-related decline in extracellular water is more pronounced than intracellular water,
particularly in females.
Decline in extracellular water might reflect age-related cross-linking in the extracellular matrix,
perhaps collagen or elastin.
Steen B (1988) Nutrition Reviews 46:45-51
Beverly Rubik* presented data showing age-related decline in body water, suggesting that
this phenomenon might arise from molecular aggregation.
The present result reinforce this suggestion and emphasize that a major aggregation event
Is cross-linking of the extracellular matrix.
This event leads to an increase in water potential (decrease in osmotic pressure)
and consequent escape of water from the extracellular space.
** An analogy is also the dehydration of insect larval cuticle during hardening (tanning),
resulting from the cuticle protein aggregation, through the formation of hydrogen and
other bonds or induced artificially by cross-linking agents.
* Beverly Rubik. Studies & Observations on a “Functional” Water, Ionized Alkaline Water.
Seventh Annual Conference on the Physics, Chemistry and Biology of Water 2012
**
Julian Vincent. If it's sclerotised it must be dry: Phenolic tanning controls hydration.
Fifth Annual Conference on the Physics, Chemistry and Biology of Water 2010
Dehydration of the extra-cellular matrix might be common to aging plant and animal tissues.
In fruit, dehydration of the extra-cellular matrix is apparently an origin of water stress
and a metabolic shift that might trigger the ripening transcriptional program.
Where to go next?
Hydration state (swelling efficacy) of
cell wall from on-the-vine ripened tomato
A literature search revealed that in the course of development,
plant cell wall (CW) undergo cyclic changes in CW relaxation
and tightening by cross-linking and accompanying hydration
and dehydration, respectively.
This scenario is relevant to CW changes occurring in ripening
fruit, as shown for (on-the-vine) ripening tomato.
H+
Acid-induced CW hydrolysis
and relaxation
HYDRATION
Oxidative Crosslinking
and CW tightening
DEHYDRATION
Wolf S et al (2012) Annu Rev Plant Biol 63:381–407
A dedicated plant cell wall integrity (CWI) maintenance mechanism monitors and
maintains functional integrity of the cell wall.
When in a relaxed hydrated state, perturbations in cell wall integrity lead to:
ROS production
Changes in cell wall composition and structure, notably oxidative cross-linking
Production of ethylene
Formation of callose (β-glucan, a β-1,3-linked glucose)
These changes are also the hallmark of fruit ripening
We propose: perturbations in cell wall integrity (CWI), distortion in cellulose
architecture brought about by turgor pressure for example, might
launch signaling cascade that trigger the ripening transcriptional program.
Turgor pressure
Hydrated CW
CWI sensing
CW perturbation
Oxidative cross-linking
Dehydration
Signaling
cascade
Ripening Transcriptional Program
The scheme suggests that loosening and an accompanying hydration of
fruit cell wall is a pre-requisite for sensing perturbation in cell wall and
subsequent events leading to the onset of ripening.
It also account for the universal expression of this developmental event
in ripening fruit.
A preliminary study revealed that citral, which disrupt the cytoskeleton
and consequent distortion in cellulose architecture, initiated ripening
in ethylene-mutant tomato fruit, even in the absence of ethylene.
MT
Localization of the cellulose synthase complex (CSC) is microtubule
(MT) dependent.
Wightman R, Turner SR (2008) The Plant Journal 54:794–805
Citral
CSC
Thank you for your attention.
Questions are welcome.
These perturbation are perceived by ‘mechano-sensing’ apparatus, resulting
in signaling cascade leading to transcriptional activation of the following:
production of ethylene
production other aging-relate phytohormones (salicylic acid or jasmonic acid)
formation of callose (β-glucan, made of β-1,3-linked glucose)
ROS production
as well as changes in cell wall composition and structure
These changes are a hall mark of fruit ripening as well as of stress responses, further attesting
that the ripening process is expression of a stress response, evoked, apparently,
by perturbation(s) in cell wall integrity.
Changes in CW structure reflect the existence of a dedicated plant cell wall integrity (CWI)
maintenance mechanism, which monitors and maintains functional integrity of the cell wall.
Perturbations in CWI, for example distortion in cellulose architecture of CW in a relaxed
hydrated state, may be brought about by turgor pressure or external forces.
Turgor pressure
Abiotic, biotic stress
When in a relaxed hydrated state, perturbations in cell wall integrity (CWI), for example
distortion in cellulose architecture, may be brought about by turgor pressure.
Cell walls and growth control
H+
Acidification, leading to hydrolysis
of CW polysaccharides and consequent
CW loosening and hydration
Plant growth is predicated on cell wall (CW)
loosening, instigated through acidity-induced
hydrolysis of CW matrix polysaccharides. The
loosened CW structure swells by taking up water.
Cross-linking
Cross-linking and consequent
CW tightening and dehydration
Growth is inhibited through cross-linking of the wall
protein Extensin and matrix polysaccharides. The
tightening of the CW results in water expulsion.
Cell wall remodeling is regulated by signaling
cascade induced by phytohormones and/ or biotic
and abiotic cues.
Wolf S et al (2012) Annu Rev Plant Biol 63:381–407
It is generally accepted that ethylene is a ripening hormone, because the gas strongly stimulate various ripening process,
and, accordingly, stimulate the expression of ripening-related genes.
However, other phytohormones (Brassinosteroids, for example) also control the ripening process.
Ethylene activity might not be important in the ripening of non climacteric fruit.
Importantly, abiotic and biotic stresses also stimulate fruit ripening.
A fundamental question is:
What are the metabolic and cellular events that might trigger hormonal or other signaling cues, which set in motions
the biosynthesis and action of ethylene and perhaps other signaling mechanism?
It is not clear, however, what might be a cellular or metabolic cue(s) that stimulate the ripening transcriptional program
Ethylene action not withstanding.
Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied by and is driven by
autonomous stress. This metabolic shift triggers, apparently, the expression of ripening related genes.
But what type of stress?
Brennan and Frenkel 1977
Frenkel and Eskin 1977
Model for regulation of climacteric ripening via
coordinated signaling
pathways. Transcription factors including LeMADSRIN, LeNOR, likely additional
MADS-box proteins, CNR and factors remaining to be
discovered (?) represent
the developmental signaling system that initiates
ripening in climacteric fruit.
Some components, such as those homologous to
LeMADS-RIN can be used in
non-climacteric species as well. The developmental
signaling system regulates
ethylene synthesis that is itself autocatalytic, in
addition to non-ethylene-mediated
ripening responses (represented by the red broken
arrow). Light influences ripening,
at least in tomato, only in relation to carotenoid
accumulation and through
activity of the DET1 (hp2) and DDB1 (hp1) gene
products.
Adams-Phillips L et al (2004) TRENDS in Plant Science Vol.9 No.7 July
2004
Functional
classification of
proteins expressed
during citrus fruit
ripening
Distribution of 101 gene products
during apricot fruit ripening
D'Ambrosio C et al (2013) J Proteom78:39-57
CW expansion:
In principle, one can distinguish the following events:
(a) hydration of newly deposited cell wall material and wall relaxation (Figure 2a)
(b) turgor-driven deformation of the cell wall
(c) mechanosensing, followed by the release of apoplastic reactive oxygen
species (ROS), cross-linking, partial
dehydration, and stiffening of the wall (Turgor-driven cell wall extension takes
place, which in turn leads to the
activation of the mechanosensing feedback loop, cell wall cross-linking, and
dehydration)
(d ) the secretion of new wall material. In what follows, these events are
discussed separately.
Monshausen GB, Gilroy S. 2009. Feeling green: mechanosensing in plants. Trends
Cell Biol. 19:228–35