In-season Heat Affects Low Temperature Sweetening, Associated
Gene Expression & Processing Quality of Stored Potato Tubers
Julian I. Jones, Daniel H. Zommick, Jacob Blauer, G.N.M. Kumar and N. Richard Knowles
Washington State University, Dept. of Hort. & Landscape Arch., Pullman
Introduction
B. Gene expression during induction of LTS
A. Background
Starch Breakdown
Sugar buildup (sweetening) during low temperature storage of potatoes results in undesirable browning (Maillard reaction) during frying
(Fig. 1). A High quality tuber with optimal storability has high specific gravity (SG) and low reducing sugars (RS). Low storage temperatures
(e.g. 4oC) can reduce disease pressure and extend marketability of potatoes, however starch is preferentially catabolized to RS in a process
known as low temperature sweetening (LTS). The effects of soil temperature (heat stress) during tuber growth on subsequent LTS and
retention of processing quality were assessed.
Potatoes grown in the southern Columbia Basin (e.g. Hermiston, OR) as part of the Tri-State Potato Breeding Program diminish in quality
more rapidly than tubers from other growing regions during storage (Fig. 1). This is most likely the result of substantially higher growing
temperatures in the southern Columbia Basin. To test this hypothesis, soil temperature was raised during different phases of tuber
development to assess the effect of heat stress on tuber physiology and retention of processing quality.
A
0
1
Starch is catabolized during LTS. The direct
involvement of starch phosphorylase (SP) in this
catabolism is controversial (Rathore et al., 2009). SP
catalyzes the reversible transfer of a glucosyl unit from
α-1,4-glucan chains (starch). Despite conflicting
reports, SP is likely involved in breaking down products
resulting from starch catabolism. Therefore, we
investigated the effects of heat stress on expression
and activity of SP during LTS.
Fig. 3: (A) Expression of the plastidic isoform of SP (Ltype) increased progressively during LTS of Ranger
tubers grown at 16oC but transcript induction was
significantly delayed in Premier, reflecting its LTS
resistance. Growth of Ranger tubers at 23 and 29oC
during bulking and maturation hastened the induction
of SP transcript during LTS. In contrast, growth of
Premier tubers at 23oC had no effect on the
subsequent induction of SP at 4oC; however, growth at
29oC resulted in increased transcript levels by 3 days
of storage at 4oC.
4oC
Oregon
4
1
B
Washington
Fig. 1: (A) Numbered line A00324-1 grown in
Othello (WA) and Hermiston (OR) stored for 3
months. Numbers in yellow are based on the
USDA fry color scale. (B) Starch is catabolized
to RS which react with Asn during frying to
form acrylamide and dark fry color.
Storage Temperature
4oC
9oC
B. Objectives
1. Assess the effects of soil temperature on retention of processing quality and LTS in cultivars Premier Russet (LTS resistant) and Ranger
Russet (LTS susceptible)
2. Characterize the influence of heat stress on transcript levels of genes for key enzymes in the LTS pathway during LTS
B
8” Depth
o
Temperature ( C)
Vine kill Heat off
1
3
6
0
1
3
6
0
1
3
6
L-type
18sRNA
16oC
23oC Bulk
23oC Matur.
29oC Matur.
Ranger Russet
Days at 4oC
0 1 3 6
0
1
3
6
0
1
3
6
0
1
3
6
L-type
18sRNA
16oC
B
23oC Bulk
Days at 4oC
0 6 12 24
23oC Matur.
29oC Matur.
Premier Russet
0
6
12
24
0
6
12
24
6
0
12
24
H
L
16oC
23oC Bulk
23oC Matur.
29oC Matur.
(B) Starch phosphorylase activity was visualized using native-PAGE on gels containing 0.3% gycogen over 24 days of storage at 4oC (10 µg
protein per lane). Two isozymes of starch phosphorylase were isolated based on affinity for glycogen: high affinity (H-type; cytosolic) and low
affinity (L-type; plastidic). Premier tubers grown at 16oC responded to cold stress with increased L-type activity. Moderate heat during tuber
maturation (23oC) resulted in LTS after 12 days (Fig. 2), but SP activity was similar to controls. Heat stress during bulking (23oC) and high heat
during maturation (29oC) resulted in lower SP activity during cold storage. Interestingly, changes in H-type SP activity of Premier correlated
well with reducing sugar accumulation (data not shown) and fry color (Fig. 2). The trend in SP activity for Ranger tubers grown at 16oC during
24 days of LTS was similar to Premier, however, in contrast to Premier, heat stress had no effect on activity during LTS.
0
20
16
12
Heat on
60
70
16 C (Ambient)
23oC
SGN-U268192
3
5
16oC)
Premier Russet (top row) - Control tubers (ambient
resisted LTS (an
inherent characteristic of Premier Russet). Moderate heat during bulking
caused an incremental decrease in SG (23oC = 1.084; 29oC (not shown) =
1.050) and induced sugar ends that increased in severity during storage at
4oC. Moderate heat during maturation (23oC) hastened sweetening and
loss of light fry color between 6 and 12 days at 4oC. Tubers at high
temperature (29oC) during maturation completely lost their LTS-resistant
phenotype and fry color darkened rapidly during storage at 4oC.
Ranger (bottom row) - Process quality of control tubers deteriorated
rapidly at 4oC (fry color darkened), reflecting the lack of resistance to LTS
in this cultivar. Growth at 23oC during maturation had no effect on
deterioration of process quality. However, growth at 29oC during
maturation resulted in sugar ends and more rapid deterioration of budend fry quality from 6 to 12 days in storage at 4oC. Soil temperature of
23oC during bulking produced significantly darker fries at harvest as well
as sugar ends. Specific gravity decreased with increasing temperature
during bulking (23oC = 1.077; 29oC (not shown) = 1.058), but was not
affected by soil temperature during maturation.
3
6
0
2
4
50 60 70 80 90 100 110 120 130 140 150 160 170 180
Days After Planting
Days After Planting
78
78
78
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
50
52
54
56
58
60
62
64 92
66
68
70
94
50
52
54
56
58
60
62
64
66
68
70
7472
76
78
78
76
76
76
74
74
6
8
1
70
0
1
3
29oC Matur.
1
3
6
0
1
3
6
0
1
5
74
88
7
90
90
72
76
10
12
14
16
70
92
86
82
90
92
88
20
22
24
0
1
0*
0
0
92
84
2
4
84
88 86
82
76
80 78
2
8
10
12
14
16
18
20
Distance Across Hill (inches)
Premier Russet (days at 4oC)
3
6
0
0
29oC Matur.
Invertase IV
0
1
3
6
0
1
3
6
0
1
2
0
0
Premier
Ranger
Days at 4oC
0 1 3 6
Invertase Inhibitor 2
0
1
3
6
0
1
3
6
0
1
12
23
1
22
24
Days at 4oC
0 1 3 6
Days at 4oC
0 1 3 6
Invertase Inhibitor 1
0
1
3
6
0
1
3
6
0
1
*USDA Stem end Fry Color
F Maturation
29ooC
Maturation
0
1
Ranger Russet (days at 4oC)
3
6
0*
0
3
4
1
1
3
3
4
12
23
3
4
3
4
16oC Ambient
1
1
2
2
23oC Bulking
1.093 a
0
0
0
1
3
4
23oC Maturation
1.090 a
1
*USDA Stem end Fry Color
1
1
2
oF Maturation
oC
29
29
Maturation
87.8 a
23oC Bulk
23oC Matur.
29oC Matur.
82.6 a
93.9 a
LSD 0.05
97.2 a
ns
6
18s rRNA
0
1
3
6
0
1
3
6
0
1
3
6
23oC
23oC
Ranger Russet
Growing
Invertase
Temperature Inhibition (%)
16oC
23oC Bulk
23oC Matur.
29oC Matur.
29oC
LSD 0.05
70.5 a
40.9 b
61.9 a
61.3 a
19.0
Conclusions
Stem
1.077 b
16oC
Bulk
Matur.
Matur.
Fig. 5: Sucrose is hydrolyzed by invertase into glc and fru and its activity has been closely associated with RS accumulation during LTS.
Invertase is also sensitive to an endogenous inhibitor (presented as avg % inhibition of total activity over 24 days at 4oC). Expression of a
cold-inducible invertase (Invertase IV) and two invertase inhibitors (Brumell et al., 2011) were followed during LTS. Premier’s resistance to
LTS was highly correlated with lack of invertase IV expression and an average of 90% inhibition of total activity by endogenous inhibitor.
Invertase IV expression in Ranger increased rapidly at the onset of LTS, consistent with high RS accumulation over the 24-day period at 4oC
(data not shown) and its LTS-susceptibility. While the higher invertase inhibition in Premier correlated with early expression of inhibitor 2, RS
content still increased in all heat-stressed tubers by 24 days at 4oC. The role of invertase in RS buildup of heat-stressed Premier tubers
warrants further investigation.
Bud
0
3
Premier
Ranger
16oC
2
1
6
Premier
Ranger
0
23oC Maturation
1
3
50
52
54
56
58
60
62
64
66
68
Ambient
0
0
6
Premier
Ranger
0
3
2
3
Premier Russet
Growing
Invertase
Temperature Inhibition (%)
70
23oC Bulking
1.089 a
6
74
90
6
Average across hill = 82oF
16oC
1.096 a
3
72
78 80
72
18
23oC Matur.
Stem
1.101 a
6
88
90
50
52
54
56
58
60
62
64
66
68
70
50
52
54
56
70 58
60
62
64
66
68
86
84
8280787674 72
86
Days at 4oC
0 1 3 6
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
Bud
1.084 b
3
Fig. 4: Sucrose-6-P synthase (SPS) is responsible for
the synthesis of sucrose-6-P from UDP-glc and fru-6-P.
Sucrose content was lowest at harvest for Premier
tubers grown under ambient conditions and with
moderate heat during maturation (23oC) (data not
shown). Expression of SPS in Premier increased within
24 h at 4oC, followed by increases in sucrose from 3
days onward (data not shown). SPS expression
increased from 0-6 days of LTS in tubers grown at 16oC.
Ranger tubers grown at high temperature had higher
SPS transcript levels at harvest than Premier tubers
and expression increased during the initial 24 h of LTS,
consistent with more rapid buildup of sucrose in this
cultivar.
Reducing Sugar Accumulation
84
72
50
52
54
56
58
60
62
64
86
66
68
707476788082 88
84
68
Distance Across Hill (inches)
74oF
1.103 a
23oC Matur.
23oC Bulk
9
Specific
Gravity
Fig. 2: Loss of processing quality (assessed using French fry planks) in
Premier Russet (top) and Ranger Russet (bottom) tubers as affected by
soil temperature during tuber development. Specific gravities (SG =
starch content) were determined at harvest (means separated by LSD
P<0.05). Each fry plank is from a different tuber selected to represent
the average fry color in a 12-tuber sample. Numbers on fries are USDA
color grades (0 light to 4 dark).
1
16oC
50 60 70 80 90 100 110 120 130 140 150 160 170 180
9
0
6
Ranger Russet
Maturation
72
Average across hill =
A. Process quality during LTS is affected by heat stress
23oC Bulk
Vine kill
Heat on
Maturation
68
Results
Days After Planting
Heat on
74
SGN-U270084
3
18sRNA
72
70
1
12
72
7
0
16
78
74
6
SPS
80
72
3
20
50
52
54
56
58
60
62
64
66
68
70
1
24
Depth (inches)
o
Temperature ( C)
F: GACCCCATATTACTCCCTATGTCTC AY864821.1
R: GGGTTCGATTTCTCTAGCTCTTTTA
16oC
80 90 100 110 120 130 140 150 160 170 180
0
0
72
74
76
0
18sRNA
28
4
66
68
70
6
SPS
16oC (Ambient)
29oC
Vine kill
8
1
70
2011
Accession #
AY864820
60
o
32
F: GGTTATCGGGTGTCCATTTG
R: TGTTGTAATATCTCCTGCTGTTTCA
50
Days After Planting
36
3
Bulking
80 90 100 110 120 130 140 150 160 170 180
2011
1
Heat on
Bulking
40
Invertase Inhibitor 1
Sucrose-6-Phosphate Synthase
Days at 4oC
Premier Russet
24
50
SGN-U270188
F: TTCAATGCTGGAGAGCACAC
R: TGTCATTCATCTGCACAGCA
Vine kill Heat off
16oC (Ambient)
29oC
28
0
Invertase IV
Sucrose-6-P Synthase
2011
16oC (Ambient)
23oC
4
Depth (inches)
Sequence
F: CCATCCATTTGGCTTGTCTT
R: TTTTCTCGATGAACCGGAAC
2011
36
8
~10” spacing
F: GGTACGGGTATGTGGGAGTG
R: GGTTATCGGGTGTCCATTTG
Plastidic Starch Phosphorylase
40
32
Changes in transcript abundance of key genes (Table 1) in
the LTS pathway over the first 6 days of storage at 4oC were
determined using reverse-transcriptase (RT)-PCR (Promega). Total RNA was extracted
from frozen tuber tissue using a modified phenol-chloroform procedure with initial
aqueous extractions at pH 9.6 to inhibit RNase activity. mRNA was selectively
synthesized into cDNA using oligo(dT)20 primer (Fermentas). All PCR reactions were run
for 35 cycles with approximately 260 ng template.
Invertase Inhibitor 2
0
Heat Cable Installation - 2010
Four soil warming cables were installed in-furrow (A) to A
ensure a uniform temperature increase across the hill.
Precise temperature control was accomplished using
rheostats and continual monitoring with temperature
probes. (B) Temperature was increased by an average of 7
and 13oC during tuber bulking (111-164 DAP, top graphs)
and maturation (151-180 DAP, bottom graphs). (C)
temperatures across the hills were measured on Sept 6,
2011. Tubers were harvested 180 DAP (Oct. 12), woundhealed for 10 days at 9oC, and then challenged to undergo
sweetening (LTS) by storing at 4oC for 24 days (Fig. 2).
Table 1. Forward (F)
and reverse primer
sequences for the
amplification of
transcripts of genes
involved in LTS
Premier Russet
oC
Days
at
4
A
0 1 3 6
Sucrose Synthesis
Materials and Methods
Gene
Starch Phosphorylase
4
4
 Increased temperature during tuber bulking (111-164 DAP) resulted in lower SG and higher sugar ends at harvest.
 Moderate heat stress (23oC) during tuber maturation had no effect on process quality of Ranger but induced LTS in Premier, which
began after ~12 days in storage at 4oC.
 Heat-stress resulted in loss of the inherent LTS-resistant phenotype from Premier tubers and exacerbated fry-darkening (LTS) in Ranger
tubers.
 Gene expression profiles and enzyme activity of Ranger tubers illustrated its LTS-susceptibility through an increase in starch catabolism
(SP), enhanced sucrose synthesis (SPS), and inversion to RS (invertase). Heat stress appeared to induce increased transcription of
enzymes responsible for LTS.
 Severe heat stress (23oC soil temperature during tuber bulking and 29 oC during tuber maturation) resulted in loss of LTS-resistance in
Premier; however gene expression and associated enzyme activities did not parallel the LTS-susceptible phenotype of Ranger.
Expression of SPS and SP corresponded more closely to sugar content at harvest. A lack of sucrose hydrolysis by invertase further
indicated that sweetening in Premier may involve alternate metabolic pathways.
References
Rathore, RS, N Garg, A Garg and A Kumar. (2009) Crit. Rev. Biotech. 29(3): 214-224
Brumell, DA, RKY Chen, JC Harris, H Zhang, C Hamiaux, AV Kralicek and MJ McKenzie. (2011) J. Exp.
Botany 62(10) 3519-3534.
Acknowledgements
This work was supported by the National Science Foundation REU Program
under grant number DBI-1156880