MODIFYING TUBER SIZE DISTRIBUTION, LOW TEMPERATURE SWEETENING and TOLERANCE to HEAT STRESS in PROCESSING POTATOES ( Solanum Tube

MODIFYING TUBER SIZE DISTRIBUTION, LOW TEMPERATURE

SWEETENING AND TOLERANCE TO HEAT STRESS IN

PROCESSING POTATOES (Solanum tuberosum L.)

DEREK JAMES HERMAN

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Horticulture

DECEMBER 2016

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of DEREK

JAMES HERMAN find it satisfactory and recommend that it be accepted.

______N. Richard Knowles, Ph.D.

______Mark Pavek, Ph.D.

______John Fellman, Ph.D.

______Nora Olsen, Ph.D.

ACKNOWLEDGEMENTS

I would like to begin by thanking Dr. Rick Knowles for allowing me to further my education by attaining my Horticulture Doctorate of Philosophy degree in his prestigious laboratory. Your guidance, mentorship and generosity have allowed me to develop into the scientist, student and young man I am today. A special thanks to Dr. Lisa Knowles and Dr.

Mohan Kumar, you are both world-class scientists and with your leadership and training I developed the skills necessary to thrive in the world of research. A special thank you to my undergraduate advisor and committee member Dr. John Fellman, your unique perspective and advice drove me to continue my education and for that I am so grateful. To the remainder of my committee, Dr. Mark Pavek and Dr. Nora Olsen, thank you for your advice on anything and everything potato. I would also like to thank all of those who helped with my research at

Washington State University, but not limited to: Dr. Daniel Zommick, Zachary Holden, Chandler

Dolezal, Dr. Rhett Spear, Dr. Jacob Blauer, Nora Fuller, Josh Rodriguez and Rudy Garza, without your assistance none of this would have been possible.

Finally, thank you to my entire family, but most importantly my parents Drs. Doug and

Wendy Herman. You instilled in me the hard work ethic and passion needed to be successful. I am forever grateful for the life you have given me, thank you and I love you.

I appreciate you all so very much, without you I would not be where I am today. Thank you and Go Cougs!

iii

MODIFYING TUBER SIZE DISTRIBUTION, LOW TEMPERATURE

SWEETENING AND TOLERANCE TO HEAT STRESS IN

PROCESSING POTATOES (Solanum tuberosum L.)

Abstract

by Derek James Herman, Ph.D. Washington State University December 2016

Chair: N. Richard Knowles

Controlling tuber size distribution and the propensity for low temperature sweetening

(LTS) of processing potatoes are key to optimizing quality and maximizing crop value. My research focused on (1) developing methods to increase tuber set and shift tuber size distribution in cv. Bondi, (2) investigating how low O 2 storage modulates LTS of cvs. Innovator and Russet

Burbank, (3) screening cultivars for tolerance to heat stress for retention of LTS-resistant phenotype and (4) determining the mechanism by which heat stress abolishes resistance to LTS.

Bondi produces vigorous foliar growth, low tuber set and high yields of large tubers that frequently exceed optimum size for seed and processing markets. We evaluated several methods for altering apical dominance, tuber set and size distribution of Bondi and its maternal parent,

Ranger Russet. Gibberellic acid (GA) applied to cut seed prior to planting reduced apical dominance, increased tuber set and decreased average tuber size; however, the optimal concentration to maximally shift tuber size distribution without decreasing marketable yield was

4-5-fold greater for Bondi than Ranger. The reduced sensitivity of Bondi to GA was likely inherited from its paternal parent Karaka, which displays similar morphological and developmental traits, indicative of high levels of endogenous GA.

Russet Burbank and Innovator are frozen processing cultivars with inherently different susceptibilities to LTS. Here we show how low O 2 storage modulates cold-induced sweetening to reveal metabolic differences in LTS metabolism intrinsic to these cultivars. While storage of tubers in 2.5 kPa O 2 greatly attenuated the initial LTS responses for both cultivars, the effect was only temporary in Innovator. Low O 2 attenuated LTS by inhibiting invertase.

Heat stress exacerbated cold-induced sweetening of Ranger and Russet Burbank tubers

(LTS-susceptible cultivars), and abolished the inherent LTS-resistance of Sage Russet, GemStar

Russet, POR06V12-3 and A02138-2 tubers. Heat stress rendered invertase cold-inducible in the

LTS-resistant but non-heat tolerant cultivars/clones. Payette and its maternal parent EGA09702-

2, however, demonstrated considerable tolerance to heat stress for retention of LTS-resistance.

Payette’s heat tolerance was conferred by the lack of cold-induction of invertase, similar to

Innate ® Russet Burbank (W8) tubers, where silenced invertase activity conferred robust tolerance to heat stress.

Table of Contents

Page

Acknowledgements………………………………………………………………………… iii

Abstract…………………………………………………………………………………….. iv

List of Tables……………………………………………………………………………….. x

List of Figures………………………………………………………………………………. xi

Abbreviations……………………………………………………………………………….. xiii

Introduction…………………………………………………………………………………. 1

References………………………………………………………………………………. 5

Chapter 1 : DIFFERENTIAL SENSITIVITIES OF GENETICALLY RELATED POTATO CULTIVARS TO TREATMENTS DESIGNED TO ALTER APICAL DOMINANCE , TUBER SET AND SIZE DISTRIBUTION

Abstract…………………………………………………………………………………. 8

Introduction……………………………………………………………………………... 10

Materials and Methods………………………………………………………………….. 13

Synopsis of Trials…………………………………………………………………… 13

Seed Preparation and Treatments…………………………………………………… 14

Field Plot Design and Maintenance………………………………………………… 15

Plant Establishment, Stem Counts, Harvesting and Sorting………………………... 15

Data Analysis and Presentation……………………………………………………... 16

Results…………………………………………………………………………………... 16

GA x BA Dose Response…………………………………………………………… 16

CV x GA Dose Response…………………………………………………………… 18

GA x Seed Age Response…………………………………………………………... 22

Discussion………………………………………………………………………………. 24

Acknowledgements……………………………………………………………………... 29

References………………………………………………………………………………. 30

Figures and Tables……………………………………………………………………… 36

Chapter 2: LOW OXYGEN STORAGE MODULATES INVERTASE ACTIVITY TO ATTENUATE COLD -INDUCED SWEETENING AND LOSS OF PROCESS QUALITY IN POTATO (Solanum tuberosum L.) Abstract……………………………………………………………………………….… 53

Introduction……………………………………………………………………………... 55

Materials and methods………………………………………………………………….. 57

Plant Materials, Storage Temperature, and Controlled Atmosphere Regimes……... 57

Whole Tuber Respiration…………………………………………………………… 58

Sprouting, Fry Process Quality, and Tissue Sample Preparation…………………... 58

Carbohydrate Analysis……………………………………………………………… 59

Invertase and Starch Phosphorylase Activities……………………………………... 60

Data Analysis and Presentation……………………………………………………... 61

Results…………………………………………………………………………………... 61

Tuber Respiration…………………………………………………………………… 61

Tuber Dormancy and Sprouting…………………………………………………….. 63

Process Quality (Fry Color, Reducing Sugars, Sucrose)…………………………… 63

Invertase and Starch Phosphorylase Activities……………………………………... 66

Discussion………………………………………………………………………………. 68

Conclusions……………………………………………………………………………... 74

Acknowledgements……………………………………………………………………... 74

vii

References………………………………………………………………………………. 75

Figures and Tables……………………………………………………………………… 84

Chapter 3: HEAT STRESS AFFECTS CARBOHYDRATE METABOLISM DURING COLD -INDUCED SWEETENING OF POTATO (S OLANUM TUBEROSUM L.)

Abstract…………………………………………………………………………………. 95

Introduction……………………………………………………………………………... 96

Materials and methods………………………………………………………………….. 98

Plant Material……………………………………………………………………….. 98

In-season Heat Stress Studies………………………………………………………. 98

Postharvest Handling and Storage………………………………………………….. 101

Tissue Sampling and Process Quality Assessment…………………………………. 101

Sucrose and Reducing Sugar Analyses……………………………………………... 102

Dormancy Break……………………………………………………………………. 103

PHHS Studies………………………………………………………………………. 103

Enzyme Analysis…………………………………………………………………… 104

RNA Extraction and qPCR…………………………………………………………. 105

Data Analysis and Presentation…………………………………………………….. 106

Results…………………………………………………………………………………... 107

In-season Heat Stress Studies………………………………………………………. 107

Tuber Set, Size, Yield and Raw Quality……………………………………………. 108

At Harvest Process Quality and Reducing Sugar Accumulation During LTS……… 109

Dormancy Break and Sprout Growth………………………………………………. 111

PHHS Studies………………………………………………………………………. 112

viii

Invertase and Starch Phosphorylase Activities……………………………………... 113

qPCR Analysis of Invertase and Invertase Inhibitors………………………………. 115

Discussion………………………………………………………………………………. 116

Acknowledgements……………………………………………………………………... 124

References………………………………………………………………………………. 125

Figures and Tables……………………………………………………………………… 136

Appendix………………………………………………………………………………... 154

List of Tables

Chapter 1

1. Table 1. Seed Treatments and Experimental Design……………………………….. 36

2. Table 2. GA and BA affect Tuber Yield and Size Distribution…………………….. 37

3. Table 3. Yield Responses to GA Concentrations in 2013…………………………... 38

4. Table 4. Yield Responses to GA Concentrations in 2014………………………….. 39

5. Table 5. Effects of GA and Seed Age on Plant Emergence and Yield in 2014…….. 40

Chapter 2

6. Table 1. P-values for 4-way ANOVA………………………………………………. 84

7. Table 2. Sprout Growth as Affected by Temperature and O 2………………………. 85

Chapter 3

8. Table 1. LTS Phenotype and Parentage of Cultivars/clones………………………... 136

9. Table 2. Effects of In-season Heat Stress on Tuber Yield and Quality…………….. 137

10. Table 3. In-season Heat Stress Affects LTS and Process Quality………………….. 138

11. Table 4. P-values for 3-way ANOVA with Planned Comparisons…………………. 139

Appendix

12. Table 1. In-season Heat Stress Affects Tuber Yield and Quality…………………... 154

List of Figures

Chapter 1

1. Fig. 1. Effects of GA ± BA on Plant Emergence in 2013…………………………... 41

2. Fig. 2. Effects of GA ± BA on Stem Numbers in 2013…………………………….. 42

3. Fig. 3. Effects of GA on Plant Emergence in 2013…………………………………. 43

4. Fig. 4. Effects of GA on Plant Growth and Tuber Development in 2013…………... 44

5. Fig. 5. GA Affects Tuber Size Distribution in 2013………………………………... 46

6. Fig. 6. Effects of GA on Plant Emergence in 2014…………………………………. 47

7. Fig. 7. Effects of GA on Plant Growth and Tuber Development in 2014…………... 48

8. Fig. 8. GA Affects Tuber Size Distribution in 2014………………………………... 50

9. Fig. 9. GA and Seed Age Affects Tuber Size Distribution in 2014………………… 52

Chapter 2

10. Fig. 1. Schematic Illustration of Factorially Designed Storage Treatments………... 86

11. Fig. 2. Temperature and O 2 Affect Tuber Respiration……………………………... 87

12. Fig. 3. Effects of Temperature and O 2 on Emergence from Dormancy……………. 88

13. Fig. 4. Fry Process Quality is Affected by Temperature and O 2……….…………… 90

14. Fig. 5. USDA Fry Color is Affected by Temperature and O 2……….……………… 91

15. Fig. 6. Temperature and O 2 Affect Sweetening of RB and Inn…………………….. 92

16. Fig. 7. Temperature and O 2-induced Invertase Activities………………………….. 93

17. Fig. 8. Cytosolic and Plastidic Starch Phosphorylase Activities…………………… 94

Chapter 3

18. Fig. 1. Soil Warming Cables and Hill Temperature Profiles……………….………. 140

19. Fig. 2. Soil Temperature Profiles…………………………………………………… 141

20. Fig. 3. Effects of In-season Heat Stress on Tuber Size Distributions………………. 143

21. Fig. 4. In-Season Heat Stress Affects Process Quality and LTS……………….…… 145

22. Fig. 5. Effects of In-season Heat Stress on Sprouting of RR and PR………………. 146

23. Fig. 6. Changes in Process Fry Color of Eight Clones as Affected by PHHS……… 148

24. Fig. 7. Effects of PHHS on LTS Depends of Genotype……………………………. 149

25. Fig. 8. PHHS Affects Invertase Activities………………………………………….. 150

26. Fig. 9. PHHS-induced Changes in Plastidic Starch Phosphorylase Activities……… 151

27. Fig. 10. Gene Expression (qPCR) as Affected by PHHS…………………………… 153

xii

Abbreviations 138-2, A02138-2 ANOVA, analysis of variance ARS, agricultural research services B, Bondi BA, benzyladenine CK, cytokinin CS, cold stress CT, cubic trend CV, cultivar DAH, days after harvest DAP, days after planting DD, degree days

DDC T, delta-delta-Ct algorithm Dev, deviations DM, dry matter DMSO, dimethyl sulfoxide Fru, fructose

GA, GA 3 gibberellic acid Glc, glucose HS, heat stress INH, acid invertase inhibitor INH1, apoplastic acid invertase inhibitor INH2, vacuolar acid invertase inhibitor INV, acid invertase LSD, least significant difference LT, linear trend LTS, low-temperature sweetening MKT, marketable yield NAA, naphthalene acetic acid NADP, nicotinamide adenine dinucleotide phosphate

xiii

NWVDP, Pacific Northwest Potato Variety Development Program OPPP, oxidative pentose phosphate pathway PGR, plant growth regulators PHHS, postharvest heat stress PR, Payette Russet PVPP, polyvinylpolypyrrolidone

Q10 , temperature coefficient qPCR, quantitative polymerase chain reaction QT, quartic trend RAR, respiratory acclimation response RB, Russet Burbank RH, relative humidity ROS, reactive oxygen species RR, Ranger Russet RS, reducing sugars RTPCR, real time polymerase chain reaction SG, specific gravity SP, starch phosphorylase (H and L) TEA, triethanolamine Tween 20, polyoxyethylene 20 sorbitan monolaurate USDA, United States Department of Agriculture V12-3, POR06V12-3 (Castle Russet)

xiv

INTRODUCTION

In 2013, nearly 64% of potatoes grown in the U.S. were used in the processing industry

(USDA NASS 2015). Potato chips and French fries dominate the process market and processors have defined strict parameters for tuber size, shape and raw product quality to meet demands of the quick-service-restaurant (QSR) industries they serve. Tubers should ideally have high specific gravity and low concentrations of reducing sugars for best French fry and chip quality.

Optimum tuber size for frozen processing (i.e. French fries) is 170-297-g tubers with L/W ratios of 1.8 or greater (Pavek and Knowles 2016). This dissertation focused on (1) evaluating the efficacies of treatments designed to alter apical dominance, tuber set and size distribution in two genetically-related frozen-processing cultivars, (2) determining how low O 2 atmosphere modulates cold-induced sweetening of a LTS-susceptible and resistant cultivar, (3) screening

LTS-resistant cultivars and advanced breeding lines for tolerance to heat stress for retention of postharvest quality, and (4) determining the mechanism by which heat stress can abolish inherent resistance to LTS.

The ideal tuber size distribution for maximum crop value is defined by the various end- use markets (seed, fresh pack, processing). Tailoring tuber set and size in relation to market requirements can add substantial value to a crop (Blauer et al. 2013). Previous research has indicated that apical dominance (stem number), tuber set and size distribution can be manipulated with plant growth regulators (PGR) (Mikitzel 1993; Smeltzer and Mackay 1963;

Struik et al. 1999; Timm et al. 1962), seed age (Knowles and Botar 1992; Knowles and Knowles

2006), or a combination of both (Blauer et al. 2013; Knowles and Knowles 2015; Knowles et al.

1985); however, the responses are highly cultivar-dependent. The cultivar Bondi, which has garnered significant interest from the potato processing industry for routinely out yielding the

1 industry standards, Russet Burbank and Ranger Russet (maternal parent), with less management inputs, is an ideal candidate for treatments designed to alter tuber set and size distribution

(Oliveira 2015). Bondi characteristically produces vigorous foliar growth, low tuber set and high yields of large tubers that often exceed the sizes desired for processing and seed production.

Accordingly, the main objective of the first chapter was to evaluate the relative responsiveness of Bondi and its maternal parent, Ranger Russet, to PGR (gibberellic acid (GA) and cytokinin) and/or age-priming seed treatments for altering apical dominance, tuber set and size distribution. GA and/or seed aging treatments were effective methods for manipulating tuber set and size distribution in both cultivars, but the sensitivities of each cultivar to GA differed. The concentrations of GA needed to maximally shift tuber size distribution without decreasing marketable yield were identified for each cultivar and potential physiological reasons for the differential responsiveness to GA are discussed in relation to the unique growth and developmental phenotypes of these cultivars.

Processing quality in potatoes is largely determined by starch, sucrose, glucose and fructose. The latter two sugars are reducing sugars that readily undergo Maillard reaction with free amino acids to produce unacceptably dark process color, off-flavors and the probable human carcinogen, acrylamide (IAREC 1994; Kumar et al 2004; Tareke et al. 2004). Tubers with optimal process quality have high starch content and low reducing sugar concentrations and can maintain this relationship during storage at 9 oC. Storage at lower temperatures (e.g. 4-6oC) will extend storage life by lowering respiration rate, limiting weight loss, inhibiting disease progression and delaying sprouting; however, a major concern with this practice is the cold- induced breakdown of starch into reducing sugars (Isherwood 1976; Sowokinos 2001; Malone et al 2006). Potato breeding programs have been successful in developing LTS-resistant varieties

(Novy et al. 2008). Furthermore, management techniques, such as low O 2 storage, have been shown to attenuate reducing sugar accumulation in cold stored potatoes of LTS-susceptible varieties (Parkin and Schwobe 1990; Schouten 1992; Mawson 1998). Little is known about the effects of low O 2 atmosphere on retention of process quality and carbohydrate interconversions in LTS-resistant varieties during full season storage.

The second chapter focused on using low O 2 storage to modulate the cold sweetening metabolism of two processing cultivars (Innovator and Russet Burbank) with differing susceptibilities to cold sweetening to characterize metabolic differences intrinsic to each cultivar.

Low O 2 storage attenuated the initial sweetening responses of both cultivars but the effect was only temporary in Innovator. The O 2-induced mitigation of LTS in both cultivars was largely the result of reduced invertase activities during cold storage, which resulted in sucrose buildup.

While low O 2 atmosphere is not an economically viable storage option for processing potatoes, it may be useful as a technique for delineating differences in the cold sweetening metabolism of susceptible and resistant varieties.

Potato breeding programs like the Pacific Northwest Variety Development Program

(NWPVD) have been successful in developing and releasing LTS-resistant varieties. Recent work, however, has demonstrated that heat stress can undermine this resistance, resulting in expression of LTS phenotype in otherwise inherently cold-resistant cultivars (Zommick et al.

2014). Understanding the mechanism(s) by which heat alters susceptibility to LTS is prerequisite to developing cultivars with more durable and robust tolerance to heat stress for retention of LTS-resistant phenotype. Therefore, the focus of chapter three was to use in-season and postharvest heat stress (PHHS) protocols to identify conventionally bred and engineered potato cultivars/clones with heat tolerance for retention of the LTS-resistant phenotype. The

3 mechanism of insensitivity to heat for retaining cold-sweetening resistance was subsequently determined for selected clones.

The sweetening phenotypes of nine cultivars/clones were classified as ‘LTS-susceptible’,

‘LTS resistant but non-heat tolerant’ or ‘LTS resistant and heat tolerant’ based on their responses to heat and cold storage treatments. Successful identification of the heat-tolerant clones was essential to determining the mechanism by which heat stress altered carbohydrate metabolism to abolish cold sweetening resistance in the heat-susceptible clones. Heat stress induces a buildup of sucrose, which is then inverted during subsequent cold storage of many LTS-resistant non- heat tolerant cultivars/clones. However, clones that exhibited tolerance to heat for retention of

LTS-resistant phenotype continued to buildup sucrose without appreciable inversion to reducing sugars during cold storage. Heat tolerance was therefore conferred by lower sensitivity of invertase to cold induction. Payette Russet, a heat tolerant, LTS-resistant cultivar showed similar low invertase activity, high sucrose accumulation and excellent process quality phenotypes as the

LTS-resistant and heat tolerant Innate ® Russet Burbank (W8) cultivar, where invertase activity has been silenced (Clark et al. 2014). Therefore, breeding for insensitivity of invertase to cold induction will likely impart durable tolerance to heat stress for retention of the LTS-resistant trait. The mechanism by which invertase activity resists cold induction in heat tolerant LTS- resistant cultivars awaits further investigation.

References

Blauer, J.M., Knowles, L.O., Knowles, N.R. 2013. Evidence that respiration is the pacemaker

of physiological aging in seed potatoes (Solanum tuberosum L.). Journal of Plant

Growth Regulation 32: 708-720.

Clark P., Habig J., Ye J., Collinge S. 2014. Petition for determination of nonregulated status for

Innate potatoes with late blight resistance, low acrylamide potential, reduced black spot,

and lowered reducing sugars: Russet Burbank event W8. J.R. Simplot Company Petition

JRS01 (USDA Petition 14-093-01p).

International Agency for Research on Cancer, 1994. Some industrial chemicals: summary of

data reported and evaluation. IARC Monographs on the Evaluation of Carcinogenic Risks

to Human 60. http://monographs.iarc.fr/ENG/Monographs/vol60/mono60-16.pdf on June

9, 2015. Accessed 14 April 2016.

Isherwood, F.A. 1976. Mechanism of sugar-starch interconversion in Solanum tuberosum.

Phytochemistry 15: 33-41.

Knowles, N.R., and G.I. Botar. 1992. Effect of altering the physiological age of potato

seed-tubers in the fall on subsequent production in a short-season environment.

Canadian Journal of Plant Science 72: 275-287.

Knowles, N.R., W.M. Iritani, and L.D. Weller. 1985. Plant growth response from aged potato

seed-tubers as affected by meristem selection and NAA. American Journal of Potato

Research 62 (6): 289-300.

Knowles, N.R., and L.O. Knowles. 2006. Manipulating stem number, tuber set, and yield

relationships for northern- and southern-grown potato seed lots. Crop Science 46: 284-

296.

Knowles, N.R., and L.O. Knowles. 2015. Optimizing tuber set and size distribution for potato

seed ( Solanum tuberosum L.) expressing varying degrees of apical dominance. Journal

Plant Growth Regulation (in press).

Kumar, D., Singh, B.P., Kumar, P. 2004. An overview of the factors affecting sugar content of

potatoes. Annals of Applied Biology 145: 247-256.

Malone, J.G., Mittova, V., Ratcliffe, R.G., Kruger, N.J. 2006. The response of carbohydrate

metabolism in potato tubers to low temperature. Plant and Cell Physiology 47: 1309-

1322.

Mawson, K. 1998. Storage of new potatoes using refrigeration, humidification, and controlled

atmospheres. Aspects of Applied Biology 52: 321-326.

Mikitzel, L.J. 1993. Influencing seed tuber yield of Ranger Russet and Shepody potatoes with

gibberellic acid. American Potato Journal 70: 667-676.

Novy, R.G. et al. 2008. Premier Russet: A dual-purpose, potato cultivar with significant

resistance to low temperature sweetening during long-term storage. Am. J. Potato Res :

85:198-209.

Oliveira, J.S. 2015. Growth and development of potato ( Solanum tuberosum L.) crops after

different cool season storage. PhD dissertation, Lincoln University, Department of

Agricultural Sciences.

Parkin, K.L., Schwobe, M.A. 1990. Effects of low temperature and modified atmosphere on

sugar accumulation and chip color in potatoes (Solanum tuberosum). Journal of Food

Science 55: 1341-1344.

Pavek, M.J., Knowles, N.R. 2016. WSU potato cultivar yield and postharvest quality

evaluations for 2015. Washington State University Special Report.

(http://potatoes.wsu.edu/wp-content/uploads/2016/01/Potato-Cultivar-Yield-and-

Postharvest-Quality-Evaluations-Research-Edition-2015.pdf).

Schouten, S.P. 1992. Possibilities for controlled atmosphere storage of ware potatoes. Aspects

of Applied Biology 33: 181-188.

Smeltzer, G.G. and D.C. Mackay. 1963. The influence of gibberellic acid seed treatment and

seed spacing on yield and tuber size profile of potatoes. American Potato Journal 40:

377-380.

Sowokinos, J.R. 2001. Biochemical and molecular control of cold-induced sweetening in

potatoes. Am. J. Potato Res. 78: 221-236.

Struik, P.C., D. Vreugdenhil, H.J. van Eck, B.W. Bachem, and R.G.F. Visser. 1999.

Physiological and genetic control of tuber formation. Potato Research 42 (2): 313-331.

Tareke, E., Rydberg, P., Karlsson, P., Eriksson, P., Tӧrnquist, M. 2004. Analysis of acrylamide,

a carcinogen formed in heated foodstuffs. Journal Agricultural Food Chemistry 50:

4998-5006.

Timm, H., L. Rappaport, J.C. Bishop, and B.J. Hoyle. 1962. Sprouting, plant growth, and tuber

production as affected by chemical treatment of white potato seed pieces. IV Responses

of dormant and sprouted seed potatoes to gibberellic acid. American Potato Journal 39:

107-115.

National Agricultural Statistics Service (USDA). 2015. Potatoes 2014 summary: September

2015. ISSN: 1949–1514.

Zommick, D.H., Knowles, L.O., Pavek, M.J., Knowles, N.R. 2014. In-season heat stress

compromises postharvest quality and low-temperature sweetening resistance in potato

(Solanum tuberosum L.). Planta 239: 1243-1263.

CHAPTER 1

Differential sensitivities of genetically related potato cultivars to treatments designed to

alter apical dominance, tuber set and size distribution 1

Abstract

The cultivar, Bondi, was selected from a cross between Ranger Russet (maternal parent) and

Karaka and is currently being evaluated as a frozen processing cultivar. Relative to Ranger,

Bondi produces vigorous foliar growth, low tuber set and high yields of large tubers that frequently exceed optimum size for seed and processing markets. We evaluated the relative efficacies of gibberellin (GA), cytokinin (benzyladenine (BA)) and seed aging for altering apical dominance, tuber set and size distribution of these genetically related cultivars. GA applied to cut seed prior to planting hastened emergence, reduced apical dominance, increased tuber set and decreased average tuber size; however, the optimal concentration to maximally shift tuber size distribution without decreasing marketable yield was 4-5-fold greater for Bondi than Ranger.

BA marginally hastened plant emergence (Bondi) and decreased apical dominance (both cultivars) only when combined with GA, but had no further effects on tuber set, yields or tuber size distributions in either cultivar. Age-priming Ranger seed for 700 degree days (DD) at 32 oC during storage shifted the tuber size distribution to a much greater extent than 2 mg L -1 GA

(optimal concentration) without reducing marketable yield. The combined age and GA treatment resulted in no further shift in size distribution for Ranger beyond that induced by the 700-DD treatment alone, but reduced the marketable yield by 9.6 MT ha -1. In contrast to GA, the mechanism by which age-priming altered tuber size distribution in Ranger could not be explained by effects on stem number/tuber set relationships alone. Bondi, however, exhibited an even greater shift toward smaller tubers with no reduction in yield with the combined 700- 8

DD/GA (2 mg L -1) treatment, reflecting its decreased sensitivity to GA. Moreover, the shift in tuber size distribution induced by aging Bondi seed for 700 DD was approximately equal to that observed by treating seed with 8 mg L -1 GA (optimal concentration). The reduced sensitivity of

Bondi to GA was likely inherited from its paternal parent Karaka, which displays similar morphological traits, including high yield of large tubers.

1Herman, D.J., Knowles, L.O., Knowles, N.R. 2016. Differential sensitivity of genetically related potato cultivars to treatments designed to alter apical dominance, tuber set and size distribution. American Journal of Potato Research 93: 331-350.

Introduction

The optimum tuber size distribution for maximum crop value depends on end-use market requirements (seed, fresh, process) and can vary regionally, nationally and internationally. Once target size classes are defined, growers have the opportunity to increase returns by instituting management techniques that will maximize yields of the most lucrative tuber size classes. Tuber size distribution is influenced by a number of factors including cultivar, degree of apical dominance (stems per plant), seed age, tuber set (i.e. number of tubers per plant), plant spacing, and length of growing season (Arsenault et al. 2001; Blauer et al. 2013b; Bussan et al. 2007;

Caldiz 2009; Iritani et al. 1972; Knowles and Knowles 2015; Knowles and Knowles 2006; Struik et al. 2006; Struik et al. 1990).

Apical dominance entails the correlative inhibition of branch stem growth by the apical meristem (Cline 1991). In potato, decreasing apical dominance leads to more stems per plant, which in turn increases tuber set and shifts size distribution toward smaller tubers (Iritani et al.

1983; Knowles and Botar 1991; Knowles and Knowles 2006). Management techniques that affect apical dominance can thus add significant value by effectively changing tuber size distribution to more closely align with market requirements (Blauer et al 2013a).

The cultivar, Bondi, was selected from progeny resulting from a cross between the parental cultivars, Ranger Russet and Karaka. Originally developed by the New Zealand Plant and Food Research group, Bondi has garnered significant interest by growers and processors around the world as a late-season, long-russet processing cultivar. Bondi produces high yields of blocky shaped tubers with excellent specific gravity, low total sugars and cream colored flesh, ideal for use in the frozen process (French fry) industry.

Much of the interest in Bondi can be attributed to its field performance relative to processing industry standards such as Ranger Russet (its maternal parent) and Russet Burbank.

Over a wide range of soil conditions, Bondi routinely out-yields Ranger Russet and Russet

Burbank by as much as 20% in many production areas (Oliveira 2015). Moreover, its high yielding ability comes with significantly lower growing costs, as Bondi requires reduced inputs of fertilizer (nitrogen and potassium) and fungicides compared with other industry standards

(Simplot Australia, 2012 https://www.simplot.com.au/pdf/simplot-sustainability-report-02.pdf.

Accessed 9 November 2015). These strengths, however, are accompanied by a number of weaknesses that have impeded its adoption. In particular, tuber set in Bondi is low, resulting in high yields of large tubers that frequently exceed optimal size for key markets. Processing contracts often stipulate premiums for tubers ≥170 g beyond a base percentage of total yield

(Pavek and Knowles 2014). On the other hand, extremely large tubers (e.g. >397 g), a characteristic of Bondi, can trigger penalty clauses (Bolotova and Patterson 2009; Thornton et al.

2007) due to increased waste during processing. Large tubers are also less than ideal for production of both single-drop and cut seed, where tubers >284-g are often considered as culls.

Large tubers yield more ‘blind’ seed-pieces, which reduces plant stand and grower returns

(Pavek and Thornton 2006). Bondi seed costs can exceed Ranger Russet and ‘Russet Burbank’ by nearly 25% due primarily to large tubers (Mark Heap, Simplot Australia Pty Ltd personal communication). Optimizing tuber set and size distribution of Bondi would potentially increase its value and help facilitate its adoption as a processing cultivar.

Apical dominance, tuber set and size distribution can be manipulated with plant growth regulators (PGR) (Mikitzel 1993; Smeltzer and Mackay 1963; Struik et al. 1999; Timm et al.

1962), seed age (Knowles and Botar 1992; Knowles and Knowles 2006), or a combination of

11 both (Blauer et al. 2013a; Knowles and Knowles, 2015; Knowles et al. 1985). Recent work with fresh market specialty varieties demonstrated that gibberellin (GA) as a seed treatment increased crop value by decreasing apical dominance, increasing tuber set and reducing average tuber size

(Blauer et al 2013a). Similar work has demonstrated the efficacy of GA at shifting tuber size distribution of chipping (Arpiwi 2003) and frozen processing cultivars (Mikitzel 1993).

Consistent through many of these reports is the observation that sensitivity to GA is highly cultivar-dependent. In addition to GA, cytokinins (e.g. benzyladenine, BA) have long been linked to the initiation of lateral bud growth and release of apical dominance in plants (Cline

1991; Pillay and Raiton 1983). Cytokinins modulate dormancy break and the sensitivity of buds to GA for eliciting sprouting from potato tubers (Eshel and Teper-Bamnolker 2012; Hartmann et al. 2011). Apical dominance in potato is also affected by auxin (e.g. naphthalene acetic acid,

NAA) but in contrast to GA, NAA restores apical dominance thereby decreasing tuber set and increasing the yields of larger tubers (Knowles and Knowles 2015; Kumar and Knowles 1993;

Knowles et al. 1985; Mikitzel 1990; Romanov et al. 2000). Auxin is therefore an appropriate treatment option where larger tubers are desirable.

The degree of apical dominance is indicative of tuber physiological age (Eshel and

Teper-Bamnolker 2012; Iritani and Thornton 1984; Knowles and Knowles 2006; Krijthe 1962).

As seed age increases, apical dominance declines. Therefore, treatments designed to advance tuber physiological age are effective at increasing stem numbers, tuber set and shifting tuber size distribution toward smaller tubers (Blauer et al 2013a; Knowles and Knowles 2006; Struik 2007;

Struik et al. 1990). Accelerating the aging process by storing seed tubers at temperatures >4 oC can alter crop value through effects on tuber size distribution (Blauer et al. 2013ab; Knowles and

Knowles 2006). Similar to the efficacy of PGRs, cultivars differ in their sensitivity to age- priming treatments (Blauer et al. 2013a; Iritani 1968; O’Brien et al. 1983; Van Loon 1987).

Bondi’s inherent growth habit and low tuber set characteristics make this cultivar an ideal candidate for treatments designed to shift tuber size distribution. Accordingly, a main objective was to evaluate the relative responsiveness of Bondi and its maternal parent, Ranger Russet, to

PGR (GA and cytokinin) and age-priming seed treatments for altering apical dominance, tuber set and size distribution. Both treatment approaches were effective for both cultivars; however, sensitivities to the treatments were vastly different, with different doses required to elicit similar responses. The concentrations of GA to maximally shift tuber size distribution without decreasing marketable yield were identified for each cultivar. Potential physiological reasons for the differential responsiveness of these cultivars to the treatments are discussed in relation to their unique growth and development phenotypes.

Materials and Methods

Synopsis of trials

The effects of seed physiological age, gibberellin A 3 (GA) and 6-benzylaminopurine

(BA) seed treatments on tuber set, yield and tuber size distributions of cvs Ranger Russet and

Bondi were evaluated in seven replicated trials (Table 1) conducted at the Washington State

University (WSU) Irrigated Research Unit (Othello, WA, 46 o 47.277’ N. Lat., 119 o 2.680’ W.

Long.) over a 2-year period. Cultivar responses to equivalent concentrations of GA and the potential synergistic effects of GA and BA were assessed in three trials in 2013. Sensitivity to

GA was highly cultivar-dependent and therefore GA dose responses were further defined with different ranges of GA concentrations in separate studies for each cultivar in 2014. Additionally,

13 the extent to which GA-induced growth and yield responses depended on the physiological age of seed tubers was explored for each cultivar in 2014.

Seed preparation and treatments

Certified (G3, generation three from nuclear) seed tubers were acquired from commercial seed growers in April and November for the 2013 and 2014 trials, respectively. The 2013 seed had been held in a commercial storage at 3-4oC except for a 3-week wound healing period at 10-

12 oC directly following harvest. Seed for the 2014 trials was subjected to treatments designed to advance seed physiological age at the WSU postharvest research and storage facility. Three seed ages were created for two of the four 2014 trials (Table 1) by storing seed at 4, 12 and 32 oC

(95% RH) for various periods to accumulate 80, 400 and 700 degree days (DD) (4 oC base), as outlined in Knowles and Knowles (2006). All age-priming treatments were given at the beginning of storage while seed was dormant to minimize their effects on sprouting during the remainder of the ca 175-d storage period. Following the DD treatments, all seed was stored at the base temperature of 4 oC (95% RH) until cutting and treating in mid-April.

In preparation for treating and planting, seed tubers (115-200 g tuber -1) were hand cut into 50- to 64-g seed pieces with apical and basal portions assigned to different blocks

(replicates) for all trials. The cut seed pieces were briefly rinsed, submerged in solutions of GA

(0-16 mg L -1), BA (0-10 mg L -1), or GA (2 mg L -1) plus BA (0-10 mg L -1) for 5 min, air dried at room temperature, and stored at 9 oC (95% RH) for 3-5 days until planting (described below).

All treatment solutions were prepared in water containing 0.1% (w/w) Tween 20

(polyoxyethylene (20) sorbitan monolaurate) from 50 mg mL -1 stock solutions of GA

(gibberellin A 3, ≥90%, Sigma-Aldrich, St. Louis, MO) in dimethyl sulfoxide (DMSO) or BA (6- benzylaminopurine, 98%, Sigma-Aldrich, St. Louis, MO) in 1 M NaOH. The concentrations of

DMSO and NaOH were held constant across all treatments, including controls (0 mg L -1 GA and/or BA).

Field plot design and maintenance

Randomized complete block designs (5 replicates) were used for all trials with treatments arranged factorially where appropriate (Table 1). Treated seed pieces were planted 20-cm deep in a Shano silt loam soil (Lenfesty 1967) with a custom two-row assist-feed planter. Treatment plots were 6.1 m in length with seed pieces spaced 25 cm apart (24 seed pieces per plot). Plots were separated by a 1.3-m alley and a single hill (i.e. one seed piece) of cv. All Blue was planted at the beginning and ends of plots to maintain interplant competition and facilitate the separation of individual plots during harvest. Rows were spaced 86 cm apart and treatment rows alternated with guard rows of non-treated seed. All trials were positioned under a center pivot irrigation system which provided water, in-season fertilizer and pesticides according to standard practices for cv. Russet Burbank potatoes in the Columbia Basin. Tensiometers and neutron probes positioned within the plots monitored soil moisture, which was maintained at a minimum of 65% field capacity throughout the season. Irrigation scheduling followed evapotranspiration models for potato on the WSU Othello Research station (WSU AgWeatherNet, http://weather.wsu.edu/awn.php Accessed 9 November 2015.). Pre-plant and in-season fertilizer applications were adjusted based on soil tests and weekly petiole analyses, respectively.

Herbicide and pesticide applications followed standard practices.

Plant establishment, stem counts, harvesting and sorting

Plant emergence counts for all studies began 27 days after planting (DAP) and continued at 2-3 day intervals until all treatments had achieved full emergence (ca. 41 DAP). The number of aboveground stems per plant (seed piece) was recorded at 53 (2013) and 50 DAP (2014),

15 approximately 1 week prior to foliar canopy row closure. Vines were mechanically mowed at

154 (2014) and 155 DAP (2013) and all trials were harvested 2 weeks later with a single-row mechanical harvester. Tubers were washed, counted, individually weighed and sorted into the following categories: <113 g, 113-170 g, 171-284 g, 285-340 g, 341-397 g, >397 g, or culls.

Total yield is the sum of all tuber weights. U.S. No. 1 yield was determined by subtracting the weights of cull tubers and undersize tubers (<113 g) from total yield. Marketable yield equals

U.S. No. 1 yield plus undersize tubers.

Data analysis and presentation

Plant development and yield data were subjected to analysis of variance (ANOVA) with sums of squares partitioned into single degree-of-freedom contrasts for main effects (cultivar,

[GA], [BA], seed age) and interactions (in the case of factorially designed trials), including polynomial trends (linear, quadratic, cubic, etc.) where appropriate (see Table 1). Means (±SE) of selected developmental and yield data are plotted versus DAP, GA or BA concentrations.

Coefficients of determination are reported, along with significance levels ( P-values) for correlation coefficients, and LSD ( P<0.05) values are provided for mean separation. Polygonal plots of yields of the six tuber size classes expressed as percent marketable yield are presented to characterize the effects of GA and seed age on shifts in tuber size distributions normalized to yield.

Results

GA x BA dose responses

Plants emerged more rapidly from non-treated Ranger Russet seed than from Bondi seed and treatment of seed with GA (2 mg L -1) greatly accelerated emergence for both cultivars (Fig.

1). BA had little effect on plant emergence from Ranger Russet seed at 32 DAP regardless of

GA treatment but it enhanced the stimulatory effect of 2 mg L -1 GA on emergence from Bondi seed (emergence increased from 65 to 82% as BA increased from 0 to 10 mg L -1) (cultivar x GA x BA LT , P<0.02) (Fig. 1BD). GA also reduced apical dominance ( P<0.01) in Ranger and Bondi seed, increasing stem numbers per seed piece by an average of 1.5 (63%) and 0.6 (31%), respectively (Fig. 2). Similar to emergence, stem numbers were not affected by BA unless combined with GA (GA x BA LT , P<0.03). On average, both cultivars produced 17% (0.5) more stems per seed piece ( P<0.05) when seed was treated with a combination of 10 mg L -1 BA and 2 mg L -1 GA.

BA had no effect on tuber set (tubers plant -1, 1000s ha -1), average tuber fresh weight (g tuber -1), yields, or tuber size distributions in either cultivar (Table 2). When averaged over BA levels, GA at 2 mg L -1 had no effect on total, U.S. No. 1 and marketable yields of Bondi, but decreased total and U.S. #1 yields of Ranger Russet by 8 and 10%, respectively ( P<0.01) with no effect on marketable yield. Non-GA treated Ranger Russet seed produced 10.7 tubers plant -1 compared with 7.3 tubers plant -1 for Bondi seed (averaged across BA treatments) and Bondi tubers were 42% larger (g tuber -1) than Ranger tubers. GA increased tuber set by 0.7 (7%) and

1.6 (22%) tubers plant -1 in Ranger Russet and Bondi, respectively ( P<0.01). These increases in tuber set were accompanied by 10% (Ranger) and 14% (Bondi) lower average tuber weights, reflecting a GA-induced shift in tuber size distribution from larger to smaller tubers for both cultivars. Yields of >340-g (Ranger) and >397-g tubers (Bondi) decreased 27 and 18%, respectively, while yields of <170-g (Ranger) and <284-g tubers (Bondi) increased 18% and

32%, respectively, with GA treatment (averaged across BA levels).

Cultivar x GA dose responses

Consistent with the results above, plant emergence in 2013 from Bondi seed was delayed relative to Ranger Russet (Fig. 3). Plant emergence from both cultivars was accelerated by GA and the responses were cultivar and dose-dependent. Maximum stimulation of emergence was achieved with 1 mg L -1 GA for Ranger Russet compared with 4 mg L -1 for Bondi (CV x [GA],

P<0.01) (Fig. 3BD). Differences in the sensitivity of these cultivars to GA were also apparent for changes in apical dominance, tuber set, average tuber weight, and marketable yields (Fig. 4A-

D, CV x [GA], P<0.01). Stem numbers increased 50% and 107% for Bondi and Ranger Russet, respectively, as GA increased from 0 to 8 mg L -1 (Fig. 4A). Tuber number plant -1, however, increased by 2.1 for Bondi but decreased by 3.1 for Ranger as GA concentration increased to 8 mg L -1 (Fig. 4B). These changes resulted in ca. 100K more tubers ha -1 for Bondi and 140K less tubers ha -1 for Ranger with 8 mg L -1 GA seed treatment. Average tuber weight decreased 28% for Bondi and 18% for Ranger Russet as GA concentration increased to 8 mg L -1 (Fig. 4C).

Despite these changes, marketable yield of Bondi averaged 84 MT/ha -1, which was substantially higher than Ranger and not affected by GA (Fig. 4D). In contrast, marketable yield of Ranger

Russet remained constant from 0 to 1.5-2 mg L -1 GA, then decreased linearly (by 24 MT ha -1) with increasing GA concentration (CV x [GA], P<0.01; MT ha -1 = 56.2 – 3.71[GA], R 2= 0.94).

Bondi also produced considerably higher total and U.S. No. 1 yields than Ranger Russet regardless of GA treatment (Table 3) and the effects of GA on yields depended on cultivar (CV x

GA, P<0.01). Total yield of Ranger Russet remained relatively constant, averaging 55.2 MT ha -1 from 0 to 2 mg L -1 GA, then decreased by 45% as GA increased from 2 to 8 mg L -1. In contrast, total yield from GA-treated Bondi seed was comparable to the non-GA treated control seed

(Table 3), as was the case for marketable yield noted previously (Fig. 4D). The GA-induced

18 changes in U.S. No. 1 yields (Table 3) were analogous to those described for total yield for each cultivar.

The effects of GA on yields of the various tuber size grades contributing to marketable yield were also cultivar dependent. For Bondi, GA increased the yields of tubers <113 g, 113-

170 g, and 170-284 g but decreased the yield of >397-g tubers (Table 3). For Ranger, the yields of <113-g, 113-170-g, 170-284-g and >284-g tubers decreased in response to 4 to 8 mg L -1 GA.

When yields of the tuber size classes are normalized as percent marketable yield (U.S. No. 1 +

<113-g), tuber size distribution from the non-treated Bondi seed was dominated (45%) by oversize (>397-g) tubers compared with Ranger where tubers <284 g in weight accounted for ca.

80% of the yield profile (Fig. 5AB). GA at 8 mg L -1 shifted the tuber size distribution of Ranger

Russet to favor higher percentage of <113-g tubers and lower percentage of >284-g tubers compared with control. By contrast, 8 mg L -1 GA shifted the tuber size distribution of Bondi tubers away from oversize (>397-g) tubers toward increased percentage of tubers <340 g, resulting in a more even distribution of tubers among all size classes. The GA-induced shift in tuber size distribution of Bondi was mainly a result of increased tuber number as a consequence of decreased apical dominance (Fig. 5 inset), with marketable yield not affected by GA. While

GA also greatly decreased apical dominance in Ranger Russet, in contrast to Bondi, it decreased the number of tubers plant -1, which contributed to the 45% reduction in marketable yield (Fig. 5 inset). Collectively, these results characterize vastly different sensitivities of Ranger Russet and

Bondi to GA.

Subsequent studies focused on optimizing GA concentration for each cultivar separately.

The objective was to determine concentrations of GA that would induce maximum increases in tuber set and shifts in tuber size distribution without decreasing marketable yields for each

19 cultivar. The decrease in marketable yield of Ranger Russet at 4 and 8 mg L -1 GA (Fig. 4D) indicated that concentrations should be lowered, while Bondi could potentially tolerate concentrations higher than 8 mg L -1 to induce an even greater shift in tuber size distribution without decreasing marketable yield. Dose/response studies were thus conducted at 0-2.5 and 0-

16 mg L -1 GA for Ranger and Bondi, respectively. Consistent with the three previous studies, plants emerged later from Bondi than from Ranger Russet seed and GA accelerated emergence for both cultivars (Fig. 6). At 28 DAP, plant emergence from Ranger seed increased almost linearly ( P<0.01) from ca. 13 to 67% as GA increased from 0-2.5 mg L -1 (Fig. 6B). For Bondi, plant emergence at 32 DAP increased with GA concentration to a maximum (79%) at 8 mg L -1

(P<0.01) and then remained relatively constant through 16 mg L -1 (Fig. 6D). Both cultivars had achieved 100% emergence by 39 DAP regardless of GA treatment (Fig. 6AC).

Cultivar-specific differences in sensitivity to GA for changes in apical dominance, tuber set, average tuber weight and marketable yields are shown in Fig. 7. Stem numbers increased to comparable levels in both cultivars (3.7 stems plant -1) with increasing GA concentration; however, much less GA was needed to reduce apical dominance by this amount in Ranger than

Bondi (Fig. 7A). Ranger seed produced maximum stem numbers at 2.1 mg L-1 GA (stem no. =

2.49 + 1.085[GA] – 0.247[GA] 2, R 2= 0.98, P<0.05). By comparison, Bondi required 16 mg L -1

GA to match this decline in apical dominance (stem no. = 2.09 + 0.27[GA] – 0.033[GA] 2 +

1.39e -3[GA] 3, R 2=0.98, P<0.01). Consistent with the three previous studies, non-treated Bondi seed set fewer tubers that ultimately grew larger than Ranger tubers (Fig. 7B and 7C). GA treatment increased the number of tubers per plant by 20% at 1 mg L -1 in Ranger and by an average of 21% from 2 to 16 mg L -1 in Bondi. The GA-induced decreases in apical dominance and increases in tuber number per plant directly affected average tuber weights. As GA

20 increased from 0 to 2.5 mg L -1, average tuber weight of Ranger Russet decreased 22% to 145 g tuber -1, while 16 mg L -1 GA was needed to decrease the tuber weight of Bondi to an equivalent level (147 g tuber -1, a 41% reduction) (Fig. 7C). Marketable yield of Ranger was unaffected

-1 -1 (LSD 0.05 = 4.9 MT ha ) by low concentrations of GA, averaging 60.4 MT ha from 0 to 1.5 mg

L-1, but yield was reduced by 5.1 MT ha -1 at 2 and 2.5 mg L -1 GA (Fig. 7D). Similarly, and consistent with the 2013 trial, market yield of Bondi was not affected by GA until the

-1 -1 concentration exceeded 8 mg L (LSD 0.05 = 9.6 MT ha ).

Consistent with the effects on marketable yield, both cultivars experienced significant decreases in total yield and yield of U.S. No. 1 tubers at the highest concentrations of GA (Table.

4). However, total yields remained relatively constant from 0 to 1.5 mg L -1 for Ranger and 0 to 8 mg L -1 for Bondi. In contrast, U.S. No. 1 yield of Ranger declined 2.7 MT ha -1 for every 0.5 mg

L-1 increase in GA concentration (MT ha -1 = 54.1 – 5.30[GA], R 2= 0.94, P<0.01) and this occurred with a concomitant increase in yield of undersize (<113 g) tubers (by 3 MT ha -1 for every 0.5 mg L -1 increase in GA concentration from 0 to 1 mg L -1, R 2= 0.99, P<0.01), resulting

-1 in little effect on marketable yield (U.S. No. 1 + <113-g tubers) until GA exceeded 1.5 mg L as noted above. Similar trends in U.S. No. 1, <113-g and marketable yields were observed for

Bondi through 8 mg L -1 GA.

Tuber size distributions were significantly influenced by GA for both cultivars. GA increased the yield of <170-g tubers at the expense of larger tubers for Ranger Russet, while the yield of tubers <284 g was increased at the expense of larger tubers for Bondi (Table 4). These shifts in tuber size distribution are depicted in polygonal plots (Fig. 8) where yields of the tuber size classes have been normalized to marketable yields for the controls, 1.5 mg L-1 GA (Ranger) and 8 mg L -1 GA (Bondi) treatments, which were the highest concentrations of GA that induced

21 significant increases in stem and tuber numbers per plant without affecting marketable yields

(Fig. 8 inset tables). At 1.5 mg L -1 GA, tuber size distribution of Ranger shifted 14% toward

<170-g tubers with 43% of marketable yield falling in this category (Fig. 8A). GA had no effect on the percentage yield of 170-284-g tubers (average 39%) and therefore the shift toward smaller tubers occurred at the expense of tubers >284 g, which decreased from 71 to 57% with treatment.

A robust GA-induced shift in tuber size distribution was also apparent in Bondi. At 8 mg L -1

GA, nearly 60% of tubers fell into the <284-g category, with a concomitant 20% reduction in tubers >284-g (Fig. 8B). Relative to Ranger, the greater tuber size distribution shift with GA in

Bondi resulted in 25% lower average weight of marketable tubers (g tuber -1), compared with an

18% decrease for Ranger (Fig. 8 insets).

GA x Seed Age Responses

Similar to the GA-induced responses reported herein, seed physiological age affects apical dominance, tuber set, tuber size distribution and yield in a cultivar-dependent manner

(Blauer et al. 2013a; Knowles and Knowles, 2006). Further studies were conducted to characterize how GA and seed age interact to affect these growth and yield responses for each cultivar and to determine whether age-priming can effectively amplify the GA-induced effects.

Seed was age-primed for brief periods at 12 or 32 oC at the beginning of storage to accumulate

80, 400 or 700 DD above 4 oC (base holding temperature) and treated with 0 or 2 mg L -1 GA immediately prior to planting. GA treatment accelerated plant emergence more than seed age for both cultivars (Table 5). Apical dominance, tuber set, and yields of the various size grades were not altered by the 400-DD age-priming treatment (Table 5), resulting in tuber size distributions

(percent marketable yield basis) identical to the 80 DD control (youngest) seed for each cultivar

(Fig. 9AC). However, age priming at 32 oC for 700 DD increased stem and tuber numbers per

22 plant and substantially shifted tuber size distributions to favor higher yields of tubers <170 g at the expense of >284-g tubers in Ranger, and <284-g tubers at the expense of >340-g tubers in

Bondi, without affecting total and marketable yields of either cultivar (Table 5, Fig. 9AC).

Treatment of the 80-DD seed with 2 mg L -1 GA induced a similar but lesser shift toward higher yields of smaller (<170-g) Ranger and (<284-g) Bondi tubers than that induced by aging for 700 DD alone on both an absolute (Table 5) and percent marketable yield basis (Fig. 9). As expected, this effect of GA on tuber size distribution from 80-DD seed was due to decreases in apical dominance, which resulted in increased tubers per plant and lower average tuber weights for both cultivars (Table 5; Fig. 9AB inset tables). GA had little effect on the percent tuber size distributions from 700-DD Ranger seed; however, the combined 700-DD and GA treatments reduced the marketable yield by 9.6 MT ha -1 (16%) compared to the non-treated 700-DD seed.

In contrast, the marketable yield of Bondi seed was unaffected by GA regardless of seed age, but

GA treatment shifted the yield profiles to favor higher percentages of <284-g tubers for all ages of seed (Fig. 9CD).

In Ranger, age-priming for 700 DD (Fig. 9A inset; Table 5) reduced apical dominance and increased tuber set to the same extent as 2 mg L -1 GA on 80-DD seed (Fig. 9B inset; Table

5), resulting in equivalent tubers per stem (average = 2.1) for both treatments. Age-priming for

700 DD alone, however, shifted tuber size distribution to a much greater extent than the 2 mg L -1

GA 80-DD treatment (Fig. 9AB). These data suggest that the mechanism by which age-priming alters tuber size distribution in Ranger is somewhat different than that induced by GA and cannot be explained by reduced apical dominance alone. This synopsis is likely also true for Bondi; however, in contrast to Ranger, 2 mg L -1 GA is four-fold less than the optimal concentration (8 mg L -1) for increasing stem and tuber numbers and shifting tuber size distribution (Figs. 7 and 8),

23 which likely explains why GA-treatment had no deleterious effect on marketable yield from the

700-DD Bondi seed (Fig. 9CD). While both cultivars were highly sensitive to the age-priming

(700 DD) treatment, their differential sensitivities to GA concentration further underscores apparent differences in the mechanisms by which age-priming and GA affect apical dominance, tuber set, yields and tuber size distributions.

Discussion

Although Bondi and Ranger Russet are genetically related, their respective growth habits differ. Relative to Ranger, Bondi produces excessive foliar growth and sets fewer tubers that grow overly large, often with an elongated phenotype. These characteristics are indicative of elevated levels of endogenous GA during plant growth (Carrera et al. 2000) (see text below).

Bondi produces high yields with relatively low agronomic inputs (fertility, pesticides); however, its low tuber set and large tuber size is a weakness for seed and processing markets (Oliveira

2015). Therefore, plant growth regulator and age-priming seed treatments were evaluated for their efficacy at increasing tuber set and shifting tuber size distribution, explicitly through altering the degree of apical dominance.

Previous work has indicated that exogenous GA and age-priming treatments can effectively increase crop value by increasing yields of desired tuber size classes for fresh- and process-market varieties (Blauer et al. 2013a; Knowles and Knowles 2015; Struik 2007; Struik et al. 1990). As apical dominance decreases, stem and tuber numbers increase, resulting in shifts in tuber size distribution toward higher yields of smaller tubers (Iritani et al. 1983; Knowles and

Botar 1992; Knowles and Knowles 2006). These responses to GA and seed age, however, are highly cultivar-dependent, with doses too high ultimately diminishing marketable yield and

24 grower returns. Consequently, treatments should be cultivar-specific and developed in relation to end use requirements for tuber size.

As expected, GA effectively hastened plant emergence, reduced apical dominance, increased tuber set and decreased average tuber size, but the optimal concentrations to achieve maximum shift in tuber size distribution without negatively affecting marketable yields varied substantially between these cultivars. Consistent with results of Mikitzel (1993), Ranger, the maternal parent of Bondi, was highly sensitive to GA, with concentrations greater than 1.5-2 mg

L-1 negatively affecting yields (Table 3, Fig. 4). Conversely, Bondi was less sensitive to GA, requiring 4- to 8-fold higher concentrations (8-16 mg L -1) to reduce apical dominance to the same extent as Ranger seed treated with 2 mg L -1 GA (Figs. 4 and 7). Regardless of GA concentration, tuber number per plant in Bondi could not be increased beyond that of the non- treated Ranger seed and remained substantially lower (by ~1.5 tubers plant -1) than Ranger seed treated with 2 mg L -1 GA (Fig. 7).

It is noteworthy that BA enhanced the stimulatory effect of GA on plant emergence from

Bondi seed but had no such effect on Ranger. BA also enhanced the effect of GA on reducing apical dominance in both cultivars (GA x BA LT , P<0.03); however, the reduced sensitivity of

Bondi to GA for increasing stem numbers was not rectified by combining BA with GA.

Differences in the sensitivity of these cultivars to GA treatment for hastening plant emergence and reducing apical dominance may be partly due to differences in endogenous cytokinin (CK) levels in buds. Hartmann et al. (2011) clearly demonstrated that cytokinins modulate the sensitivity of tuber buds to GA. Sprout growth is only triggered by GA when endogenous cytokinin levels are sufficient. Differences in the responsiveness of these cultivars to GA may be dictated by differences in threshold cytokinin levels needed for GA response, or in the

25 concentrations of cytokinins in buds at the time of GA treatment. Bondi has longer dormancy than Ranger, which manifests in delayed emergence and plant establishment. This difference is also indicative of lower endogenous cytokinins in buds of Bondi and could partly explain the stimulation of plant emergence by BA when combined with GA on Bondi seed. Further work to compare bud cytokinin levels and the CK/GA relationships needed to trigger sprout growth and alter apical dominance of these cultivars is warranted.

Differences in cultivar response to age-priming with and without GA treatment were also evident. Age-priming for 400 DD at moderate temperature (12 oC) had no effect on apical dominance, tuber set and size distribution for either cultivar. Age-priming for 700 DD at high temperature (32 oC), however, effectively reduced apical dominance, increased tuber set, and decreased average tuber size in both cultivars, without decreasing marketable yields (Table 5).

Age priming for 700-DD combined with 2 mg L -1 GA treatment significantly reduced the marketable yield of Ranger Russet. Bondi, however, showed an even greater shift in tuber size distribution (at no expense to marketable yield) with this combined treatment than with either treatment alone, reflecting its decreased sensitivity to GA compared with Ranger. These results indicate that tuber size distribution in Bondi could likely be shifted even more without decreasing marketable yield by further aging (e.g. >700 DD at 32 oC) and/or higher GA concentrations.

GA plays a key role in regulating foliar growth, stolon growth and tuber set (Askenova et al 2012; Bou-Torrent et al 2011; Carrera et al 2000). It is therefore reasonable to speculate that the endogenous levels of GA may be inherently higher in Bondi than Ranger, which would account for Bondi’s extremely vigorous vine growth, elongated internodes, fewer tubers set and elongated tuber phenotype. GA promotes stem elongation and inhibits tuber formation (Booth

1963; Ewing 1987; Kloosterman and Bachem 2014; Kumar and Wareing 1974). Concentrations of bioactive GA’s are highest during the initial stages of plant establishment, which stimulates foliar canopy development and stolon elongation prior to tuberization.

GA is synthesized via the terpenoid pathway in shoot apical meristems and leaves. The most mobile form of GA, inactive GA 20 , is translocated to the stolon where it is converted into its most bioactive form, GA 1, by GA 3 oxidase (encoded by StGA3ox2 ), resulting in stolon elongation (Prat 2010). Once inductive conditions for tuberization (e.g. daylength and/or day/night temperature regimes) are perceived, StGA3ox2 in the stolon is downregulated, suppressing GA 3 oxidase activity (Kloosterman and Bachem 2014). Simultaneously, StGA2ox1 is expressed resulting in increased GA 2 oxidase activity and the catabolism of bioactive GA 1.

The resulting suppression of GA biosynthesis (down regulation of StGA3ox2 ) coupled with increased GA catabolism (up regulation of StGA2ox1 ) reduces bioactive GA concentrations in subapical and internode regions of the stolon tip, greatly attenuating stolon elongation. The drop in GA concentration, in concert with an increase in several other hormones (auxin, abscisic acid, cytokinins), stimulates radial expansion and the deposition of starch in cortical parenchyma cells in the subapical region of the stolon, which constitutes the initial stages of tuber initiation and formation (Bou-Torrent et al. 2011; Kloosterman and Bachem 2014; Prat et al. 2010). Inherent differences in the modulation of foliar and tuber growth by GA could thus at least partly explain the differences in plant growth and tuber set of these cultivars. Bondi produces on average 50% more above ground dry matter than the industry standard, Russet Burbank (Oliveira 2015).

Bondi’s excessive vine growth (at times in excess of 2.2 meters in length) can be partly attributed to increased internode elongation; a characteristic response to elevated levels of GA.

In potato, high activity of StGA20ox1 can increase translocation of GA 20 from foliage to stolons with subsequent conversion to bioactive GA 1 (Carrera et al. 2000). Transgenic lines overexpressing GA 20 oxidase produced taller plants with elongated internodes, petioles and stolons, reduced tuber number and yield (Hartmann et al. 2011). If this scenario is true for

Bondi, an alternative approach to mitigating low tuber set and oversize tubers would be the use of GA biosynthesis inhibitors applied to foliage in-season. The GA biosynthesis inhibitors, paclobutrazol and ancymidol, have been shown to increase tuber set in select cultivars of potato

(Hussey and Stacey 1984; Jackson and Prat 1996; Menzel 1980). Further work to characterize the involvement of GA in modulating the plant growth and tuber set responses of Bondi is needed.

The use of GA as a seed treatment to increase tuber set and decrease tuber size would seem counterintuitive given GA’s role in stimulating stolon elongation and inhibiting tuberization as discussed above. However, applying GA to seed tubers likely has no residual effect on the regulation of endogenous GA levels in plants (esp. stolons) at the time of tuberization; rather its main effect on tuber set and subsequent size distribution appears mediated solely through modulating the degree of apical dominance, resulting in more stems per seed piece. As previously discussed and shown, reducing apical dominance (increasing stems plant -1) increases tuber set per plant and when the total sink demand remains constant, the result is a decrease in average tuber size (Iritani et al. 1983; Knowles and Knowles, 2006). Hence, one way to increase tuber set and shift size distribution is to increase the number of stems plant -1 without inducing a proportional decrease in the number of tubers stem -1 or marketable yield. For example, over a wide range of GA concentrations (0-8 mg L -1) (Fig. 4), Bondi averaged 3.4 tubers stem -1 and experienced no significant reductions in this yield component as stem numbers

28 per plant increased with GA concentration. Ranger Russet, on the other hand, responded to GA with a significant reduction in tubers per stem, dropping nearly 2.7 tubers stem -1 as GA increased from 0 to 8 mg L -1 GA ( P<0.01). While stem numbers also increased for Ranger Russet over those GA concentrations, the decline in apical dominance was insufficient to offset the reduction in tuber number per stem, resulting in lower (-23.6 MT ha -1) marketable yield and further characterizing the differential sensitivities of these two cultivars to GA. The decreased sensitivity of Bondi to GA was likely inherited from its paternal parent, Karaka, which displays similar morphological traits, including high yield of large tubers

(http://www.eurogrow.co.nz/varieties/karaka. Accessed 9 November 2015.).

Acknowledgements

Financial support was provided by the USDA Specialty Crop Block Grant program through the Washington State Department of Agriculture, Simplot Australia Pty Ltd., and the Northwest

Potato Research Consortium. We thank Jacob Blauer, Agronomy Scientist Manager, J.R.

Simplot Co. and Mark Heap, Biosciences Manager, Simplot Australia Pty. Ltd. for their insight and advice during the course of this research.

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Table 1. Seed treatments and experimental designs for seven field trials conducted at Othello, WA. The three 2013 trials were vine killed 155 DAP and harvested 172 DAP. Vine kill and harvest dates for the four trials in 2014 were 154 and 168 DAP, respectively.

a -1 -1 b Trial Cultivars Planting Randomized Block Designs GA (mg L ) BA (mg L ) Seed Age (DD)

1 RR, B 4/11/13 Factorial – 2 cultivars x 6 [GA] 0, 0.5, 1, 2, 4, 8 n/a c n/a 2 B 4/11/13 Factorial – 2 [GA] x 3 [BA] 0, 2 0, 5, 10 n/a 3 RR 4/11/13 Factorial – 2 [GA] x 3 [BA] 0, 2 0, 5, 10 n/a 4 B 4/14/14 Single factor – 6 [GA] 0, 2,4 , 8, 12,16 n/a n/a 5 RR 4/14/14 Single factor – 6 [GA] 0,0.5,1,1.5,2,2.5 n/a n/a 6 B 4/14/14 Factorial – 2 [GA] x 3 seed ages 0, 2 n/a 80, 400, 700 7 RR 4/14/14 Factorial – 2 [GA] x 3 seed ages 0, 2 n/a 80, 400, 700 aRR, Ranger Russet; B, Bondi bDD, storage degree days (4 oC base) cn/a, not applicable

Table 2. Effects of gibberellic acid and cytokinin seed treatments on tuber set, yields, tuber size distributions and average tuber size of Ranger Russet and Bondi potatoes grown at Othello, WA. Seed was planted April 11, 2013. Vines were mowed 155 days after planting. Results of analysis of variance are summarized for each cultivar (LSD, least significant difference at P<0.05; LT, linear trend; Dev, deviations from linearity). Marketable (Mkt) yield includes yield of U.S. No. 1 plus <113-g tubers.

Tuber Yield (MT ha -1) Marketable Tubers GA BA Tubers Cultivar Total U.S. #1 <113g 113-170g 170-284g 284-340g 340-397g >397g Mkt Yld g tuber -1 1000s ha -1 (mg L -1) (mg L -1) plant -1

Ranger 0 0 91.5 74.9 12.1 13.3 29.0 9.7 7.4 15.7 86.8 177 10.9 498 5 91.1 70.4 12.4 11.7 25.6 8.3 9.2 15.7 82.5 172 10.6 485 10 90.6 74.9 11.7 13.6 25.9 11.5 7.2 16.9 86.1 178 10.7 488 2 0 81.9 65.2 15.1 14.8 23.6 9.2 6.1 11.9 80.3 161 11.1 504 5 87.3 68.8 15.8 14.3 25.6 9.4 5.4 14.2 84.8 163 11.6 527 10 82.5 64.1 16.0 15.1 25.6 8.8 5.2 9.7 80.3 153 11.5 526

LSD 0.05 8.8 8.8 2.2 2.7 5.6 2.7 2.9 7.2 7.9 14.1 0.8 35 GA 0.01 0.01 0.01 0.05 ns ns 0.01 0.05 ns 0.01 0.01 0.01 BA LT ns ns ns ns ns ns ns ns ns ns ns ns

BA DEV ns ns ns ns ns ns ns ns ns ns ns ns GA x BA LT ns ns ns ns ns ns ns ns ns ns ns ns

GA x BA DEV ns ns ns ns ns ns ns ns ns ns ns ns

Bondi 0 0 79.9 70.8 5.8 6.7 17.2 9.4 7.6 30.0 76.6 256 6.8 310 5 87.3 76.9 5.2 6.5 21.2 11.2 8.3 29.6 81.9 242 7.6 345 10 89.6 79.7 5.4 7.4 19.9 11.7 10.6 30.0 85.1 252 7.6 356 2 0 89.6 78.8 7.9 9.4 22.6 10.1 10.8 25.7 86.4 222 8.7 395 5 88.0 75.7 9.0 10.6 21.9 8.8 9.7 24.8 84.6 208 8.9 407 10 90.3 79.9 8.1 9.7 26.9 10.3 9.9 23.0 87.8 219 8.9 406

LSD 0.05 11.9 13.3 1.8 3.4 5.4 3.8 3.8 10.3 12.4 31.7 0.9 40

GA ns ns 0.01 0.01 0.01 ns ns 0.10 ns 0.01 0.01 ns BA LT ns ns ns ns ns ns ns ns ns ns ns ns

BA DEV ns ns ns ns ns ns ns ns ns ns ns ns GA x BA LT ns ns ns ns ns ns ns ns ns ns ns ns

GA x BA DEV ns ns ns ns ns ns ns ns ns ns ns ns

Table 3. Effects of gibberellic acid seed application on tuber yields of Ranger Russet and Bondi potatoes grown at Othello, WA. Seed was planted April 11, 2013. Vines were mowed 155 days after planting. Results of analysis of variance are summarized beneath the table (LSD, least significant difference at P<0.05; LT, linear trend; QT quadratic trend, CT, cubic trend). U.S. No. 1 yield includes all size categories except <113-g tubers.

Tuber Yield (MT/ha -1) Cultivar GA (mg L -1) Total U.S. #1 <113g 113-170g 170-284g 284-340g 340-397g >397g

Ranger 0 55.1 37.2 14.6 11.7 15.5 3.6 2.5 4.0 0.5 58.0 39.0 16.8 13.3 17.2 2.5 2.9 2.9 1 56.0 37.9 16.1 11.7 15.5 3.4 2.9 4.3 2 51.7 34.5 15.5 12.4 12.6 2.5 2.9 4.5 4 38.1 21.5 15.7 9.4 7.9 1.6 0.9 1.8 8 30.2 16.8 11.7 6.1 7.9 1.3 0.4 0.9

Bondi 0 89.1 80.1 4.3 5.2 17.2 9.7 9.4 38.5 0.5 88.5 78.8 4.3 6.7 20.8 12.8 9.2 29.6 1 89.1 77.3 5.6 8.3 23.0 9.7 9.7 26.6 2 98.1 85.8 6.7 9.4 23.5 10.3 9.9 32.7 4 84.6 73.1 7.9 11.2 20.6 10.1 11.5 19.7 8 83.1 69.9 9.7 11.9 20.6 10.6 7.2 19.7

LSD 0.05 10.3 10.8 2.0 2.7 4.9 3.4 2.9 7.2 CV 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 GA LT 0.01 0.01 ns ns 0.01 ns 0.05 0.01 GA QT ns ns 0.01 0.05 ns ns ns ns GA CT 0.05 0.10 ns ns 0.05 ns ns ns CV x GA LT 0.01 0.05 0.01 0.01 0.01 ns ns 0.01 CV x GA QT ns ns ns ns 0.05 ns 0.10 ns CV x GA CT ns ns ns ns ns ns ns ns

Table 4. Effects of gibberellic acid seed treatment on tuber yields of Ranger Russet and Bondi potatoes grown at Othello, WA. Seed was planted April 14, 2014. Vines were mowed 154 days after planting. Results of analysis of variance are summarized beneath the table (LSD, least significant difference at P<0.05; polynomial trends are indicated; ns, not significant). U.S. No. 1 yield includes all size categories except <113-g tubers.

Tuber Yield (MT ha -1) GA Cultivar Total U.S. #1 <113g 113-170g 170-284g 284-340g 340-397g >397g (mg L -1)

Ranger 0 62.9 54.4 6.7 11.0 24.8 7.6 5.4 5.6 0.5 61.8 52.2 9.2 14.2 21.5 6.5 5.4 4.9 1.0 60.9 47.7 12.8 13.7 20.6 4.9 3.1 5.6 1.5 58.9 46.4 11.9 13.3 21.7 5.6 2.2 3.4 2.0 55.1 41.7 12.8 13.5 18.3 4.7 2.9 2.5 2.5 56.0 42.4 13.3 13.7 18.8 4.9 2.9 1.8

LSD 0.05 5.2 6.5 2.5 2.2 5.2 2.7 2.2 3.1 Linear 0.01 0.01 0.01 ns 0.05 0.05 0.01 0.01 Quadratic ns ns 0.05 ns ns ns ns ns Cubic ns ns ns ns ns ns ns ns Quartic ns ns ns ns ns ns ns ns Deviations ns ns ns ns ns ns ns ns Control vs GA ns ns ns 0.01 ns ns ns ns

Bondi 0 65.2 58.4 4.3 5.2 15.1 10.3 8.5 19.4 2 65.4 56.6 7.2 7.6 18.1 7.9 6.1 16.5 4 67.2 59.1 6.5 9.4 20.6 7.9 7.2 14.2 8 61.6 50.4 7.9 8.8 17.7 5.2 7.0 11.9 12 56.2 45.1 8.8 10.1 18.3 6.7 3.1 6.7 16 51.5 38.3 9.7 9.7 17.7 4.3 1.8 5.2

LSD 0.05 10.1 10.3 3.1 3.8 4.3 2.7 3.4 6.1 Linear ns ns ns ns ns ns ns ns Quadratic ns ns ns ns ns ns ns ns Cubic 0.01 0.01 0.01 ns ns 0.01 0.01 0.01 Quartic ns ns ns 0.05 ns ns ns 0.01 Deviations ns ns ns ns ns ns ns ns Control vs GA ns ns ns 0.01 0.05 ns ns ns

Table 5. Effects of gibberellic acid seed treatment and seed age on emergence, stem numbers, tuber set, yields, tuber size distributions and average tuber size of Ranger Russet and Bondi potatoes grown at Othello, WA. Seed was planted April 14, 20 14 and emergence was recorded 28 (Ranger) and 32 DAP (Bondi.) Vines were mowed 154 days after planting. Results of analysis of variance are summarized for each cultivar (LSD, least significant difference at P<0.05; LT, linear trend; Dev, deviations from linearity). Marketable (Mkt) yield includes yield of U.S. No. 1 plus <113-g tubers.

Tuber Yield (MT ha -1) Marketable Tubers GA Seed Emerg. Stem Tubers Cultivar Total U.S. #1 <113g 113-170g 170-284g 284-340g 340-397g >397g Mkt Yld g tuber -1 1000s ha -1 (mg L -1) Age (DD) % No. plant -1

Ranger 0 80 7 2.8 65.9 59.8 4.5 8.8 22.6 10.1 6.3 12.1 64.3 226 6.5 388 400 13 2.3 61.8 56.2 4.7 9.2 21.7 7.9 7.6 9.7 60.9 211 6.5 383 700 33 4.4 60.2 45.3 14.8 13.9 19.0 4.5 2.9 4.9 60.0 155 9.0 536 2 80 69 4.0 65.4 52.8 11.2 13.0 21.9 5.4 3.1 9.7 64.1 171 8.6 507 400 51 4.1 61.4 51.7 9.2 11.9 17.2 6.7 3.8 12.1 60.7 188 7.6 448 700 76 4.9 50.8 36.5 14.2 12.4 15.5 3.1 2.0 3.6 50.4 138 8.2 487

LSD 0.05 15 1.1 7.6 8.1 3.4 3.4 5.6 3.4 1.6 7.0 7.0 24 0.8 58 40 GA 0.01 0.01 ns 0.01 0.01 ns ns 0.05 0.01 ns ns 0.01 0.01 0.05

Age LT 0.01 0.01 0.01 0.01 0.01 ns 0.5 0.01 0.01 0.05 0.01 0.01 ns 0.01

Age DEV 0.01 0.05 ns 0.05 0.01 ns ns ns 0.01 ns ns 0.01 ns 0.01 GA x Age LT ns ns ns ns 0.01 0.05 ns ns 0.05 ns ns 0.05 ns 0.01

GA x Age DEV ns ns ns ns ns ns ns ns ns ns ns ns ns ns

Bondi 0 80 22 2.1 68.1 65.6 1.8 3.4 15.1 9.2 9.2 28.9 67.7 307 4.9 223 400 25 2.0 64.7 61.8 2.0 3.6 13.9 8.3 7.6 28.4 63.8 299 4.7 215 700 50 3.7 70.1 64.5 4.9 7.4 21.2 11.7 6.3 17.9 69.5 229 6.7 303 2 80 73 2.5 75.1 69.9 2.9 6.7 19.4 10.1 7.6 26.0 72.8 271 6.0 272 400 52 2.3 69.9 65.6 3.4 6.3 16.8 8.8 7.2 26.6 69.0 266 5.8 263 700 74 3.8 63.8 57.3 6.1 11.5 20.6 7.0 7.2 11.2 63.4 198 7.0 320

LSD 0.05 17 0.3 10.8 11.0 1.3 1.8 5.4 3.8 4.3 9.2 10.3 31 0.7 30

GA 0.01 0.01 ns ns 0.01 0.01 ns ns ns ns ns 0.01 0.01 0.01 Age LT 0.05 0.01 ns ns 0.01 0.01 ns ns ns 0.01 ns 0.01 0.01 0.01

Age DEV 0.01 0.01 ns ns 0.01 ns 0.05 ns ns 0.05 ns 0.01 0.01 0.01 GA x Age LT 0.05 ns ns ns ns ns ns 0.05 ns ns ns ns ns ns

GA x Age DEV ns ns ns ns ns ns ns ns ns ns ns ns ns ns

A B 100 Ranger 100 Ranger 32 DAP a a a BA 90 90 0 BA 5 80 80 bcd BA 10 70 70 d d 60 60 50 50 40 Control BA 5 40 Emergence(%) 30 BA 10 30 20 GA 2 20 GA BA 10 2 5 GA BA 10 0 2 10 0 C 25 30 35 40 45 D 0 2 100 Bondi 100 32 DAP Bondi 90 90 BA 0 abc ab 80 BA 5 80 cd BA 70 70 10 60 60 50 50 Control e 40 40 BA 5 e e Emergence(%) 30 BA 10 30 20 GA 2 20 GA BA 10 2 5 GA BA 10 0 2 10 0 25 30 35 40 45 0 2 -1 Days After Planting GA (mg L )

Fig. 1. Effects of gibberellin (GA) and cytokinin (BA) seed treatments on plant emergence (%) of cultivars Ranger Russet (A, B) and Bondi (C, D). Subscripts in legends denote concentrations

(mg L -1). Cut seed (50-64 g seed-pieces) was treated by immersing for 5 min in solutions of GA and/or BA and planted at the WSU Othello Research Unit. Bars are ±SE (n=5). For (B) and

(D): cultivar, P<0.01; GA, P<0.01; cultivar x GA, P<0.01; cultivar x GA x BA linear , P<0.02.

Letters indicate LSD, P<0.05 across cultivars.

AB 5 5 Ranger Russet Bondi BA ab BA 0 a 0 BA 5 BA 5 4 4 BA 10 b BA 10

3 3 c c cde cd cde cdef def 2 2 ef f Stems per seed-piece per Stems 1 1

0 0 0 2 0 2 GA (mg L -1 ) GA (mg L -1 )

Fig. 2. Effects of GA and BA seed treatments (see Fig. 1) on stem numbers of cultivars Ranger

Russet (A) and Bondi (B). Subscripts in legends denote concentrations (mg L -1). Seed was treated and planted as described in Fig. 1. Bars are ±SE (n=5). Cultivar, P<0.01; GA, P<0.01; cultivar x GA, P<0.01; GA x BA linear , P<0.03. Letters indicate LSD, P<0.05 across cultivars.

A B 100 Ranger 100 Ranger 90 90 80 80 70 70 60 60 50 50 Control 40 40 GA 0.5

Emergence(%) 30 30 GA 1

20 GA 2 20 GA 10 4 10 GA 0 8 0 29 DAP 0 1 2 4 8 C 25 30 35 40 45 D 100 Bondi 100 Bondi 90 90 80 80 70 70 60 60 50 50 Control 40 40 GA 0.5

Emergence(%) 30 30 GA 1

20 GA 2 20 GA 10 4 10 GA 0 8 0 32 DAP

25 30 35 40 45 0 1 2 4 8 -1 Days After Planting GA (mg L )

Fig. 3. Effects of GA seed treatments on plant emergence (%) of cultivars Ranger Russet (A, B) and Bondi (C, D). Subscripts in legends denote concentrations. Seed was treated and planted as described in Fig. 1. Bars are ±SE (n=5). For (B) and (D): cultivar, P<0.01; GA, P<0.01; cultivar x GA, P<0.01; emergence was analyzed at 29 and 32 DAP for Ranger and Bondi, respectively.

A B 5.0 11 500 Ranger 475 4.5 10 450 Ranger 2 4.0 R = 0.85* 425 9 400

3.5 375 (1000's) 8 -1 350 3.0 7 325 Tubersper plant

300 Tubersha Stems per seed-pieceper Stems 2.5 Bondi Bondi 6 R2= 0.94** 275 2.0 250 5 C 0 2 4 6 8 D 0 2 4 6 8 300 100

Bondi -1 280 90 )

-1 260 240 80 Bondi R2= 0.28ns 220 70 R2= 0.94** 200 60 180 160 50 Ranger 140 40 Ranger R2= 0.94** 120 2

Tuber fresh weight (g tuber (g Tuberweight fresh R = 0.78* 30 100 Market yield (US #1 + <113 g) MT ha <113MT +g) #1 (US yield Market 80 20 0 2 4 6 8 0 2 4 6 8 GA (mg L -1 ) GA (mg L -1 )

Fig. 4. Effects GA seed treatments on stems per seed-piece (A), tubers per plant and per hectare

(B), tuber fresh weight (C) and marketable yield (D) of cultivars Ranger Russet and Bondi. Seed was treated as described in Fig. 1. Bars are ±SE (n=5). Cultivar x GA, P<0.01 for all yield components. *P<0.05 and ** P<0.01 for correlation coefficients.

Fig. 5. Polygonal plots depicting the shifts in tuber size distribution induced by treating Ranger

Russet (A) and Bondi (B) seed with 8 mg L -1 GA. Seed was treated as described in Fig. 1. The yields of <113-g, 113-170-g, 170-284-g, 284-340-g, 340-397-g, and >397-g U.S. No. 1 tubers are plotted as percent marketable yield on each axis. Marketable yields (Mkt), stems per plant, tubers per plant, tubers ha -1, and tuber fresh weights are compared in the inset tables ( ** P<0.01; ns, not significant). Yields with ANOVA results are given in Table 3.

< 113 g 40 A 35 Ranger Russet % Yield 30 25 >397 g 8 mg L -1 113-170 g 20 15 GA (mg L -1) 10 0 8 5 Mkt Yield (MT ha -1) 51.9 28.3** Stems plant -1 2.3 4.7** 0 Tubers plant -1 9.1 6.0** Tubers ha -1 413K 275K** g tuber -1 126 103** 0 mg L -1

340-397 g 170-284 g

284-340 g

< 113 g 40 B Bondi 35 % Yield 30 >397 g 25 113-170 g 0 mg L -1 20 15 8 mg L -1 GA (mg L -1) 10 0 8 5 Mkt Yield (MT ha -1) 84.5 79.8 ns 0 Stems plant -1 2.0 3.1** Tubers plant -1 6.8 8.9** Tubers ha -1 309K 407** g tuber -1 273 196**

340-397 g 170-284 g

284-340 g

A B 100 Ranger 100 Ranger 90 90 80 80 70 70 60 60 50 50 40 Control 40 GA 0.5

Emergence(%) 30 30 2 GA 1 R =0.98**

20 GA 1.5 20 GA 10 2 10 GA 0 2.5 0 28 DAP C 20 25 30 35 40 45 D 0.0 0.5 1.0 1.5 2.0 2.5 100 Bondi 100 Bondi 90 90 80 80 70 70 60 60 50 50 40 Control 40 GA 2

Emergence(%) 30 30 GA 4 20 20 GA 8

10 GA 12 10 GA 0 16 0 32 DAP

20 25 30 35 40 45 024 8 12 16 Days After Planting GA (mg L -1 )

Fig. 6. Effects of GA seed treatments on plant emergence (%) of cultivars Ranger Russet (A,B) and Bondi (C,D). Subscripts in legends denote concentrations of GA (mg L -1). Seed was treated and planted as described in Fig. 1. Note the different GA concentrations for each cultivar. Bars are ±SE (n=5). ** P< 0.01 for correlation coefficient.

A B 5.0 10 450 425 4.5 9 400 Ranger 4.0 375 8 Ranger 350

3.5 (1000's) 7 325 -1 Bondi 300 3.0 6 275 Bondi Tubers per plant Tubers ha Tubers

Stems per seed-piece perStems 2.5 250 5 225 2.0 200 4 0 2 4 6 8 10121416 0 2 4 6 8 10121416 C D 300 80 -1 280

260 Bondi 70 2 Bondi R = 0.96** 2 240 R = 0.98** 60 220

200 50 R2= 0.85** 180 Ranger

160 40 Tuber fresh weight (g/tuber) freshTuberweight 2 140 R = 0.94**

Ranger ha g) <113MT (US #1 + yield Market 120 30 0 2 4 6 8 10121416 0 2 4 6 8 10121416 GA (mg L -1 ) GA (mg L -1 )

Fig. 7. Effects of GA seed treatments on stems per seed-piece (A), tubers per plant and per hectare (B), tuber fresh weight (C) and marketable yields (D) of cultivars Ranger Russet and

Bondi. Seed was treated as described in Fig. 1. Bars are ±SE. ** P<0.01, for correlation

-1 -1 coefficients. For marketable yields, LSD 0.05 = 4 MT ha (Ranger) and 9.6 MT ha (Bondi).

Fig. 8. Tuber size distributions of Ranger Russet (A) and Bondi (B) as affected by 1.5 and 8 mg

L-1 GA seed treatments, respectively. Seed was treated as described in Fig. 1. Yields of the tuber size classes (see Table 4) are plotted as percent marketable yield. Only the GA concentrations that induced the greatest shifts in tuber size distribution without affecting marketable yields are shown for each cultivar. Marketable (Mkt) yields, stems per plant, tubers per plant, tubers ha -1 and tuber fresh weights are compared in the inset tables (** P<0.01; ns, not significant). Yields with ANOVA results are given in Table 4.

< 113 g 40 A 35 Ranger Russet % Yield 30 25 >397 g -1 113-170 g 20 1.5 mg L 15 GA (mg L -1) 10 0 1.5 5 Mkt Yield (MT ha -1) 61.3 58.4ns Stems plant -1 2.5 3.6** 0 Tubers plant -1 7.4 8.6** Tubers ha -1 335K 390K** g tuber -1 185 152**

0 mg L -1

340-397 g 170-284 g

284-340 g

< 113 g 40 B Bondi 35 % Yield 30 25 >397 g 113-170 g 20

15 8 mg L -1 GA (mg L -1) 10 0 8 Mkt Yield (MT ha -1) 62.9 58.4ns 5 Stems plant -1 2.1 2.9** 0 Tubers plant -1 5.9 7.1** Tubers ha -1 269K 323K** g tuber -1 251 187**

0 mg L -1 340-397 g 170-284 g

284-340 g

Fig. 9. Tuber size distributions of Ranger Russet (A,B) and Bondi (C,D) as affected by seed age and GA. Seed tubers were age-primed by storing at 12 and 32 oC for 80, 400 and 700 DD (4 oC base) following harvest and subsequently held at 4 oC (95% RH) until planting. Seed was then cut and treated with 2 mg L -1 GA (B,D) as described in Fig. 1. Yields of the tuber size classes are plotted as percent marketable yield. Marketable (Mkt) yields, stems per plant, tubers per plant, tubers ha -1 and tuber fresh weights are compared in the inset tables for the 80- and 700-DD non-treated (A,B) and GA-treated (B,D) seed (** P<0.01; ns, not significant). Growth and yield component data for all treatments are given in Table 5 with ANOVA.

< 113 g < 113 g A 40 C 40 Ranger Russet 35 Bondi 35 % Yield % Yield GA (0 mg L -1) 30 GA (0 mg L -1) 30 25 >397 g 25 >397 g 113-170 g 113-170 g 20 20 700 DD 400 DD 15 15 80 DD Seed Age 10 Seed Age 10 80 DD 700 DD 80 DD 700 DD -1 5 5 Mkt Yield (MT ha ) 64.3 60.0ns Mkt Yield (MT ha -1) 67.7 69.5ns -1 Stems plant 2.8 4.4** 0 Stems plant -1 2.1 3.7** 0 -1 Tubers plant 6.5 9.0** Tubers plant -1 4.9 6.7** 700 DD -1 Tubers ha 388K 536K** Tubers ha -1 223K 303K** -1 g tuber 226 155** 400 DD g tuber -1 307 229** 80 DD

340-397 g 170-284 g 340-397 g 170-284 g 52 284-340 g

284 -340 g < 113 g < 113 g B D 40 Ranger Russet 40 Bondi 35 35 GA (2 mg L -1) % Yield GA (2 mg L -1) % Yield 30 30 25 25 >397 g 113-170 g >397 g 113-170 g 20 20 700 DD 400 DD 15 400 DD 15 Seed Age 10 Seed Age 10 80 DD 700 DD 80 DD 700 DD 5 5 700 DD Mkt Yield (MT ha -1) 64.1 50.4** Mkt Yield (MT ha -1) 72.8 63.4ns Stems plant -1 4.0 4.9ns 0 Stems plant -1 2.5 3.8** 0 Tubers plant -1 8.6 8.2ns Tubers plant -1 6.0 7.0** Tubers ha -1 507K 487Kns Tubers ha -1 272K 320K** g tuber -1 171 138** g tuber -1 271 198** 80 DD 80 DD

340-397 g 170-284 g 340-397 g 170-284 g

284-340 g 284-340 g

CHAPTER 2

Low oxygen storage modulates invertase activity to attenuate cold-induced sweetening and

loss of process quality in potato ( Solanum tuberosum L.) 2

Abstract

Russet Burbank and Innovator are mid- to late-season frozen-processing cultivars with inherently different dormancy periods and susceptibilities to low temperature-induced sweetening (LTS).

In contrast to Russet Burbank, which is highly prone to accumulation of reducing sugars (Glc +

Fru) when stored below 8-9 oC, Innovator tubers exhibit moderate resistance to LTS and retain process quality longer at lower temperatures (4-6 oC). However, Innovator’s LTS resistance is not robust and often varies across production regions. Here we show that low O 2 storage modulates LTS to reveal metabolic differences intrinsic to these cultivars. Changes in tuber respiration, process quality, reducing sugars, sucrose, starch phosphorylase and invertase

o activities were compared at 4 and 8 C in 2.5 and 21 kPa O 2 over a 212-d storage period. Tuber

o respiration declined rapidly as O 2 level decreased from 21 to 2.5 kPa during acclimation at 8 C.

Respiration rates then fell further as the temperature was lowered from 8 to 4 oC, but this response was greatly muted for tubers at 2.5 versus 21 kPa O 2. Tubers at 21 kPa O 2 completed their cold-induced respiratory acclimation response (RAR) within 7 d compared with 13 d at 2.5 kPa O 2, and the RAR was much greater for tubers at 2.5 kPa O 2. While reducing sugars increased most rapidly in tubers over the first 30 d at 4 oC, Innovator tubers had lower invertase activity and sweetened less than Russet Burbank tubers, characterizing its LTS-resistant phenotype. Low O 2 greatly attenuated these initial LTS responses for both cultivars; however, the effect was only temporary in Innovator. LTS resumed in Innovator tubers from 93 to 212 d

with reducing sugar levels increasing to equal that of Russet Burbank tubers stored at 4 oC and

21 kPa O 2. Activities of α-1,4 glucan phosphorylase (SP) were higher in Russet Burbank than

Innovator and increased progressively over the storage period regardless of temperature and oxygen concentration. Innovator tubers stored at 2.5 kPa O 2 had higher SP activities from 30 to

o 153 d at 4 and 8 C compared with tubers stored at 21 kPa O 2, which correlated well with increased sucrose buildup and earlier sprouting. The low O 2-mediated inhibition of LTS was largely a consequence of reduced invertase activities.

2Herman, D.J., Knowles, L.O., Knowles, N.R., 2016. Low oxygen storage modulates invertase activity to attenuate cold-induced sweetening and loss of process quality in potato ( Solanum tuberosum L.). Postharvest Biology and Technology 121: 106-117.

1. Introduction

Maintaining raw product quality during long-term storage of potatoes destined to frozen processing (French fries) is highly dependent on cultivar and storage temperature. Storage at less than 9 oC effectively extends storage life by reducing tuber respiration, fresh weight loss, disease pressure and sprouting (Burton 1966; Schippers 1977; Burton 1978; Wiltshire and Cobb

1996). Retention of process quality at lower temperatures, however, is compromised by the induction of low-temperature sweetening (LTS) in susceptible cultivars. LTS involves the catabolism of starch into reducing sugars (Glc + Fru) (Isherwood 1976; Marquez and Anon

1986; Sowokinos 2001; Malone et al. 2006), which then serve as substrates for the Maillard reaction during frying (Denny and Thornton 1941; Habib and Brown 1956; Mazza 1983; Fuller and Hughes 1984; Roe and Faulks 1991; Mottram et al. 2002; Stadler et al. 2002; Kumar et al.

2004). In potato, products of the Maillard reaction can give rise to undesirable off-flavors, dark pigments and acrylamide. This latter product has been identified as a ‘probable human carcinogen’ in many processed foods (IARC 1994; Tareke et al. 2000; Tareke et al. 2002; Stadler and Scholz 2004). Breeding for resistance to LTS can mitigate these problems; however, the degree of cold tolerance expressed by LTS-resistant cultivars is often modulated significantly by management, environmental conditions and stress (Zommick et al. 2014ab), and this translates into variation in retention of process quality under cold storage conditions. Understanding the metabolic bases for variation in LTS-resistant phenotypes of cultivars important to the processing industry will lead to more effective breeding for stability of the LTS-resistant trait, as well as improved management recommendations to maximize retention of process quality during storage.

Innovator and Russet Burbank are dual purpose (fresh and frozen processing) russet skin potato cultivars that vary in dormancy and resistance to LTS. Russet Burbank is a late season, long dormancy cultivar, which remains the industry standard for frozen processing in North

America. By contrast, Innovator has early- to mid-season maturity, shorter dormancy and moderate resistance to LTS. While Russet Burbank tubers are highly susceptible to the accumulation of reducing sugars when stored below 8-9oC, Innovator tubers can maintain relatively low reducing sugar concentrations and thus acceptable process quality when stored at lower temperatures (4-6oC). However, the degree of LTS resistance expressed by Innovator is often variable across production regions, which is no-doubt attributable to poorly understood management x environment interactions that affect tuber physiology in general and sweetening metabolism in particular. The mechanism of resistance to LTS in Innovator is not known and is critical to understanding how management and environment interact to alter the expression of this important phenotype.

Previous work has indicated that low-oxygen storage can attenuate sweetening of potatoes stored at low temperatures (Pflug et al. 1964; Butchbaker et al. 1967; Workman and

Twomey 1969; Harkett 1971; Sherman and Ewing 1983; Parkin and Schwobe 1990; Schwobe and Parkin 1990; Schouten 1992; Schouten 1993; Zhou and Solomos 1998). However, the efficacy of this technique is highly dependent upon oxygen concentration, timing, and cultivar

(Butchbaker et al. 1967; Workman and Twomey 1969; Sherman and Ewing 1983; Parkin and

Schwobe 1990; Schouten 1992; Mawson 1998). Here we use storage in 2.5 and 21 kPa O 2 atmospheres to manipulate the LTS responses of Russet Burbank and Innovator tubers to gain a better understanding of the metabolic bases for their differential sensitivities to sweetening.

While not economically feasible for commercial storage, low O 2 modulated cold sweetening to

reveal cultivar-dependent differences in LTS-resistant metabolism and associated changes in process quality.

2. Materials and Methods

2.1. Plant materials, storage temperature and controlled atmosphere regimes

Potato tubers ( Solanum tuberosum L., cvs. Russet Burbank and Innovator) were obtained from a commercial grower (Burley, ID) on October 1, 2013 (harvested September 26) and stored at 10 oC (95% RH) for two weeks to facilitate wound healing. The effects of storage temperature

o (4 vs 8 C), oxygen concentration (21 vs 2.5 kPa O 2), and storage duration (0, 30, 93, 153, and

212 d) on retention of process quality, low temperature sweetening and associated enzyme activities were evaluated for each cultivar. Treatments were arranged factorially as indicated in

Fig. 1. Tubers of each cultivar (170-284 g per tuber) were initially blocked for size (fresh weight) into four replicates (3 tubers per replicate) and placed into 13-L sealed storage chambers

(Gamma Plastic Company, San Diego, CA, USA) equipped with inlet and outlet ports (Fig. 1C).

Each chamber contained 12 tubers of each cultivar (4 replicates of 3 tubers per cultivar). The tubers rested on an internal grate situated 6 cm above the floor of each chamber to allow incoming air to enter and circulate from beneath the pile (Fig. 1A and B). The chambers were connected to separate manifolds which supplied CO 2-free air (21 kPa O 2, balance N 2) or 2.5 kPa

-1 O2 (balance N 2) at 1.5 mL s for the duration of storage. The 2.5 kPa O 2 atmosphere was generated using a custom-built Permea nitrogen generator (Permea Inc., St. Louis, MO, USA) as described in Blauer et al. (2013). All tubers were acclimated initially for 8 d at 8 oC (95% RH) prior to lowering the temperature to 4 oC (95% RH) for half the chambers. Chambers were removed from the manifolds and tubers sampled at the designated storage interval (Fig. 1) to

assess changes in sprout development, fry process quality, tuber carbohydrates, invertase, invertase inhibitor and starch phosphorylase activities.

2.2. Whole tuber respiration

Tuber respiration rates were monitored to determine the effects of O 2 concentration on the initial respiratory acclimation responses (RAR) to decreasing temperature (8 to 4 oC) and as an indicator of changes in metabolic rate over 155 d of cold-sweetening. The outlet atmosphere from two of the 212-d, 21 and 2.5 kPa O 2 chambers was directed through an LI-6262 infrared

CO 2 gas analyzer (LI-COR Inc., Lincoln, NB, USA) as described previously (Zommick et al.

2014b). Carbon dioxide concentrations were quantified at 2.5-h intervals through the initial 8-d acclimation period at 8 oC and continuing through 155 d of storage at 4 oC. Tuber respiration

-1 -1 rates (CO 2 evolution) are reported as µg kg s . Respiration rates are thus the average of 12

Russet Burbank and 12 Innovator tubers (24 tubers total; equivalent mass for each cultivar) at each O 2 concentration. No attempt was made to characterize potential cultivar-dependent differences in respiration rates, which have been shown to be minor relative to the effects of decreased O 2 (Blauer et al. 2013) and temperature (Zommick et al. 2014b).

2.3. Sprouting, fry process quality, and tissue sample preparation

Sprout fresh weight (grams per tuber) was determined individually for all 24 tubers in a treatment chamber at 30, 93, 153 and 212 d, and samples of tuber tissue were taken for analysis of glucose (Glc), fructose (Fru), sucrose (Suc), invertase and starch phosphorylase activities

(described below). For each cultivar, tubers of the three replicates (four tubers per replicate) were cut in half along the apical to basal axis slightly off center, and a complete 1.5-mm-thick

slice (periderm intact) was taken from the cut surface of the smaller half with a mandoline cutter to represent the whole tuber. The single slices from each of the three tubers per replicate were combined, collectively frozen (-80 oC) and lyophilized. The lyophilized samples were ground with mortar and pestle, sieved through a 60-mesh screen (0.246 mm) and stored for further analysis after all samples had been collected and processed.

French fry process quality was also evaluated at each sampling date using methods detailed in Zommick et al. (2014a). French fry planks (9.5-mm-thick x 2.9-cm-wide x length of tuber) were cut longitudinally along the apical to basal axis from the larger half (see above) of each tuber (one plank per tuber) and the 12 planks representing a cultivar from a particular treatment chamber were collectively fried for 3.5 min at 191 oC in vegetable oil. A Photovolt reflectance meter (Model 577, Photovolt Instruments Inc., Indianapolis, IN, USA) was used to measure light reflectance from the apical and basal ends of each fry plank as an indicator of the extent of Maillard browning (fry darkening) associated with buildup in reducing sugars (RS; Glc

+ Fru). Reflectance data were plotted versus days in storage and translated into USDA 0 (light) to 4 (dark) color values. Photovolt reflectance unit readings ‡ 31 = USDA 0; 25-30 = USDA 1;

20-24 = USDA 2; 15-19 = USDA 3; and £ 14 = USDA 4 (Stark et al., 2016). USDA values >2 and differences in reflectance values ≥9 between apical and basal portions of fries constitute unacceptably dark and/or non-uniform fry color, respectively (Driskill et al. 2007).

2.4. Carbohydrate analysis

Tuber carbohydrates were extracted from lyophilized tissue by modifying the methods of

Zommick et al. (2014a). Lyophilized tissue (250 mg) was extracted by vortexing with 3 mL

60% (v/v) methanol in triethanolamine·HCl (TEA) buffer (30 mM, pH 7.0). The homogenate

was centrifuged (10,000g, 15 min) and the supernatant analyzed for Glc, Fru and sucrose using enzymatic methods (Bergmeyer et al. 1974; Bernt and Bergmeyer 1974) modified for analysis on microplates (Knowles et al. 2009). The assay is based on the stoichiometric reduction of NADP

(monitored at A340 ) resulting from the enzymatic conversion of Glc and Fru to 6- phosphogluconate (via glucose-6-phosphate, hexokinase and phosphoglucose isomerase). Total

Glc (free Glc plus that hydrolyzed from sucrose) was determined by incubating a separate aliquot of extract with acid invertase (Bergmeyer and Bernt 1974). Sucrose, Glc and Fru were quantified based on standard curves (0.05-2.4 mM).

2.5. Invertase and starch phosphorylase activities

Acid invertase and starch phosphorylase were extracted from 200 mg lyophilized tissue at

o 4 C with 1 mL HEPES buffer (50 mM, pH 7.5) containing 15 mM MgCl 2·6H 2O, 2 mM

Na 2EDTA·2H 2O, 5 mM NaHSO 3, 10% (v/v) glycerol, 2% (w/v) PVPP and 2 µL protease inhibitor cocktail (2.5 mL L-1 leupeptin, chymostatin, pepstatin and antipain, and 20 mL L -1 4- aminobenzamindine and benzamidine). The extracts were centrifuged (10,000g, 15 min, 4 oC) and protein precipitated from the supernatant with 60% (w/v) (NH 4)2SO 4. The pelleted protein

(10,000g, 20 min, 4 oC) was resolubilized in the original extraction buffer (without PVPP) and stored at -18 oC until further analysis. Acid invertase activity was measured colorimetrically in the presence (basal activity) and absence (total activity) of its endogenous inhibitor by a microplate modification of Brummell et al. (2011) as outlined in Zommick et al. (2013).

Activities were expressed as μmol sucrose hydrolyzed s -1 kg -1 protein (U kg -1). Tuber soluble protein was quantified (Bradford, 1976) using protease-free BSA (Sigma-Aldrich, St. Louis,

MO) as a standard.

Changes in activities of the plastidic (L-type; low glycogen affinity) and cytosolic (H- type; high glycogen affinity) isoforms of starch phosphorylase ( a -1,4-glucan phosphorylase) over the 212-d storage period were assessed by activity gel electrophoresis (Steup 1990). The two isoforms were separated on 8% native polyacrylamide gels containing 0.65% (w/v) glycogen

(100 µg protein per lane). Following electrophoresis, the gels were incubated (37 oC for 120 min) in 100 mM MES buffer (pH 6.2) containing 12 mM Glc-1-phosphate and 0.5 g L -1 soluble potato starch as a primer for starch synthesis. The gels were then stained with KI/I to visualize and compare starch synthesizing activities of the two isoforms.

2.6. Data analysis and presentation

Treatment effects were analyzed in an overall 4-way factorial analysis of variance

(ANOVA) with sums of squares partitioned into single degree-of-freedom contrasts for main effects (cultivar, temperature, oxygen, storage duration) and their interactions (Table 1).

Similarly, 3-way factorial ANOVA’s analyzed the effects of cultivar, temperature and oxygen concentration on sprouting, process fry color (Photovolt reflectances of apical and basal ends of fry planks), Suc, reducing sugars and invertase activity (±inhibitor) separately at each sampling date. Data are plotted with least significant interval bars (i.e. ±0.5LSD, P<0.05; Mason et al.

2003) versus time in storage.

3. Results

3.1. Tuber respiration

o Tubers were initially acclimated to 2.5 or 21 kPa O2 atmospheres at 8 C for 8 d (Fig. 2A) before dropping the temperature to 4 oC for the LTS-phase of study. Tubers responded to the

lower O 2 with an immediate and substantial decline in respiration rate. Respiration rate (CO 2

-1 -1 o evolution) fell from 1.39 to 0.25 µg kg s during the initial 5 d of storage in 2.5 kPa O 2 at 8 C

(Fig. 2A). Respiration rates of tubers in both O 2 atmospheres remained relatively constant over the next 3 d at 8 oC, and then fell rapidly over the next 35-47 h in response to decreasing the temperature to 4 oC. On an absolute basis, the temperature-induced decrease in respiration rate was 4.3-fold greater in tubers held at 21 kPa O 2 compared with 2.5 kPa O 2. However, the percentage decline in respiration rate during this time period was approximately 32% regardless of O 2 atmosphere. Respiration rates then increased to a maximum at ca. 7 (21 kPa O 2) and 13 d

(2.5 kPa O 2) before declining to relatively low levels over the remainder of the 155-d monitoring period at 4 oC.

The cold-induced (4 oC) respiratory acclimation response (RAR) is the difference between the initial minimum and maximum rates of respiration at 4 oC. To directly compare the effects of O 2 atmosphere on tuber RAR’s, respiration rates were normalized to the initial rates

(=100%) at the transition to the LTS phase (i.e. zero-time at 4 oC) and plotted (Figs. 2B and C).

Differences in the rates of establishment, time to completion (from trough to peak) and magnitude (percent increase from trough to peak) of the RAR’s were affected by O 2 concentration (Fig. 2C). Tubers at 21 kPa O 2 completed their RAR about 8 d earlier than tubers

-1 -1 at 2.5 kPa O 2. The changes in respiration rate (µg kg s ) during acclimation (from trough to peak) were 17 and 7% for tubers at 2.5 and 21 kPa O2, respectively. Following the RAR, the respiration rate of tubers at 21 kPa O 2 decreased through 55 d and then remained relatively constant (at ca. 55% of the zero-time rate) through the remainder of the 155-d monitoring period.

By contrast, the respiration rate of tubers held at 2.5 kPa O 2 declined following completion of the

RAR to ca. 65% of the zero-time rate, which was maintained from 40 to 90 d. Respiration then

increased through 155 d as the low-oxygen tubers emerged from dormancy and began to sprout

(Fig. 2B).

3.2. Tuber dormancy and sprouting

No sprout growth was evident from either cultivar following 30 d of storage; however, the low-oxygen environment stimulated sprouting as early as 93 d in both cultivars at 8 oC (Table

2), and even at 4 oC in Innovator where growth was insufficient for fresh weight determination but sprout initiation was clearly visible (Fig. 3). As expected, tubers broke dormancy and

o o sprouted earlier when stored at 8 C compared with 4 C regardless of cultivar and O 2 atmosphere (Table 2; Fig. 3). Innovator tubers emerged from dormancy earlier than Russet

Burbank at both temperatures and oxygen concentrations. These data characterized the cultivar x temperature x O 2 concentration interaction ( P<0.05) on dormancy break at 93 d of storage (Table

2). By 153 d at 8 oC, Innovator tubers had 1.7- and 6.3-fold more sprout growth than Russet

Burbank tubers when stored at 2.5 and 21 kPa O 2, respectively.

3.3. Process quality (Fry color, Reducing Sugars, Sucrose)

o Regardless of O 2 concentration and cultivar, storage of tubers at 4 C resulted in darkening of fries and reduced process quality (Figs. 4 and 5, P<0.001) due to significant buildup in reducing sugars when compared with tubers held at 8 oC (Fig. 6AB). While these cold- induced changes were greatest during the initial 30 d of storage, the extent of LTS and associated changes in fry color over the 212-d storage period differed by cultivar and depended on temperature and O 2 concentration (cultivar x temperature x O 2 x duration, P<0.01; Table 1).

Russet Burbank tubers were more susceptible to LTS than Innovator tubers. The reducing sugar

concentrations in Russet Burbank tubers stored at 4 oC increased 70- and 48-fold over the initial

30 d of storage at 21 and 2.5 kPa O 2, respectively (Fig. 6A). By contrast, the cold-induced increase in reducing sugars of Innovator tubers was less (52-fold at 21 kPa O 2 and 28-fold at 2.5 kPa O 2), culminating in 26% (21 kPa O 2) and 42% (2.5 kPa O 2) lower concentrations when compared with Russet Burbank tubers at 30 d, and characterizing the superior LTS-resistant phenotype of Innovator tubers (Fig. 6AB). Following the initial increase, reducing sugar concentrations in Russet Burbank tubers at 4 oC remained constant from 30 to 212 d regardless of O 2 atmosphere and this was also the case for Innovator tubers stored at 21 kPa O 2. However, in contrast to Russet Burbank, the reducing sugar content of Innovator tubers stored at 2.5 kPa

O2 increased 2.4-fold from 93 to 212 d to equal that of Russet Burbank tubers stored at 21 kPa

O2 (Fig. 6B). Hence, over the first 93 d of storage, low O 2 inhibited the LTS response of

Innovator more than Russet Burbank, but this effect dissipated with further time in storage (Fig.

6AB), resulting in progressive deterioration (darkening) of process (fry) color (Figs. 4 and 5).

Innovator tubers, however, maintained acceptable fry color (USDA 0-1, basal end) regardless of temperature and O 2 concentration throughout the 212-d storage period (Figs. 4 and 5). Color of the basal ends of Russet Burbank fries became unacceptable (USDA 3) by 30 and 153 d for tubers stored at 21 and 2.5 kPa O 2, respectively (Figs. 4 and 5). Storage of tubers in 2.5 kPa O 2 was therefore more effective in slowing the cold-induced loss of process quality in Innovator than Russet Burbank tubers.

While fry color values in the USDA 0-2 range constitute acceptable fry quality, uniformity of color from apical to basal ends of fry planks is also an important criterion.

Uniformity of fry color was considered unacceptable when the absolute difference in Photovolt reflectance (ref) between basal and apical ends was equal to or greater than nine reflectance

units. Innovator tubers maintained uniform fry color (difference = 2.6-7.4 ref units) over the

212-d storage period regardless of storage temperature and O 2 concentration (Fig. 4).

o Conversely, except for the zero-time samples, Russet Burbank tubers held at 4 C and 21 kPa O 2 produced non-uniform fry color (basal minus apical reflectance units ≥9) throughout the 212-d storage period. Low O 2 atmosphere delayed the development of non-uniform fry color in Russet

Burbank tubers through the initial 30 d, beyond which color uniformity became unacceptable.

Uniformity of fry color for Russet Burbank tubers stored at 8 oC was acceptable at all sampling dates and O 2 concentrations except when stored for 153 d at 21 kPa O 2 (color difference = 9 ref units).

Changes in tuber sucrose concentrations during storage differed by cultivar and depended on temperature and O 2 concentration (cultivar x temperature x oxygen x duration, P<0.05)

(Table 1). The sucrose concentration of Russet Burbank tubers was equal to Innovator tubers at

o zero-time (Fig. 6). From 0 to 30 d at 4 C and 2.5 kPa O 2, concentrations increased 41 and 63% in Russet Burbank and Innovator tubers, respectively. By comparison, storage at 21 kPa O2 (4 oC) reduced this initial buildup in sucrose to 11% for Russet Burbank and 45% for Innovator tubers. These trends in initial sucrose accumulation were opposite to those for reducing sugar

o buildup in both cultivars at similar O 2 concentrations. From 30 to 212 d at 4 C, sucrose concentrations in Russet Burbank tubers declined 26% at 2.5 kPa O 2 compared with 20% in tubers stored at 21 kPa O 2. A similar trend was evident for Innovator tubers stored at 21 kPa O 2

o o (4 C) where sucrose concentration declined by 36% from 30 to 212 d. At 2.5 kPa O 2 and 4 C, however, sucrose content of Innovator tubers fell 33% from 30 to 153 d and then increased by an equivalent amount through 212 d. Sucrose concentrations in tubers stored at 8 oC decreased

progressively in both cultivars over the storage period (55% on average) to comparable levels at

212 d and were not affected by O 2 atmosphere.

3.4. Invertase and starch phosphorylase activities

Changes in basal (inhibitor present) and total (inhibitor absent) invertase activities over the 212-d storage period were affected by cultivar, storage temperature and O 2 concentration

(Table 1). During the initial 30 d of storage at 4 oC, activities increased more in tubers stored at

21 kPa O 2 than 2.5 kPa O 2 in tubers of both cultivars (Fig. 7). The basal invertase activity in

o Russet Burbank tubers at 4 C and 21 kPa O 2 then remained relatively constant through 212 d, but total activity continued to increase linearly (R 2=0.99, P<0.01), reflecting a significant increase in the activity of invertase inhibitor in tubers from 30 to 212 d. By contrast, over the

o same storage period, basal invertase activity in tubers stored at 2.5 kPa O 2 (4 C) decreased linearly (by 57%; R 2= 0.90; P<0.05) while total activity remained relatively constant through

212 d, which also reflects an influence of the inhibitor in regulating invertase activity. On

o average, the basal invertase activities in Russet Burbank tubers stored at 8 C (21 kPa O 2) were

o lower than in tubers stored at 4 C (21 kPa O 2) from 30 to 212 d of storage but temperature had

o little effect on basal activities at 2.5 kPa O 2. Total invertase activities in Russet Burbank at 8 C were higher than basal activities, reflecting the influence of invertase inhibitor; however, in contrast to 4 oC storage, total activities remained relatively constant throughout the 212-d storage

o period at 8 C for tubers stored at both O 2 levels.

The initial low temperature-induced increase in basal invertase activity of Innovator tubers held at 21 kPa O2 was attenuated relative to Russet Burbank tubers, resulting in 44% lower activity by 30 d at 4 oC (Fig. 7). However, basal invertase activity continued to increase in

o Innovator tubers through 93 d (4 C, 21 kPa O 2), reaching levels comparable to Russet Burbank

o (4 C, 21 kPa O 2) through the remainder of the 212-d storage period. Like Russet Burbank, total

o invertase activity in Innovator tubers stored at 4 C increased more at 21 kPa O 2 than 2.5 kPa O 2 and was significantly higher than the basal activity at most sampling times. At 8 oC, basal invertase activity in Innovator tubers was low and constant with no effect of O 2 level through

153 d of storage. The basal activity at 8 oC then increased from 153 to 212 d to become significantly higher in Innovator tubers held at 21 kPa O 2 compared with 2.5 kPa O 2. In contrast

o to Russet Burbank, total invertase activity in Innovator tubers stored at 8 C and 21 kPa O 2 increased over the 212-d storage period, but remained relatively constant when stored at 2.5 kPa

O2.

Activities of the cytosolic (SPH; high glycogen affinity) and plastidic (SPL; low glycogen affinity) isoforms of a -1,4-glucan phosphorylase increased with storage time, regardless of temperature and O 2 level (Fig. 8). Russet Burbank tubers had higher activities of both isoforms than Innovator tubers at harvest and throughout storage, which correlated with higher reducing sugars (Fig. 6) and darker fry colors (Figs. 4 and 5), particularly for tubers stored

o at 4 C. Effects of temperature and O 2 concentration on activities of the SPL isoform from

Russet Burbank tubers were difficult to resolve due to inherently high activity; however, tubers

o stored at 4 C and 21 kPa O 2 had higher activity of the cytosolic (SPH) isoform than tubers held

o at 8 C and 21 kPa O2 (Fig. 8). In contrast to Russet Burbank, storage of Innovator tubers at 2.5

o kPa O 2 induced higher activities of both isoforms from 30 to 153 d at 4 and 8 C compared with tubers stored at 21 kPa O 2 (Fig. 8), which correlated well with the low O2-induced stimulation of sprouting (earlier dormancy break) observed for this cultivar (Fig. 3; Table 2). Similarly, activities of the plastidic isoform in Innovator tubers stored at 8 oC increased more rapidly and

were generally higher than in tubers stored at 4 oC (Fig. 8), likely also reflecting earlier dormancy break at the higher temperature.

4. Discussion

The LTS responses of Russet Burbank and Innovator tubers, two mainstream frozen processing (French fry) cultivars with respective low and moderate resistances to LTS, were modulated by low O 2 storage for a better understanding and characterization of differences in their cold sweetening behavior. Reports of the effects of low O 2 storage on reduction of sweetening and retention of postharvest quality of LTS-susceptible potato cultivars have been mixed, largely due to the range of O 2 concentrations used. Oxygen levels too high do not mitigate LTS as much as lower concentrations (Workman and Twomey 1969; Harkett 1971).

Conversely, atmospheres too deplete of O 2 induce anaerobic metabolism, which leads to the production of lactate, ethanol, off-flavors, increased decay and a complete loss of process quality

(Lipton 1967; Sherman and Ewing 1983; Shouten 1992). The O 2 concentration in our studies

(2.5 kPa) was chosen based on previous work suggesting maximum reduction of LTS without invoking anaerobic metabolism (Harkett 1971; Sherman and Ewing 1983; Parkin and Schwobe

1990; Khanbari and Thompson 1994).

As expected, lowering the O 2 concentration from 21 kPa to 2.5 kPa greatly reduced tuber respiration, which fell even further when the temperature was lowered from 8 to 4 oC.

Respiration rates then increased as tubers acclimated to 4 oC, characterizing cold-induced respiratory acclimation responses (RAR) as defined by Zommick et al. (2014b). Zommick et al.

(2014b) established a positive correlation between RAR and total sugar buildup in cold resistant

o and susceptible cultivars stored at 4 C and 21 kPa O 2 over a 30-d period of sweetening. Here

we demonstrate that O 2 concentration greatly affected the kinetics of the cold-induced RARs

(averaged over cultivars). When assessed as percent increase in respiration from trough to peak

(Zommick et al. 2014b), the RAR for tubers at 2.5 kPa O 2 was 17% compared with only 7% for tubers at 21 kPa O 2 where the buildup in total sugar concentration (RS + Suc) was greatest. Low

O2, however, slowed development of the RAR relative to tubers stored at 21 kPa O 2, which likely reflects the limited availability of substrate (O2) and need for energy to fuel the attenuated sweetening metabolism. These results are in contrast to Zhou and Solomos (1998) where no

o RAR was evident from tubers stored at 1.5 kPa O 2 during LTS at 1 C. Absence of a RAR under these conditions may have been due to the extremely low temperature or the induction of anaerobic metabolism, which would not have invoked increased CO 2 production initially, as the first formed product of anaerobiosis in potato tubers is lactate (Barker and Saifi 1952; Barker and

o Kahn 1968). Indeed, storage of potato tissue at 1.5 kPa O 2 and 4 C induced lactate formation within 65 h of storage (Knowles, unpublished results).

o The increase in respiration observed for low-O2 stored tubers at 4 C versus those held at

21 kPa O 2 beyond 90 days is likely linked to the stimulation of early sprouting. Low O 2 hastens emergence from dormancy (Burton 1958; Shouten 1993). The low O 2-induced stimulation of sprouting was cultivar-dependent, with Innovator tubers sprouting earlier than Russet Burbank tubers at both storage temperatures, reflecting the inherent differences in dormancy of these two cultivars. However, the resumption of LTS (i.e. buildup in reducing sugars) in low-O2-stored

Innovator tubers at 4 oC from 93 to 212 d (see discussion below) is likely not directly attributed to the stimulation of sprouting, as reducing sugar levels remained constant despite equivalent or greater sprout development in tubers from other treatments (e.g. both cultivars at 8 oC and 2.5

o and 21 kPa O 2; Innovator at 4 C and 21 kPa O 2 at 212 d). Given that each chamber contained

tubers of both cultivars, cultivar-dependent respiratory responses to low O 2 during LTS and sprouting cannot be deduced. Further work is needed to characterize the significance of low O 2 atmosphere on development of the RAR during cold sweetening of tubers expressing LTS- susceptible and resistant phenotypes. Sprout inhibitors would be useful in such studies for resolving the respiratory responses associated with low O 2-induced sprouting versus LTS.

As anticipated, Innovator tubers sweetened less and maintained superior process fry color

o at 4 C and 21 kPa O 2 than Russet Burbank tubers, reflecting its LTS-resistant phenotype. While hypoxia attenuated sweetening and deterioration of fry color in both cultivars, the effect was initially greater for Innovator, resulting in lower Glc and Fru levels through 93 d at 4 oC. LTS then resumed for Innovator tubers stored at 2.5 kPa O 2, with reducing sugar concentrations increasing linearly (2.4-fold) through 212 d to a level substantially higher than in Russet

Burbank tubers held under hypoxia and equal to Russet Burbank tubers stored at 21 kPa O 2 and 4 oC. This resumption of LTS in Innovator was attended by progressive deterioration in process fry color. By contrast, 2.5 kPa O 2 restricted the LTS-induced buildup of reducing sugars in

Russet Burbank tubers to a maximum of approximately 11 g kg -1 dry weight, which was maintained from 30 to 212 d of storage. Low O 2 clearly affected the progression of LTS differently in these two cultivars, providing an opportunity to assess the extent to which invertase and starch phosphorylase were modulated by hypoxia.

The sucrose and reducing sugars that accumulate during LTS of potato are ultimately derived from starch breakdown (Isherwood 1973; Isherwood 1976). Storage temperature affects the equilibrium between synthesis and catabolism of starch and sucrose, which ultimately dictates reducing sugar levels in tubers (Davies and Viola 1992; Hill et al. 1996; Blenkinsop et al. 2004). Relative to Russet Burbank, the higher resistance of Innovator tubers to sweetening at

o 4 C and 21 kPa O 2 may reflect reduced starch breakdown, enhanced starch synthesis (i.e. recycling), or both. The mechanism of low temperature-induced starch catabolism in tubers is complex and details of the initial steps are poorly understood (Bethke 2013) but the process involves the concerted action of a number of enzymes (glucan water dikinase, a and b -amylases, debranching enzyme, limit dextrinases, starch phosphorylase, and others) with sucrose being the first free sugar to accumulate before Glc and Fru (Sowokinos 1990; Davies and Viola 1992;

Classen et al. 1993; Zhou and Solomos 1998). Regardless of the O 2 concentration, Glc/Fru ratio in Russet Burbank tubers stored at 4 oC averaged 1.1, which implicates inversion of sucrose as the immediate source of reducing sugars (Zrenner et al. 1996) during LTS at both O 2 levels.

o This was also the case for Innovator tubers stored at 2.5 kPa O 2 and 4 C (Glc/Fru = 1.1).

However, in contrast to Russet Burbank, the Glc/Fru ratio of Innovator tubers during LTS at 21 kPa O 2 was higher ( P<0.05), averaging 1.35 from 153 to 212 d, which suggests greater contribution of Glc directly from starch (Wu and Chen 1997) and/or potentially more rapid metabolism of the Fru produced from sucrose inversion, as proposed by Wiberley-Bradford et al.

(2014). Hence, in addition to invertase, low O 2 undoubtedly selectively modulates the activities of other enzymes involved in regulating carbohydrate metabolism during LTS of resistant and susceptible varieties.

While low O 2 modulated invertase activity to limit reducing sugar buildup and improve process quality, the effect was cultivar-dependent. Basal and total invertase activities increased

o in tubers stored at 4 C and 21 kPa O 2. However, this response was substantially suppressed by

2.5 kPa O 2 over the 212-d storage period in both cultivars. Our results agree with Zhou and

Solomos (1998) who demonstrated reduced expression and activity of acid invertase in Russet

o Burbank tubers during 30 d of storage in 1.5 kPa O 2 at 1 C. However, as noted above, the low-

O2 inhibition of LTS in Innovator was only temporary through 93 d; reducing sugars increased sharply thereafter despite very low invertase activities. Sucrose concentrations also remained

o relatively high in Innovator tubers at 4 C and 2.5 kPa O 2 from 93 to 212 d. Collectively, these data reflect increased starch catabolism in Innovator tubers over this period of resumed LTS at

2.5 kPa O 2.

Modulation of invertase by its endogenous inhibitor is a key regulatory mechanism in

LTS (Greiner et al. 1999; Brummell et al. 2011; Liu et al. 2013; McKenzie et al. 2013).

Invertase inhibitor activities, derived as the difference between total and basal activities, were affected by cultivar, O 2 concentration and temperature. After the initial and well documented

(Pressey and Shaw 1966; Zhu et al. 2014; Zommick et al. 2014ab) increase in invertase activity

o during the first 30 d of cold storage at 4 C and 21 kPa O 2, basal activity remained relatively constant over the remaining 180 d of storage for both cultivars. Total activity however, continued to increase linearly (140%) in Russet Burbank tubers from 30 to 212 d, indicating equivalent increase in the activity of endogenous inhibitor (total minus basal activity). In contrast to Russet Burbank, the increase in invertase inhibitor in Innovator tubers was delayed,

o with a 96% increase occurring from 153 to 212 d at 21 kPa O 2 and 4 C. Invertase inhibitor

o activities in tubers of both cultivars stored at 2.5 kPa O 2 and 4 C remained relatively low and constant from 30 to 212 d compared with tubers stored at 21 kPa O 2. Pressey and Shaw (1966) were the first to demonstrate a role for invertase inhibitor in regulating invertase activity during

LTS of susceptible cultivars (Red Pontiac and Kennebec). Storage of tubers at different levels of

O2 in the studies reported herein revealed that the extent and time course of modulation of invertase by its endogenous inhibitor likely differ for LTS resistant versus susceptible cultivars.

Recent work demonstrated that the plastidic isoform of starch phosphorylase (SP) is not required for mobilization of starch from leaves (Zeeman et al. 2004; Lloyd et al. 2005). The high affinity of SP for small linear maltodextrins and low affinity for high molecular weight branched chain glucans indicate a downstream role (if any) in the catabolism of starch in leaves.

Similarly, silenced SPL did not attenuate total sugar (sucrose + RS) buildup during LTS of potato, suggesting the plastidic isoform is not required for cold-induced starch catabolism; however, the isoform was induced in non-SPL-silenced heat-stressed tubers undergoing LTS

(Knowles, unpublished results), supporting work by Zeeman et al. (2004) that indicates a possible role for SPL in stress tolerance. Notwithstanding these results, SP activities increased during the first 30 d of storage at 4 oC in both Russet Burbank and Innovator tubers, a period coincident with the greatest cold-induced increases in sucrose and reducing sugars at both levels of O 2. The activities of both isoforms increased progressively over the 212-d storage period in

Russet Burbank tubers regardless of temperature and O 2 concentration. This was also evident for

o Innovator tubers stored at 4 and 8 C in 2.5 kPa O2. However, at 21 kPa O2, the increase in SPL activity occurred from 93 to 212 d at 8 oC and from 153 to 212 d at 4 oC. Increases in the activities of SP isoforms during LTS were also shown by Zommick et al. (2014a). In addition to a potential downstream role in starch catabolism, plastidic SP can participate in starch synthesis and functions at relatively low temperatures (Fettke et al. 2010; 2012). Therefore, the effects of

O2 and temperature on SPL activity may partly reflect cultivar-dependent changes in the ability to re-synthesize starch during LTS. While more detailed analyses are needed to elucidate the role of SP (if any) in the LTS-responses of susceptible and resistant cultivars, using low O2 storage to modulate gene expression and activities of SP isoforms during LTS may be a useful approach to clarifying its function.

5. Conclusion

Comparative evaluation of the cold sweetening responses of Russet Burbank and

Innovator tubers at 2.5 and 21 kPa O 2 effectively defined differences in their LTS phenotypes.

Low O 2 storage decreased tuber respiration rate, delayed the cold-induced RAR, and attenuated sweetening. The initial low O 2–induced reduction in LTS was greater for Innovator than Russet

Burbank tubers; however, this effect was only temporary for Innovator tubers, with robust resumption of LTS from 3 to 7 months of storage. The mitigation of LTS by low O 2 was largely attributable to reduced invertase activity, which led to higher sucrose levels in both cultivars.

However, invertase inhibitor activity, starch phosphorylase activities, and the extent of starch breakdown (as indicated by total sugar buildup) were also differentially modulated by O 2 concentration, further characterizing the intrinsic differences in LTS phenotype of these cultivars.

Acknowledgements

We gratefully acknowledge financial support from the USDA-ARS, USDA Specialty Crop

Block Grant program, and McCain Foods Ltd. We thank John Walsh, Potato Storage Specialist,

McCain Foods Ltd, NB Canada for his insight and advice during the course of this research.

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Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves process quality

by decreasing the frequency of sugar-end defects. PLoS ONE 9: e93381.

doi:10.1371/journal.pone.0093381.

Zrenner, R., Schuler, K., Sonnewald, U., 1996. Soluble acid invertase determines the hexose-to-

sucrose ratio in cold-stored potato tubers. Planta 198: 246-252.

Zommick, D.H., Kumar, G.N.M., Knowles, L.O., Knowles, N.R., 2013. Translucent tissue defect

in potato (Solanum tuberosum L.) tubers is associated with oxidative stress accompanying an

accelerated aging phenotype. Planta 238:1125-1145.

Zommick, D.H., Knowles, L.O., Pavek, M.J., Knowles, N.R., 2014a. In-season heat stress

compromises postharvest quality and low-temperature sweetening resistance in potato

(Solanum tuberosum L.). Planta 239: 1243-1263.

Zommick, D.H., Knowles, L.O., Knowles, N.R., 2014b. Tuber respiratory profiles during low

temperature sweetening (LTS) and reconditioning of LTS-resistant and susceptible potato

(Solanum tuberosum L.) cultivars. Postharvest Biology and Technology 92: 128-138.

Table 1 . Sources of variation and levels of significance ( P- values) for the effects of cultivar, storage temperature (4 vs 8 oC), oxygen atmosphere (2.5 vs 21 kPa O 2) and storage duration (0, 30, 93, 153, and 212 d) on process quality (French fry color), sugar concentrations and invertase activities in Russet Burbank and Innovator potato tubers (see Figs. 2-5). Sources of Fry Reducing Invertase activity Variation Color 1 Sugars Sucrose +Inh -Inh Significance levels ( P values) Cultivar (C) 0.01 0.01 ns 0.05 0.05 Temperature (T) 0.01 0.01 0.01 0.01 0.01 Oxygen (O) ns 2 0.01 0.01 0.01 0.01 Duration (D) 0.01 0.01 0.01 0.01 0.01 C x T ns 0.01 ns ns ns C x O ns 0.05 ns ns ns C x D 0.01 0.01 0.01 0.01 0.01 T x O ns 0.01 0.01 0.01 0.01 T x D 0.01 0.01 0.01 ns 0.01 O x D 0.01 0.01 ns 0.01 0.01 C x T x O 0.05 0.01 ns ns ns C x T x D ns ns ns ns 0.01 C x O x D 0.05 0.01 ns ns ns T x O x D 0.01 0.01 0.05 0.05 0.05 C x T x O x D 0.01 0.01 0.05 ns ns 1Average of apical and basal fry color (Photovolt reflectance). 2ns, not significant.

Table 2. Sprout growth from Russet Burbank and Innovator potato tubers as affected by storage temperature and oxygen concentration. Days in Storage Cultivar Temp O2 93 153 212 Sprout fresh weight oC kPa (g/tuber) Burbank 4 2.5 0c 0c 0.37 b 21 0c 0c 0.03 b 8 2.5 0.23 b 3.37 b ND 1 21 0c 0.78 c ND

Innovator 4 2.5 0c 0.20 c 2.08 a

21 0c 0c 0.75 b

8 2.5 0.63 a 5.69 a ND

21 0.09 bc 4.89 ab ND

2 LSD 0.05 0.16 1.66 1.07 Cultivar (C) <0.01 3 <0.01 <0.01 Temperature (T) <0.01 <0.01 - Oxygen (O) <0.01 <0.05 <0.01 C x T <0.01 <0.01 - C x O <0.05 ns ns T x O <0.01 ns - C x T x O <0.05 ns -

1Not determined 2Least significant difference, P<0.05. Letters indicate mean separation within a column by LSD ( P<0.05). 3P-values for indicated sources of variation.

Fig. 1. Schematic diagram of the factorial arrangement of storage treatments (temperature, O 2 concentration, and storage duration) for Innovator and Russet Burbank tubers. Each respiration chamber was fitted with an internal grate (A) to elevate tubers above the air inlet port. Twelve tubers of each cultivar were sealed together in each chamber (B) and oxygen atmospheres (CO 2- free air, balance N 2) were established via the inlet and outlet ports (C). Carbon dioxide was quantified in the outlet stream from each chamber at 2.5-h intervals through 155 d of storage (see

Fig. 2). Chambers were removed from the system at 30, 93, 153 and 212 d of storage to assess tuber sprouting, process quality (fry color), reducing sugars, sucrose, invertase, invertase inhibitor and starch phosphorylase activities. 86

8oC 4oC A 1.5 1.4 4oC ) 1.3 -1

s 1.2 -1 1.1 kg

g 1.0 m 0.9 0.8 21 kPa O C 0.7 2 90 0.6 RAR 0.5 o 85 0.4 4 C 0.3 2.5 kPa O TuberRespiration ( 0.2 2 80 0.1 21 kPa O 0.0 2 75 B 110 4oC 100 70

90 RespirationTuber Initial % 65 2.5 kPa O 2 2.5 kPa O 2 80 60 70 0 5 10 15 20 Days at 4 oC 60 21 kPa O 2 TuberRespiration%Initial 50

40 0 20 40 60 80 100 120 140 160 Days at 4 oC

Fig. 2. (A) Effects of low O 2 and temperature on tuber respiration (CO 2 evolution) rates during

o LTS. Tubers were initially acclimated to 2.5 and 21 kPa O 2 for 8 d at 8 C prior to lowering the temperature to 4 oC (zero-time) to induce LTS. (B) Tuber respiration rates were normalized to

o 100% of the zero-time (switch to 4 C) rate to directly compare changes in response to O 2 level.

o kPa O 2 over the initial 17 d at 4 C. Respiration rates are the average of Innovator and Russet

Burbank tubers.

Fig. 3. Emergence from dormancy and sprouting of Russet Burbank (RB) and Innovator (Inn)

o tubers during a 212-d storage period at 4 and 8 C in 21 and 2.5 kPa O 2 atmospheres. The four tubers shown for each treatment were chosen to represent the average sprout development from a

12-tuber sample at each date (see Table 2 for an analysis of sprout fresh weights).

Fig. 4. Changes in the processing quality (French fry color) of Russet Burbank (left column) and

Innovator (right column) tubers as affected by storage temperature and O 2 concentration over a

212-d storage interval. Color was measured with a Photovolt reflectance meter as light reflectance from the apical (middle row) and basal ends (bottom row) of fry planks and plotted along with the average reflectance (top row). High numbers indicate lighter fry color. USDA fry color ratings of 0 (light) to 4 (dark) are defined by the USDA axis and indicated by the color shading (values >USDA 2 constitute unacceptably dark color by industry standards). ANOVA results are presented in Table 1. Non-overlapping bars at each sampling (days in storage) indicate significant difference (LSD, P<0.05) between means (n=12). Fry samples are shown in

Fig. 5.

Russet Burbank Innovator 50

o 8 C, 2.5 kPa O 2 o 8 C, 21 kPa O 2 0 40 o 8 C, 21 kPa O 2 o 8 C, 21 kPa O 2 30 1 o 4 C, 2.5 kPa O 2 o 4 C, 21 kPa O 2 o 2 rating USDA color 20 4 C, 2.5 kPa O 2 Avg Fry Color (refunits) Color Fry Avg o 3 4 C, 21 kPa O 2 4 10 Russet Burbank Innovator 50

o 8 C, 2.5 kPa O 2 o 0 8 C, 21 kPa O 2 40 o 8 C, 2.5 kPa O 2 o 8 C, 21 kPa O 2

30 o 1 4 C, 2.5 kPa O 2 o, o 4 C, 21 kPa O 2 4 C, 2.5 kPa O 2 2 o USDA color rating 20 4 C, 21 kPa O 2 3 Apical Fry Color (ref units) Color Apical(ref Fry 4 10 Russet Burbank Innovator

50 o 8 C, 2.5 kPa O 2 o 8 C, 21 kPa O 2 0 40

o 8 C, 2.5 kPa O 2 o 8 C, 21 kPa O 30 2 1

o kPa 4 C, 2.5 O 2 2 o 4 C, 21 kPa O USDA colorrating 20 2 3 Basal Fry Color (ref units) (ref Color BasalFry o 4 C, 2.5 kPa O 2 o 4 C, 21 kPa O 2 4 10 0 30 93 153 212 0 30 93 153 212 Days in Storage Days in Storage

Fig. 5. Changes in the process color and uniformity of sample French fly planks from Russet

o Burbank and Innovator tubers stored at 4 and 8 C in 21 and 2.5 kPa O 2 atmospheres over time.

Fry samples are oriented with the apical ends up. *Numbers indicate USDA color ratings of the basal ends of fries (see Fig. 4) (USDA values >2 are unacceptable by industry standards). Each fry plank is from a different tuber and the 4 planks shown were selected to represent the average color from a 12-tuber sample for each treatment.

22 22 Russet Burbank A Innovator B 20 20 4oC, 21 kPa O 18 2 18 16 16 o dry weight) dry dry weight) dry 4 C, 21 kPa O 2 -1 14 14 -1

12 o 12 4 C, 2.5 kPa O 2 10 10 8 8 4oC, 2.5 kPa O 6 o 2 6 8 C, 2.5 kPa O 2 o 4 8 C, 2.5 kPa O 2 4

2 o o 2 8 C, 21 kPa O 2 8 C, 21 kPa O 2 Reducing Sugars (g kg Reducing(gSugars 0 0 kg Reducing(gSugars 22 22 Russet Burbank CDInnovator 20 20 18 18 16 16 14 14 o o 4 C, 2.5 kPa O 2 4 C, 2.5 kPa O dry weight) dry 12 2 12 weight) dry -1 -1 10 10 4oC, 21 kPa O 8 2 8 4oC, 21 kPa O 6 2 6 o 8 C, 2.5 kPa O 2 4 o 4 Sucrose (g kg Sucrose 8 C, 21 kPa O 2 kg Sucrose (g 2 o 2 8 C, 21 kPa O o 2 8 C, 2.5 kPa O 2 0 0

0 30 93 153 212 0 30 93 153 212 Days in Storage Days in Storage

Fig. 6. Changes in reducing sugar (Glc + Fru) and sucrose concentrations of Russet Burbank (A

o and C) and Innovator (B and D) tubers during 212 d of storage at 4 and 8 C in 21 and 2.5 kPa O 2 atmospheres. ANOVA results are presented in Table 1. Non-overlapping bars at each sampling

(days in storage) indicate significant difference (LSD, P<0.05) between means (n=12).

Basal Activity Total Activity Basal Activity Total Activity (inhibitor present) (inhibitor absent) (inhibitor present) (inhibitor absent) 7 Russet Burbank Russet Burbank Russet Burbank Russet Burbank 7 ) ) 6 6 -1 4oC 4oC 8oC 8oC -1 kPa 5 21 O2 5

4 21 kPa O2 4

3 2.5 kPa O 2 3 21 kPa O2 2 2 21 kPa O2 Invertase Activity (U kg Activity (U Invertase Invertase Activity (U kg InvertaseActivity 1 2.5 kPa O 2 1 2.5 kPa O 2 2.5 kPa O 0 2 0 0 30 93 153 212 0 30 93 153 212 0 30 93 153 212 0 30 93 153 212 7 Innovator Innovator Innovator Innovator 7 ) 6 ) -1 6 -1 93 4oC 4oC 8oC 8oC 21 kPa O2 5 5

21 kPa O 4 2 4

3 3

kPa 2 21 O2 21 kPa O2 2 Invertase Activity (U kg Activity (U Invertase 2.5 kPa O kg Activity (U Invertase 1 2 1 2.5 kPa O 2 2.5 kPa O 2 2.5 kPa O 2 0 0 0 30 93 153 212 0 30 93 153 212 0 30 93 153 212 0 30 93 153 212 Days in Storage Days in Storage Days in Storage Days in Storage

Fig. 7. Changes in the activities of acid invertase in the presence (basal activity) and absence (total activity) of its endogenous

o inhibitor during storage of Russet Burbank (top row) and Innovator (bottom row) tubers stored at 4 and 8 C in 21 and 2.5 kPa O 2.

ANOVA results are presented in Table 1. Non-overlapping bars at each sampling (days in storage) indicate significant difference

(LSD, P<0.05) between means (n=12).

Fig. 8. Changes in the starch-synthesizing activities of cytosolic (SPH) and plastidic (SPL) starch phosphorylase in Russet Burbank (A) and Innovator (B) tubers during storage at 4 and 8 o C in 2.5 and 21 kPa O 2 for 212 d. The high (SPH) and low affinity (SPL) isoforms were separated on glycogen-containing native gels, which were then incubated with G-1-P and starch to stimulate starch synthesis. Each lane represents a pooled sample of four replicates representing 12 tubers.

CHAPTER 3

Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato

(Solanum tuberosum L.)

Abstract

Heat stress exacerbated cold sweetening (buildup of reducing sugars) of the LTS-susceptible potato ( Solanum tuberosum L.) cultivars, Ranger Russet and Russet Burbank, and completely abolished the resistance to cold sweetening in the LTS-resistant cultivars/clones, Sage Russet,

GemStar Russet, POR06V12-3 and A02138-2. Payette Russet and EGA09702-2, however, demonstrated considerable tolerance to heat stress for retention of their LTS-resistant phenotype.

Heat-primed Payette Russet and EGA09702-2 tubers accumulated 4-fold more sucrose when subsequently stored at 4 oC, while reducing sugar concentrations also increased marginally but remained low relative to the non-heat tolerant LTS-resistant clones, resulting in light colored fries. By contrast, sucrose concentrations in heat-primed tubers of the non-heat tolerant clones remained unchanged during LTS, but reducing sugars increased 5-fold, resulting in darkening of processed fries. Acid invertase activity increased in the LTS-susceptible and non-heat tolerant

LTS-resistant cultivars/clones during cold storage. However, Payette Russet tubers maintained very low invertase activity regardless of heat stress and cold storage treatments, as was the case for Innate Russet Burbank (W8) tubers, where silenced invertase conferred robust tolerance to heat stress for retention of LTS-resistant phenotype. Importantly, heat stressed tubers of Payette

Russet, EGA09702-2 and Innate Russet Burbank (W8) demonstrated similar low reducing sugar and high sucrose accumulating phenotypes when stored at 4 oC. Tolerance to heat stress for retention of LTS-resistant phenotype in Payette Russet and likely its maternal parent,

EGA09702-2, is therefore conferred by the ability to maintain low invertase activity during cold storage of heat-stressed tubers.

Introduction

Fry process quality in potatoes ( Solanum tuberosum L.) is largely determined by specific gravity (dry matter and starch content) and reducing sugar content (glucose and fructose; Glc +

Fru). Tubers of optimum process quality have low reducing sugars and relatively high specific gravities at harvest and can retain these characteristics for a prolonged period when stored at 9 oC.

Storage at lower temperatures (e.g. 4-6oC) can preserve quality and extend marketability by limiting tuber respiration, fresh weight loss, disease progression, and by prolonging dormancy

(Burton 1966; Schippers 1977; Burton 1978; Wiltshire and Cobb 1996); however, process quality (French fries and chips) is often sacrificed by the cold-induced breakdown of starch into reducing sugars (Isherwood 1976; Sowokinos 2001a; Malone et al. 2006). During the frying process, reducing sugars react with free amino acids in the Maillard reaction, producing dark colored pigments, off-flavors and acrylamide, a probable human carcinogen (IAREC 1994;

Tareke et al. 2000; Mottram et al. 2002; Stadler et al. 2002; Kumar et al 2004; Stadler and

Scholz 2004; Tareke et al. 2004). Therefore, a major goal of breeding programs is to develop varieties that resist low temperature sweetening (LTS) during cold storage (Novy et al. 2008).

High temperatures during tuber development can alter plant source-sink relationships, disrupt tuber initiation, reduce specific gravity, and increase tuber reducing sugar concentrations and associated physiological disorders, which can greatly compromise yield and overall process quality (Yamaguchi, 1964; Krauss and Marschner 1984; Wolf et al. 1991; Midmore 1992;

Midmore and Prange 1992; Midmore and Roca 1992; Struik and Ewing 1995; Lafta and

Lorenzen 1995; Stevenson et al. 2001; Timlin et al. 2006; Sowokinos et al. 2007; Thompson et al. 2008). High sugar (sucrose, Glc, and Fru) content, sugar end development (Thompson et al.

2008; Thornton et al. 2010), mottling, translucent tissue defect (Zommick et al. 2013) and other sugar-related disorders can either manifest at harvest or develop later during storage as a consequence of in-season temperature stress at key stages of tuber development (Zommick et al.

2014). The threshold tolerance to heat stress for inducing sweetening-related disorders and loss of process quality varies significantly by cultivar.

While potato breeding programs have been successful in developing LTS-resistant varieties (Novy et al. 2008; 2010), recent work has demonstrated that heat stress can attenuate this resistance, resulting in LTS-susceptibility (Zommick et al. 2104). The heat-induced loss of process quality due to sweetening is only expressed when tubers are stored at cold temperatures

(data reported herein). Heat stress somehow modifies the response to cold, inducing changes in carbohydrate metabolism that compromise quality during low temperature storage.

Understanding the mechanism(s) by which heat stress alters susceptibility to LTS is prerequisite to developing cultivars with more durable and robust tolerance to high temperature for retention of the LTS-resistant phenotype.

In studies reported herein, cultivars/clones with varying degrees of resistance to LTS were challenged with in-season and postharvest heat stress to identify those with tolerance to heat stress for retention of LTS-resistant phenotype. These approaches facilitated classification of eight conventionally bred cultivars/clones and the genetically engineered Innate Russet

Burbank (W8) cultivar (provided by the J.R. Simplot Co., Boise, ID) into one of three categories:

‘LTS-susceptible’, ‘LTS-resistant but non-heat tolerant’, or ‘LTS-resistant and heat-tolerant’.

Subsequent evaluation of the LTS phenotypes, invertase/invertase inhibitor activities and starch

phosphorylase activities of a subset of these cultivars/clones in response to heat and cold treatments demonstrated that heat stress alters how LTS-resistant but heat susceptible clones perceive cold to induce invertase activity and accumulation of reducing sugars during storage at low temperature. By contrast, retention of the LTS-resistant phenotype in heat tolerant cultivars/clones was conferred by the resistance of invertase to cold induction.

Materials and methods

Plant material

Certified seed tubers (G3 from nuclear) of cvs. Ranger Russet, Russet Burbank, GemStar

Russet and Sage Russet were acquired directly from commercial seed growers in the fall (Table

1). Seed tubers of LTS-resistant advanced clones (numbered lines) were provided by the Pacific

Northwest Variety Development Program and included Castle Russet (POR06V12-3), A02138-

2, Payette Russet (A02507-2LB) and its maternal parent, EGA09702-2. EGA09702-2 was originally selected from true potato seed provided by Dr. Ewa Zimnoch-Guzowska, Młochów

Research Center, Plant Breeding and Acclimatization Institute (IHAR), Młochów, Poland.

Seedling tubers from the Polish seed were generated at Aberdeen, ID in 1997, with field selection of EGA09702-2 in Corvallis, OR in 1998 (Novy et al. 2016). Innate Russet Burbank

(W8) seed tubers possessing robust resistance to LTS by virtue of silenced (RNAi) invertase

(Clark et al. 2014) were provided by the J.R. Simplot Co. (Boise, ID). All seed was stored at 4 oC

(95% RH) until cutting and planting in mid-April for the studies described below.

In-season heat stress studies

To assess the effects of in-season heat stress on subsequent retention of postharvest process quality and LTS, tubers of Ranger Russet (LTS susceptible) and the LTS-resistant clones, POR06V12-3, A02138-2 and Payette Russet were subjected to elevated soil temperatures 98

for 20 and 40 days during both the bulking (80-120 DAP) and maturation (120-160 DAP) stages of tuber development. The five treatments for each cultivar included 2 durations of heat x 2 stages of development plus an ambient (non-heat stressed) control (3 replicates). Seed tubers

(120-180 g) of each cultivar/clone were hand cut into 50- to 64-g seed pieces, blocked for portion (apical and basal portions assigned to different replicates) and wound healed at 9 oC (95%

RH) for 3 days prior to planting.

Seed pieces were planted 20 cm deep and 25 cm apart in a Shano silt loam soil (Lenfesty

1967) at the Washington State University Irrigated Research and Extension Unit, Othello, WA

(46.8 oN Lat, -119.0 oW Long) on April 16, 2014. Plots of each cultivar/clone (4.5-m-long; 18 seed pieces per plot) were arranged in a randomized block design in 61-m rows flanked by guard rows of cv. Ranger Russet. Heat treated rows were equipped with two 61-m, 1000-W soil heating cables (Redi-Heat, Wrap-On Co., Inc., Bedford Park, IL, USA) installed 20-25 cm apart at seed depth (20 cm after hilling), as described by Zommick et al. (2014). Control (non-heated) rows lacked heat cables. Seed pieces in the heated rows were hand planted (Fig. 1a) and then hilled with a two-row assist feed planter that simultaneously planted and hilled the adjacent guard row. Rows were spaced 86 cm apart.

Water was provided by a solid-set irrigation system. Soil moisture was monitored by neutron probes and was maintained at a minimum of 65% field capacity throughout the growing season. Irrigation scheduling followed evapotranspiration models for potato production at the

WSU Othello Research Unit (WSU AgWeatherNet, http://weather.wsu.edu/awn.php, accessed

23 May, 2016). Pre-plant and in-season fertilizer applications (fertigation through solid set) were adjusted based on soil tests and weekly petiole analyses, respectively. Herbicide and

pesticides were applied as needed following standard practices for production of long-season frozen-processing potatoes in the Columbia Basin.

Soil temperature of the heated rows was controlled with rheostats (Redi-Heat TM

Thermostat, Phytotronics Inc., Earth City, MO, USA) attached to each heat cable. Soil temperatures were recorded (13-cm depth) hourly from 57 to 165 days after planting (DAP) with

Watchdog temperature sensors (Spectrum Technologies Inc., Aurora, IL, USA) installed ca. 10 m in from the beginning and ends of each treatment row. Seasonal temperature profiles for the five treatment rows are shown in Fig. 2. Heated rows averaged 27.0±1.8 oC from 80 to 160 DAP compared with 19.3±2.7 oC for control. The average difference in soil temperature between non- heated control and heated plots was 8.2±2.3 oC from 80 to 160 DAP. A 30.5-cm long temperature probe (Oakton Instruments, Vernon Hills, IL, USA) was used to map the temperature of hills in cross section (Fig. 1bc). Hill temperatures were recorded in a grid pattern at 5-cm intervals from the top of the hill to a depth of 23 cm and at 8.9-cm intervals across the

63-cm wide hills ( n=40) for control and heated rows (Zommick et al. 2014).

Plots were harvested with a single-row harvester on 2 October 2014 (169 DAP). The tubers were washed, counted and individually weighed with a custom-built automated cup-type sorter. Tubers were sorted into the following size classes: <113 g, 113-170 g, 170-284 g, 284-

340 g, 340-397 g, >397 g, or culls. Total yield is the weight of all tubers including culls.

Marketable yield equals total yield minus culls. The 170- to 340-g tubers were used in postharvest studies (see below) to assess the effects of in-season heat stress treatments on LTS and process quality.

100

Postharvest handling and storage

Tubers of each cultivar and treatment were initially stored at 9 oC (95% RH) for 14 days to wound heal following harvest. The tubers were then blocked for size into three replicates and specific gravity (SG) was measured by the weight in air/weight in water method (Gould 1999) (4 tubers per replicate). Prior to placing tubers at 4 oC (95% RH) for the LTS phase of storage, zero- time samples were taken to compare initial French fry process quality (fry color), percent dry matter, and sucrose, glucose (Glc) and fructose (Fru) concentrations. Tubers were then stored at

4oC (95% RH) with subsequent sampling at 10, 20 and 30 days (LTS phase of storage) to assess the effects of in-season heat treatments on changes in process quality (fry color) and sweetening over time. There were three replicates (4 tubers per replicate) of each treatment (5 in-season heat treatments) for each cultivar at each sampling date (including zero-time).

Tissue sampling and process quality assessment

The procedures for sampling tubers for French fry process quality and carbohydrate analyses are described in Herman et al. (2016). Briefly, at 0, 10, 20 and 30 d of storage (4oC), tubers (3 replicates of 4 tubers per replicate) were halved along the apical to basal axis. A

French fry plank (9.5-mm-thick x 2.9-cm-wide x length of tuber) was cut from one of the halves and a 1.5-mm-thick tissue slice (periderm intact) was shaved from the cut surface of the other half with a mandoline slicer. Slices from each of the four tubers per replicate were stacked and halved again along the apical to basal axis. Half the tissue was dried at 60 oC for 72 h in a forced- air drying oven and dry matter was determined as percentage fresh weight. The remaining tissue

(collectively representing four tubers per sample) was frozen (-80 oC) and lyophilized. The lyophilized samples were ground with mortar and pestle and sieved through a 60-mesh screen

101

(0.246 mm) for analysis of sucrose and reducing sugars (see below) following completion of the

30-d LTS study.

French fry process quality was evaluated at each sampling time as described by Zommick et al. (2014). French fry planks from each of the 12 tubers (see above) per treatment were fried collectively for 3.5 min at 191 oC in soybean oil. Color of the apical and basal ends of each plank was then quantified with a Photovolt reflectance meter (Model 577, Photovolt Instruments Inc.,

Indianapolis, IN, USA). Basal reflectance data were plotted versus days at 4 oC to characterize the effects of in-season heat treatments on changes in process color during LTS for each cultivar/clone. The basal reflectance values were also translated into USDA color ratings (≥31 =

USDA 0; 25-30 = USDA 1; 20-24 USDA 2; 19-15 USDA 3; and ≤14 USDA 4) (Stark et al.

2016). Photovolt reflectance values less than 20 (USDA 3 or greater) are unacceptable by industry standards (Driskill et al. 2007).

Sucrose and reducing sugar analyses

Sucrose, Glc and Fru were extracted from 250-mg samples of ground lyophilized tissue by vortexing with 3 mL 60% (v/v) methanol in triethanolamine (TEA) buffer (30 mM, pH 7.0) as described in Herman et al. (2016). The extracts were centrifuged (10,000 g, 15 min) and Glc,

Fru and sucrose were quantified in the supernatant by methods of Bergmeyer et al. (1974) and

Bernt and Bergmeyer (1974) as modified for microplate determination (Knowles et al. 2009). In this assay, the conversion of Glc and Fru to 6-phosphogluconate was coupled to the stoichiometric reduction of NADP (monitored at A340 ) via the action of glucose-6-phosphate dehydrogenase, hexokinase and phosphoglucose isomerase. A separate aliquot of extract was incubated with acid invertase to hydrolyze sucrose, which was then quantified as the difference between moles total Glc and moles free Glc (Bergmeyer and Bernt 1974). Sugar concentrations

102

were determined based on standard curves containing equimolar (0.05-1.8 mM) mixtures of Glc,

Fru, and sucrose.

Dormancy break

The effects of timing (bulking vs. maturation) and duration (20 vs. 40 days) of in-season heat stress on sprouting of tubers were assessed over an 87- and 165-d postharvest storage period for Ranger Russet and Payette Russet, respectively. Twenty (170- to 340-g) tubers of each cultivar from each of the five in-season heat treatments were stored at 9 oC (95% RH) in the dark directly following harvest. Length of the longest sprout on each tuber was measured approximately every 10 days beginning 30 days after harvest (DAH). Sprout length ±SE is plotted versus time. Final total sprout fresh weights (g tuber -1) were compared at the end of the storage period.

Postharvest heat stress (PHHS) studies

Additional studies were conducted to screen cultivars and advanced breeding lines for tolerance to heat stress (HS) for retention of LTS-resistant phenotype using the PHHS protocol described in Zommick et al. (2014). All LTS susceptible and resistant cultivars/clones (Table 1) except EGA09702-2 were grown full season (up to 7 seasons, 2014 data shown as representative) at the Washington State University Irrigated Research and Extension Unit using standard commercial practices for production of long-season processing russets in the Columbia

Basin. Tubers of EGA09702-2 (the maternal parent of Payette Russet) were grown at Aberdeen,

ID and provided by R. Novy (USDA ARS). Following harvest, the tubers were washed, sorted for size (described previously) and initially wound healed for 16 d at 9 oC (95% RH). The 170- to 284-g tubers of each cultivar were blocked for size into four replicates (three tubers per replicate) and subjected to four storage temperature treatments (control, HS, cold stress (CS),

103

HS+CS). Control tuber samples were stored at 9 oC; HS tubers were held at 32 oC for 21 days; CS tubers were held at 9 oC for 21 days and then 4 oC for 32 days to invoke LTS; and tubers subjected to the combination treatment were heat primed at 32oC for 21 days followed by 32 days at 4 oC

(HS + CS). Fry plank and tissue samples were taken at the end of each treatment for analysis of process quality, sucrose and reducing sugars as described above for the in-season heat stress study. Acid invertase and starch phosphorylase activities were also compared, along with expression levels of the vacuolar acid invertase and apoplastic and vacuolar invertase inhibitor genes.

Enzyme analysis

Acid invertase activity was determined using methods detailed in Herman et al. (2016).

Invertase was extracted from 200 mg lyophilized tissue (composite of 3 tubers per replicate; 4 replicates) with 1 mL HEPES buffer (50 mM, pH 7.5) containing 15 mM MgCl 2·6 H 2O, 2 mM

Na 2EDTA·2 H 2O, 5 mM NaHSO 3, 10% (v/v) glycerol, 2% PVPP and 2 µL protease inhibitor cocktail (2.5 µL mL -1 leupeptin, chymostatin, pepstatin and antipain, and 20 µL mL -1 4- aminobenzamindine and benzamidine) at 4 oC. The homogenate was centrifuged (10,000g, 15

o min, 4 C) and the supernatant combined with 60% (NH 4)SO 4 to precipitate the protein. Once pelleted (10,000g, 20 min, 4 oC), the protein was re-solubilized in the original extract buffer

(minus PVPP) and stored at -18 oC. Invertase activity was determined colorimetrically by a microplate adaptation of Brummell et al. (2011) as described in Zommick et al (2013) in the presence (basal activity) and absence (total activity) of endogenous inhibitor. Basal and total activities are expressed as nmol sucrose hydrolyzed mg -1 protein h -1. Soluble protein was quantified (Bradford 1976) using protease-free BSA (Sigma-Aldrich, St. Louis, MO) as a standard.

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Activity gel electrophoresis (Steup 1990) was used to evaluate relative changes in activities of the plastidic (L-type; low glycogen affinity) and cytosolic (H-type; high glycogen affinity) isoforms of starch phosphorylase (α-1,4-glucan phosphorylase) in response to PHHS treatments. One hundred milligrams of lyophilized tissue (25 mg combined from each of 4 replicates representing 12 tubers) for each treatment was homogenized with 1 mL of 0.1 M Tris buffer (pH 6.8) containing 10% (w/v) glycerol, 1% NaHSO 3, 2 µL protease inhibitor cocktail (as above), and 1% (w/v) Triton TM X-100 (Sigma-Aldrich, St. Louis, MO). The plastidic and cytosolic isoforms were separated on 8.5% native polyacrylamide gels containing 0.65% (w/v) glycogen (115 µg protein per lane). Following electrophoresis (180 min, 150 V), gels were washed and subsequently incubated (37 oC for 120 min) in 100 mM MES buffer (pH 6.2) containing 0.5 mg mL -1 soluble potato starch and 12 mM Glc-1-phosphate. The gels were stained with KI/I to compare starch synthesizing activities of the two isoforms. Data are presented for the plastidic isoform only since activity of the cytosolic isoform was not affected by the PHHS treatments.

RNA extraction and qPCR

Changes in gene expression of acid invertase (INV) and its apoplastic (Inh1) and vacuolar (Inh2) inhibitors in response to PHHS treatments were compared for Russet Burbank,

A02138-2, Payette Russet and Innate Russet Burbank (W8) tubers. Primers were designed and synthesized as described previously (Zommick et al. 2013; 2014). Forward and reverse primer sequences, annealing temperatures and amplified PCR product lengths for invertase (SGN-

U270188), Inh1 (AY864820) and Inh2 (AY864821) are detailed in Zommick et al. (2013) and

(2014).

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Nucleic acids were extracted from tuber tissue as described by Kumar et al. (2015). First strand cDNA was synthesized from DNase-treated samples (TURBO DNA-freeTM Kit, Ambion

Inc., Foster City, CA, USA) using the Verso cDNA Synthesis Kit with anchored oligo (dT) primer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Real-time quantitative PCR

(qPCR) analysis was performed using the LightCycler® 480 High Resolution Melting Master kit

(Roche Diagnostics Corp., GmbH, Germany) on the LightCycler® 480 Real-Time PCR System version 1.5 (Roche Diagnostics Corp., Indianapolis, IN, USA). Reaction mixtures were prepared according to kit specifications and consisted of 50 ng cDNA, 5 µM forward and reverse primer,

1.5 µM MgCl 2 and 10 µL master mix (2x) to a final volume of 20 µL. The PCR amplification ran for 10 min at 95 oC, followed by 40 cycles of 45 sec (95 oC), 45 sec (56 oC) and 45 sec (72 oC), ending with 10 min at 72 oC for final extension. INV, Inh1 and Inh2 mRNA levels were normalized to EF-1-α (Nicot et al. 2005) using the DDC T method (Livak and Schittgen 2001).

Final qPCR products were visualized on a 1% (w/v) agarose gel contain EtBr.

Data analysis and presentation

Tuber number, weight, yield, specific gravity, dry matter and sprout fresh weight data for each cultivar were subjected to analysis of variance (ANOVA) with single degree-of-freedom contrasts partitioned for main effects (stage of application and duration of in-season heat stress) and their interaction (stage x duration). Means are separated by LSD ( P<0.05). Effects of stage and duration of heat stress on tuber size distribution were similar across cultivars/clones.

Therefore, data for yields of the six tuber size classes were averaged across clones, expressed as percent marketable yield, and summarized in polygonal plots to depict the effects of stage and duration on shifts in tuber size distribution. Fry color (basal-end Photovolt reflectance), sucrose

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and reducing sugar data were plotted ±SE versus time over the 30-d interval of storage at 4 oC

(LTS phase of study).

The effects of PHHS treatments (control, HS, CS, HS+CS) on process quality (basal fry color), sucrose, reducing sugars and invertase (±inhibitor) were analyzed by one-way (for each cultivar) and three-way (cultivar x HS x CS) ANOVA. The 3-way ANOVA included planned comparisons between groups of LTS-susceptible and resistant cultivars/clones. PHHS treatment effects on expression (qPCR) of invertase, Inh1 and Inh2 were analyzed separately by cultivar.

All data are plotted ±SE with means separated by LSD ( P<0.05).

Results

In-season heat stress studies

Soil warming cables installed in-furrow (Fig. 1a) were used to implement heat stress during the bulking (80-120 DAP) and maturation (120-160 DAP) phases of tuber development.

Soil temperatures at 13-cm depth were effectively increased from 20.2±1.9 oC (ambient) to

27.8±1.1 oC during the bulking period (Fig. 2a). Similarly, temperatures during maturation averaged 18.5±3.1 oC and 27.1±1.7 oC for ambient and heated rows, respectively (Fig. 2b). The temperature treatments were averaged over the 80-160 DAP bulking and maturation periods and are henceforth denoted as 19 and 27 oC for the ambient (control) and heated plots, respectively.

Cross-sectional temperature profiles (heat maps) of the hills were prepared at 98 DAP to assess temperature uniformity of heated versus control rows. The non-heated (ambient) row averaged 18 oC through most of the hill except the outermost 6.5 cm, which was 2 oC higher

(20 oC) (Fig. 1b). Consistent with location of the heat cables, the heated row (Fig. 1c) averaged

30 oC from 16- to 23-cm depth in the central portion of the hill and temperature decreased to

24 oC at the outer periphery of the hill.

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Tuber set, size, yield and raw quality

Effects of the elevated soil temperature treatments on yield and tuber quality (specific gravity, dry matter) depended on stage (bulking vs. maturation) and duration of heat and were similar across the four cultivars/clones (see supplementary Table). Data are therefore averaged across cultivars/clones and presented in Table 2. The effect of early heat (bulking phase) on tuber number per plant depended on duration. Twenty days of growth at 27 oC during bulking

(80-100 d) increased tuber number per plant by 1.3 tubers over control (ambient 19 oC) but 40-d at 27 oC (80-120 DAP) had no effect on tuber number. High soil temperature during maturation decreased tuber number per plant relative to the control (19 oC) and 27 oC bulking treatments, and the effect was greater for 40- versus 20-d exposure. On average, stage of exposure to high soil temperature (bulking vs. maturation) had a greater effect on tuber number per plant than the duration of exposure and no interaction between stage and duration was apparent.

Elevated soil temperature reduced tuber yields (total and marketable), size (fresh weight), specific gravity and percent dry matter and the extent of reduction depended on stage and duration of heat (Table 2). Averaged over duration, tuber yields fell by 25 and 46% when soil temperature was maintained at 27 oC during the bulking and maturation stages, respectively.

Increasing the duration of heat from 20 to 40 d further reduced yields by the same amount for both developmental stages, as evident by the lack of interaction between these treatments. On average, yield losses were 28% higher for the 40- versus 20-d duration of high soil temperature

(averaged over stage).

While 27 oC soil temperature during bulking was less detrimental to yield than during maturation, tuber specific gravity and dry matter were more negatively affected by heat during bulking (Table 2). Under ambient conditions, specific gravity and dry matter averaged 1.088 and

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24.2%, respectively. High soil temperature during bulking reduced specific gravity to 1.068 and dry matter to 19.5% (averaged over duration), compared with 1.080 specific gravity and 22.7% dry matter when heat was applied during tuber maturation. However, specific gravity was the only component affected by an interaction between stage and duration. Duration of heat had no effect on specific gravity during the maturation phase, but 40 d at 27 oC during bulking reduced tuber specific gravity more than 20 d.

The heat-induced decrease in marketable yields and effects on tuber number resulted in a general reduction in average tuber fresh weight for all cultivars/clones in response to elevated soil temperature (Table 2 and supplementary Table). Averaged across 20 and 40 d duration, tuber fresh weights were 36% and 28% lower when grown at 27 oC during bulking (146.5 g tuber -

1) and maturation (164 g tuber -1), respectively, compared with control (228 g tuber-1). Duration of heat also affected average tuber size during both stages of development. Tubers grown at

27 oC for 20 d averaged 171 g tuber -1 compared with 139.5 g tuber -1 for 40-d duration. These decreases in average tuber size reflect effects of the high soil temperature on tuber size distribution. As percent marketable yield, tuber size distribution shifted away from a relatively high percentage of tubers >284 g to increased percentage of <170-g tubers when grown at 27 oC during bulking and maturation (Fig. 3). The 170-284-g tubers remained relatively constant at ca.

35% of marketable yield regardless of treatment. Moreover, the heat-induced shift in tuber size distribution was greatest during bulking versus maturation and for the 40- versus 20-d duration of heat at both stages of development.

At-harvest process quality and reducing sugar accumulation during LTS

Relative to the other clones, Ranger Russet had the highest reducing sugar concentrations and darkest zero-time (following wound healing at 9oC) fry color across all in-field heat

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treatments (Table 3, Fig. 4). While reducing sugar concentrations increased and fry colors darkened over the 30-d interval of cold sweetening for all cultivars/clones, the rates and magnitudes of these changes depended on clone and in-season heat stress treatment. Non-heat stressed (ambient, 19 oC) Ranger Russet tubers accumulated reducing sugars (i.e. sweetened) at a faster rate than non-heat treated POR06V12-3, A02138-2 and Payette Russet tubers during storage at 4 oC, reflecting the LTS-resistant phenotypes of the latter three clones (Fig. 4).

Relative to tubers grown at ambient temperature, late season heat (27 oC) during maturation for

20 d had no appreciable effect on reducing sugar buildup and fry colors during LTS for all clones except POR06V12-3 where reducing sugar levels were lower and fry colors lighter in tubers from this treatment (Fig. 4, Table 3).

Heat stress of Ranger tubers during bulking (20 and 40 d) and maturation (40 d) resulted in unacceptable USDA 3 color fries (basal end) by 10 d and USDA 3-4 color fries by 30 d at 4 oC

(Fig. 4). Importantly, A02138-2 tubers from the 40-d, 27 oC bulking treatment accumulated slightly higher reducing sugar concentrations (28.3 mg g -1 dry wt) during 30 d of LTS than the non-heat stressed (19 oC) tubers of Ranger Russet (26.5 mg g -1 dry wt) (Table 3, Fig. 4).

Similarly, POR06V12-3 tubers from the high temperature 40-d bulking treatment sweetened the most relative to Ranger tubers grown at ambient temperature. In-season heat stress therefore abolished or greatly attenuated the LTS-resistant phenotypes of these two cultivars.

In contrast to the other clones, Payette Russet tubers maintained the lowest reducing sugar concentrations and lightest fry colors (USDA 0) regardless of in-season heat treatment when challenged with LTS for 30 d (Fig. 4, Table 3). Conversely, Payette Russet tubers built up higher sucrose concentrations than the other clones by 30 d of LTS for most of the in-season treatments. The lower reducing sugar, lighter fry color and higher sucrose content of Payette

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Russet tubers following 30-d storage at 4 oC indicate superior tolerance to heat stress for retention of LTS-resistant phenotype when compared with POR06V12-3 and A02138-2 and suggests that this heat tolerance may involve inhibition of cold-induced sucrose inversion, a possibility evaluated further in the PHHS studies described below.

Dormancy break and sprout growth

Stage and duration of heat affected the length of dormancy of Ranger Russet and Payette

Russet tubers when stored at 9 oC (95% RH). Non-heat treated Ranger Russet tubers grown at ambient soil temperature (19 oC) emerged from dormancy (days to 2.5-mm sprouts) approximately 56 days after harvest (DAH) compared with 112 days for Payette Russet tubers, characterizing intrinsic differences in dormancy length of these two cultivars (Fig. 5ab). Heat

(27 oC) during maturation of Ranger tubers accelerated emergence from dormancy regardless of duration (20 or 40 d) but the subsequent rate of sprout elongation was greatest for tubers from the 40-d maturation treatment (Fig. 5a). Heat during tuber bulking had little effect on emergence from dormancy of Ranger Russet tubers. By contrast, Payette Russet tubers from the 27 oC, 40-d bulking treatment and 20- and 40-d maturation treatments sprouted considerably earlier than non-heat stressed (19 oC) tubers (Fig. 5b). However, bulking for 20 d at 27 oC had no effect on dormancy break and subsequent sprout growth of Payette Russet tubers. Total sprout fresh weight per tuber at 65 d (Ranger) and 165 d (Payette) of storage was equivalent for tubers from all treatments except those exposed to 27 oC for 40 d during maturation (Fig. 5cd). Tubers from this latter treatment produced the greatest final sprout fresh weights in accordance with the earliest dormancy break from these tubers. Moreover, 40 days of heat during maturation also significantly reduced apical dominance in both cultivars, as evidenced by an increase in the number of eyes with sprouts and multiple sprouts within each eye (data not shown).

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PHHS studies

The PHHS protocol (Zommick et al 2014) was evaluated as a screening tool for heat stress tolerance in LTS resistant cultivars/clones. The LTS responses of heat-primed (21 d,

32 oC) tubers of Russet Burbank, Ranger Russet (both LTS-susceptible) and the LTS-resistant cultivars/clones, Sage Russet, POR06V12-3, A02138-2, Payette Russet, EGA090702-2, Innate

Russet Burbank W8 (Table 1) and GemStar Russet (data not shown) were compared. As expected, all cultivars/clones produced light (USDA 0, average 42 reflectance units) and uniform fry color (apical to basal end) following an initial 16 d of wound healing (9 oC control, Fig. 6ab) and extending through the entire 69-d storage period at 9 oC (data not shown). However, consistent with their higher reducing sugar concentrations (Fig. 7a), Russet Burbank, Ranger

Russet and Sage Russet tubers produced significantly darker fries than the other clones following harvest (Fig. 6b). Process quality (basal fry color) improved slightly (lightened 16%) for Russet

Burbank and Ranger Russet tubers in response to heat priming (21 d at 32 oC) but decreased slightly (darkened 13% but remained USDA 0) for five of the six LTS-resistant cultivars/clones for which fry color data for the heat-primed tubers was measured (Fig. 6b; no data for Sage

Russet). Heat priming had no effect on reducing sugar concentrations except in Russet Burbank tubers where reducing sugars declined 62% in response to the heat treatment (Fig. 7a). However, heat priming significantly increased tuber sucrose concentrations in six of eight cultivars/clones

(Fig. 7b). Russet Burbank and Ranger Russet tubers accumulated 5.7- and 2.4-fold more reducing sugars during LTS (32 d at 4 oC) than the average of POR06V12-3, A02138-2, Payette

Russet, EGA09702-2 and Innate Russet Burbank (W8) tubers, resulting in significantly darker

(USDA 2-3) fries and demonstrating the robust LTS-resistant phenotypes of the latter five clones/cultivars ( P<0.05, Fig. 7a, Fig. 6ab). The reducing sugar concentration of Sage Russet

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tubers, however, was equal to Ranger Russet tubers following 32 d at 4 oC (Fig. 7a) but fry color remained 17% lighter (USDA 1) (Fig. 6ab). Importantly, heat priming substantially increased the sensitivity of Russet Burbank, Ranger Russet, Sage Russet, POR06V12-3, A02138-2 (Fig.

7a) and GemStar Russet (data not shown) tubers to cold sweetening. The reducing sugar concentrations of heat-primed tubers increased 1.2- (R. Burbank), 2.9- (Ranger), 1.8- (Sage), 2.3-

(POR06V12-3), 8.3- (A02138-2) and 1.9-fold (GemStar Russet) during LTS over the respective non-heat stressed cold-stored tubers, with associated loss of process color (USDA 3-4 fry color,

Fig. 6). Heat stress thus exacerbated LTS and loss of process quality in the LTS susceptible cultivars, Russet Burbank and Ranger Russet, and extinguished the LTS resistant phenotypes of

Sage Russet, POR06V12-3 and A02138-2 (Figs. 7a and 6). The LTS resistance of these latter three cultivars/clones can thus be classified as heat labile (non-heat tolerant) for retention of LTS resistance (Fig. 6a). By contrast, during LTS, heat-primed Payette Russet, EGA09702-2 and

Innate Russet Burbank (W8) tubers accumulated significantly lower concentrations of reducing sugars (Fig. 7a) but much higher levels of sucrose (Fig. 7b) than all other cultivars/clones. The buildup of sucrose rather than reducing sugars allowed heat-primed tubers of these clones to maintain acceptably light-colored fries (USDA 0-1) during LTS (Fig. 6). Payette Russet,

EGA09702-2 and Innate Russet Burbank (W8) were therefore classified as heat tolerant for retention of their LTS-resistant phenotypes. Moreover, consistent with the LTS responses of

Payette Russet to in-season heat stress, the PHHS data further suggests that heat tolerance may involve invertase.

Invertase and starch phosphorylase activities

Changes in basal (endogenous inhibitor present) and total (endogenous inhibitor absent) invertase activities in response to the PHHS treatments depended on cultivar/clone. While total

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invertase activity (Fig. 8b) was higher than basal activity (Fig. 8a), the treatment-induced trends for each cultivar/clone were identical regardless of the presence of endogenous inhibitor. At

9oC, the LTS-susceptible cultivars (Ranger Russet and Russet Burbank) had 7.9 and 10.6-fold higher basal invertase activities, respectively, than the average of the three LTS-resistant clones

(A02138-2, Payette Russet, Innate Russet Burbank W8). Heat stress alone had no effect on invertase activity. However, activities increased markedly during 32 d of LTS at 4 oC (CS) in

Ranger Russet, Russet Burbank and A02138-2 tubers, but not in Payette Russet and Innate

Russet Burbank (W8) tubers, which maintained the lowest activities across all storage temperature treatments. Heat stress prior to CS resulted in lower invertase activity in Ranger

Russet tubers relative to CS alone, but increased the invertase activity in A02138-2 tubers.

Relative to the control non-stressed tubers (9 oC), basal invertase activities of heat-primed tubers of Ranger, Payette Russet, Russet Burbank and Innate Russet Burbank (W8) did not increase when subjected to LTS (HS+CS). The lack of induction of invertase by CS or HS+CS in Payette

Russet and Innate Russet Burbank (W8) tubers correlates well with the significant increases in sucrose accumulation induced by these treatments (Fig. 7b).

Relative changes in activities of the plastidic and cytosolic isoforms of α-1,4-glucan phosphorylase in response to the PHHS treatments were assessed in Russet Burbank, Payette

Russet, and Innate Russet Burbank (W8) tubers. While treatments had no discernable effect on activities of the cytosolic isoform (data not shown), activities of the plastidic isoform depended on PHHS treatment and cultivar. Regardless of storage treatment, activity was not detected in

Innate Russet Burbank tubers due to gene silencing (Clark et al. 2014) (Fig. 9). By contrast, activity was relatively low but detectable in non-heat stressed Russet Burbank tubers stored at

9oC (control) and was not affected by heat stress (21 d at 32 oC) alone. While Russet Burbank

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tubers subjected to LTS at 4 oC for 32 d (CS) showed a marginal increase in activity, activity was greatly stimulated if tubers were first heat-stressed and then subjected to LTS (HS+CS). Heat- priming and LTS treatments alone had no effect on plastidic starch phosphorylase activities in

Payette Russet tubers, which were barely detectable and lower than the activities observed for

Russet Burbank tubers. However, similar to Russet Burbank, activity increased substantially when heat stressed Payette Russet tubers were subsequently stored at 4 oC, which correlates well with the buildup in sucrose (Fig. 7b) and relatively low invertase activity (Fig. 8) and reducing sugar concentration (Fig. 7a) observed in these tubers in response to the combined HS+CS treatment. qPCR analysis of invertase and invertase inhibitors

Changes in the expression of vacuolar acid invertase and apoplastic (Inh1) and vacuolar

(Inh2) invertase inhibitor genes in response to LTS (CS) of non-heat stressed and heat stressed tubers were compared for Russet Burbank, A02138-2, Innate Russet Burbank (W8) and Payette

Russet tubers. Cold storage and HS prior to CS (HS+CS) treatments had no effect on expression of invertase in Russet Burbank and Innate (W8) tubers (Fig. 10). However, invertase expression increased significantly ( P<0.05) in heat-primed tubers of A02138-2 in response to

LTS (Fig. 10), which concurred with the increased invertase activity (Fig. 8) and higher reducing sugar concentration observed in these tubers (Fig. 7a). Interestingly, CS alone increased invertase expression in Payette Russet tubers; however, this response did not translate to increased invertase activity (Fig. 8), resulting in very low buildup in reducing sugars (Fig. 7a) and no change in sucrose levels (Fig. 7b).

Expression of apoplastic invertase inhibitor (Inh1) was substantially higher in Innate

W8 tubers than the other clones and increased when heat-primed tubers were subjected to LTS at

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4oC (HS+CS) ( P<0.05; Fig. 10). Similarly, Russet Burbank tubers exposed to the combined

(HS+CS) treatments showed a significant (average 3.4-fold) increase in Inh1 expression relative to tubers stored at 9 oC or subjected to CS (32 d at 4 oC) alone ( P<0.05). The combined HS+CS treatment decreased expression of Inh1 in Payette Russet tubers. Inh1 expression in A02138-2 tubers, however, was highest in tubers subjected to CS alone followed by HS+CS and tubers stored at 9 oC.

The expression levels of vacuolar invertase inhibitor (Inh2) were 100-fold lower than invertase and Inh1. Inh2 expression was unaffected by heat and/or cold stress in A02138-2 and

Innate W8 tubers (Fig. 10). In contrast, expression of Inh2 in Russet Burbank tubers was 1.6- and 2.3-fold less in tubers subjected to the CS and CS+HS treatments, respectively, than in tubers stored at 9 oC. Payette tubers subjected to CS and HS+CS also had reduced expression of

Inh2 relative to tubers stored at 9 oC.

Discussion

Zommick et al. (2014) showed that increased soil temperatures effectively potentiated cold sweetening in the LTS-susceptible cultivar, Ranger Russet, and the LTS-resistant cultivar and clone, Premier Russet and AO02183-2, respectively. Moreover, tubers of Premier Russet and AO02183-2 varied slightly in their sensitivity to the high temperature treatments for loss of

LTS-resistant phenotype, suggesting inherent differences in tolerance to in-season heat stress for preservation of this trait. Similar to heat stress applied in-season, storage of tubers for 21 d at

32 oC (heat priming) directly following harvest (PHHS) exacerbated LTS in Ranger Russet and abolished the intrinsic resistance of Premier Russet tubers to subsequent cold sweetening

(Zommick et al. 2014). These data suggested that both the in-season and PHHS protocols may

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have potential for screening cultivars/clones for tolerance to heat stress for retention of LTS- resistant phenotype. The metabolic basis for tolerance to heat stress for retention of cold sweetening resistance is not known. Accordingly, the studies reported herein screened up to nine cultivars/clones with varying degrees of cold sweetening resistance to determine: (1) sensitivity to different durations of high soil temperature applied during bulking and maturation for retention of LTS-resistant phenotype, (2) efficacy of the PHHS treatment protocol for identifying

LTS-resistant clones with heat tolerance, and (3) the mechanism of tolerance to heat stress for retention of the cold sweetening resistant trait.

Soil warming cables installed in-furrow increased soil and thus tuber pulp temperatures by 8 oC above ambient (19 oC) for 20 and 40 days during bulking and maturation of Ranger

Russet (LTS-susceptible), POR06V12-3, A02138-2 and Payette Russet (LTS-resistant clones) tubers. The tubers were then screened for retention of LTS-resistant phenotype over a 30-d storage period at 4 oC. While ambient soil temperatures in many areas of the Columbia Basin of

WA and OR often reach and even exceed 27 oC for short periods during the growing season (U.S.

Dept. of Interior, Bureau of Reclamation, http://www.usbr.gov/pn/agrimet/graphs.html ), such temperatures are not sustained for the durations imposed here. The in-season high-heat treatments therefore constituted a relatively severe stress for evaluating effects on LTS phenotypes.

The yield responses to in-season heat stress varied with duration and timing. High soil temperature for 40 days, in general, was more detrimental to total and marketable yields than 20 days and this effect was consistent for all cultivars. Moreover, yields and tuber number per plant were substantially lower when heat was applied during tuber maturation compared with bulking.

The lower yields may have been due to a combination of heat-induced tuber resorption early in

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the maturation period while plants were actively bulking, coupled with increased tuber decay later while tubers matured in warm soil under senescing or fully-senesced vines. Resorption of tubers to maintain canopy development, plant water relations and provide substrate to support high rates of respiration has been linked to physiological disorders like heat necrosis, internal brown spot and hollow heart (Werner 1936; Larsen and Albert 1945; Friedman 1955; Wolcott and Ellis 1959; Crumbly et al. 1973; Hiller et al 1985). Reabsorption of tubers during periods of environmental stress is symptomatic of altered carbohydrate metabolism (Levy and Veilleux

2007; Hancock et al. 2014), resulting in a shift in plant source/sink relationships. That is, tubers assume the role of ‘source’ to help maintain foliar (canopy) growth (Yamaguchi et al. 1964;

Crumbly et al. 1973; Rykaczewska 2015). Increased tuber decay was observed during harvest of the high heat, 40-d maturation plots and was likely a contributor to the lower tuber number and yields relative to the other treatments.

While trends in yield responses to in-season heat treatments were similar among all clones, the heat-induced changes in specific gravity of Payette Russet tubers differed from the other clones. Heat in general decreased the overall gravity of Payette tubers uniformly (19 vs

27 oC, P<0.01), regardless of stage (early vs late, ns) and duration (20 vs 40 d, ns) (stage x duration, ns). By contrast, the heat-induced decreases in tuber gravities for the remaining cultivars and clones depended on stage and duration (Supplemental Table). Further work is needed to determine whether these differences somehow relate to the more robust heat tolerant phenotype of Payette Russet.

While evaluation of the effects of increased soil temperature on plant productivity provides insight into cultivar-dependent tolerance to in-season heat stress, the overall goal was to screen cultivars/clones to potentially identify any with heat tolerance for retention of LTS-

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resistant phenotype. To facilitate these studies, morphologically indistinguishable tubers of each cultivar/clone were sampled from each of the in-season heat treatments to evaluate effects on dormancy, process quality, LTS phenotype and associated sweetening metabolism during storage. All cultivars produced acceptably light colored fries (USDA 0-1) regardless of treatment when stored at 9 oC through wound-healing (Table 3) and the entire 44-d storage period

(data not shown). However, prior to storage at 4 oC, tubers grown at elevated soil temperatures averaged higher levels of reducing sugars and sucrose and lower specific gravity and dry matter relative to non-heat stressed tubers, which collectively reflects the inhibition of starch synthesis in tubers during periods of heat stress (Krauss and Marshner 1984; Mohabir and John 1988;

Geigenberger et al. 1998; Zommick et al 2014). Dropping the storage temperature from 9 to 4 oC to invoke LTS induced declines in process quality coincident with increases in reducing sugars and sucrose in control and heat-stressed tubers and the extent of these changes was clone- dependent (Table 3, Fig. 4). Relative to the other cultivars/clones, Payette Russet tubers built up significantly higher levels of sucrose, lower reducing sugars and maintained the lightest (most optimal) process color (USDA 0) regardless of exposure to elevated soil temperature. Payette

Russet thus tolerated in-season heat stress for retention of LTS-resistant phenotype and process quality better than the other LTS-resistant clones, POR06V12-3 and A02138-2. These results suggested that heat tolerance may involve invertase and spurred additional screening studies using a PHHS protocol (Zommick et al. 2014) to characterize the metabolic basis of tolerance to heat stress for retention of LTS-resistant phenotype in Payette Russet.

The PHHS studies examined the effects of heat stress on subsequent LTS of nine cultivars/clones with LTS susceptible and resistant phenotypes. Heat stress prior to cold storage exacerbated cold-induced sweetening of the LTS-susceptible cultivars, Russet Burbank and

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Ranger Russet, resulting in high reducing sugar concentrations and unacceptable dark colored fries (Figs. 6 and 7). Consistent with their LTS-resistant phenotypes (Table 1), non-heat stressed tubers of Sage Russet, POR06V12-3, A02138-2 and GemStar Russet (data not shown) maintained relatively low reducing sugar concentrations and produced light colored fries when stored for 32 d at 4oC. However, heat stress prior to cold storage abolished the LTS-resistance of these cultivars/clones, resulting in significant deterioration of process quality. In contrast, heat- stressed tubers of Payette Russet and its maternal parent, EGA09702-2, maintained their LTS- resistant phenotypes, producing light color fries in response to the HS+CS treatments. This was not the case for Payette’s paternal parent, GemStar Russet, which as described above lost its inherent ability to resist LTS when subjected to heat stress. Therefore, it is likely that Payette

Russet inherited its robust tolerance to heat stress from EGA09702-2.

The tolerance of Payette (and likely EGA09702-2) to heat for retention of LTS-resistant phenotype and thus process quality (Fig. 6) appears to be conferred by reduced sensitivity of invertase to cold induction (Fig. 8), resulting in the buildup of sucrose in heat-primed tubers during cold-sweetening (Fig. 7). Indeed, heat-primed tubers of Innate Russet Burbank (W8) tubers in which acid invertase has been silenced (Clark et al. 2014) displayed a sucrose- accumulating/low reducing-sugar phenotype similar to Payette Russet tubers during LTS (Figs.

7, Table 4). The total and basal activities of acid invertase in Payette Russet tubers remained low and comparable to the activities observed in Innate Russet Burbank (W8) tubers regardless of

PHHS treatment (Fig. 8, Table 4). However, despite the low invertase activities of these clones, distinct differences in invertase expression levels were observed. Innate Russet Burbank (W8) tubers, as anticipated, had little detectable expression of acid invertase, consistent with RNAi silencing (Clark et al. 2014). Payette Russet on the other hand readily expressed the vacuolar

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acid invertase gene which was cold-inducible (Fig. 10), but the increased transcript did not translate to protein functionality and invertase activity remained low and unaffected by the

PHHS treatments relative to the LTS-susceptible but heat-labile tubers of A02138-2 (Fig. 8).

These data indicate inherent differences in the regulation of invertase activity in heat tolerant versus non-heat tolerant clones for retention of LTS-resistant phenotype. The disconnect between invertase gene expression and activity in Payette is not surprising given the many examples of incongruence between expression and enzyme function throughout the literature in general, and more specifically, work by Ou et al. (2013) demonstrating weak correlation between transcript levels and acid invertase activities across eight potato genotypes. The insensitivity of invertase in Payette Russet tubers to PHHS treatments suggests some form of posttranscriptional regulation affecting invertase protein synthesis/function (Pressey and Shaw 1966; McKenzie et al 2005; Brumell et al. 2011; Tauzin et al. 2014). The mechanism by which Payette Russet tubers maintain low activity of invertase in response to heat stress and cold storage is key to the heat tolerance of this cultivar and warrants further investigation.

Heat priming alone had no effect on buildup in reducing sugars in any of the clones; however, consistent with results shown by Zommick et al. (2014), it significantly increased the concentration of sucrose in nearly all clones (Fig. 7, Table 4), demonstrating heat-induced effects on tuber carbohydrate metabolism. The increases in sucrose and/or reducing sugars in response to PHHS treatments reflect increased starch catabolism. Interestingly, heat priming greatly increased the activity of plastidic starch phosphorylase during storage of Russet Burbank and

Payette Russet tubers at 4 oC (Fig. 9), which correlated with the substantial increases in sucrose and/or reducing sugars induced by this treatment. By contrast, starch phosphorylase activity was non-detectable in Russet Burbank Innate (W8) tubers regardless of PHHS treatment. These

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data demonstrate plasticity in the pathways that mediate starch catabolism in response to heat stress, cold storage and their combination. While starch catabolism can be mediated both hydrolytically (e.g., α- and β-amylases) and phosphorolytically (Smith et al. 2005; Bethke 2013), the phosphorolytic pathway is clearly not necessary, as evidenced by the cold-induced buildup of sucrose in Russet Burbank Innate (W8) tubers (Figs. 7 and 9).

The LTS-resistant clones, Sage Russet, POR06V12-3, A02138-2, GemStar Russet,

Payette Russet and EGA09702-2, resist starch degradation during cold storage, which is reflected in their abilities to maintain low reducing sugar and sucrose concentrations and thus light color fries. Differences in susceptibility to LTS have been linked to the regulation of UDP-glucose pyrophosphorylase (Zrenner et al. 1993; Sowokinos 2001b; McKenzie et al. 2005), acid invertase (Pressey and Shaw 1966; Richardson et al. 1990; Zrenner et al. 1993; McKenzie et al

2013; Zhu et al 2014), sucrose-phosphate-synthase (Hill et al. 1996; Reimholz et al. 1997) and β- amylase (Nielsen et al. 1997, Reimholz et al. 1997), key enzymes in carbohydrate metabolism.

Clones which exhibit high resistance to LTS potentially have lower activities of one or many of the enzymes that mediate starch and/or sucrose catabolism (McKenzie et al. 2005; 2013). Starch catabolism seemingly increases in response to heat priming, as evidenced by increases in sucrose levels (Fig. 7b). The cold induction of acid invertase (Bethke 2013, Zommick et al 2014;

Herman et al. 2016) then facilitates inversion of sucrose to reducing sugars during subsequent cold storage of LTS-resistant but heat susceptible clones. The temperature-insensitive and low- invertase activities of Payette Russet and Innate (W8) tubers (Fig. 8) prevents this inversion

(Fig. 7b), underscoring the importance of maintaining a low level of acid invertase to confer tolerance to heat stress for retention of the LTS-resistant phenotype. Similarly, RNAi silencing of acid invertase in Ranger Russet and Russet Burbank tubers minimized sugar end development

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(Zhu et al. 2014), a common defect that manifests in response to heat and other environmental stresses (Thompson et al. 2008).

Increased activity of starch phosphorylase in the leaves of Arabidopsis has been linked to improved tolerance to abiotic stress (Zeeman et al. 2004). Researchers postulated that increases in phosphorolytic starch degradation in leaves provided additional substrate to fuel the oxidative pentose phosphate pathway (OPPP), which generates reducing equivalents to mitigate stress- induced increases in reactive oxygen species (ROS) (Smirnoff 1993; Mittler 2002; Zeeman et al.

2004). Respiration is a major source of ROS in plants. Blauer et al. (2013) demonstrated with seed-tubers that heat priming (21 d at 30 oC) permanently increased tuber respiration rate, which would contribute to increased oxidative stress. Hence, the increased activity of plastidic starch phosphorylase in Russet Burbank and Payette Russet tubers could potentially reflect an increased need for reducing power to catabolize ROS in the heat-stressed/cold-stored tubers.

In conclusion, we demonstrate that in-season and/or postharvest heat stress can undermine resistance to LTS in many conventionally bred clones/cultivars. Heat stress exacerbated the LTS-susceptibility of Ranger Russet and Russet Burbank and abolished the LTS- resistance in Sage Russet, GemStar Russet, POR06V12-3 and A02138-2 by stimulating starch catabolism and increasing the available substrate (sucrose) for inversion to reducing sugars during storage at 4 oC. Heat stressed tubers of Payette Russet and Russet Burbank Innate (W8) retained their LTS-resistance by maintaining low invertase activity. However, the total sugar

(sucrose + Glc + Fru) build up (i.e. hexose equivalents) in these clones was comparable with that from the LTS susceptible and resistant cultivars/clones that had no tolerance to heat stress.

Payette Russet’s tolerance to heat stress (and likely EGA09702-2) is conferred by the ability to maintain low invertase activity during cold storage (similar to the silenced activity of RB Innate

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W8 tubers). The mechanism by which invertase activity resists cold induction following heat stress in Payette Russet warrants further investigation.

Acknowledgements

We gratefully acknowledge financial support from the USDA-ARS, Northwest Potato

Research Consortium and the Washington State Potato Commission. We thank the J.R. Simplot

Company, Boise, ID for providing Innate Russet Burbank (W8) seed potatoes and Dr. Richard

G. Novy (USDA-ARS, Aberdeen, ID) for providing tubers of EGA09702-2.

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Table 1. Low-temperature sweetening (LTS) susceptible (S) and resistant (R) cultivars and clones screened for tolerance to in-season and postharvest heat stress for effects on LTS phenotype.

Parentage LTS Cultivar/clone Seed source maternal paternal phenotype Reference a Ranger Russet commercial Butte A6595-3 S Pavek et al. 1992 Russet commercial Sport mutation of Burbank S Herman et al. 2016a Burbank Knowles and Pavek Sage Russet commercial A89384-10 A91194-4 R 2007 136 GemStar commercial Gem Russet A8341-5 R Love et al. 2006 Russet EGA09702- GemStar Payette Russet NWVDP R Novy et al. 2016 2 Russet Pavek and Knowles POR06V12-3 NWVDP PA00V6-4 PA01N22-1 R 2016 Knowles and Pavek A02138-2 NWVDP A96563-8 Premier Russet R 2013 EGA09702-2 NWVDP PS 1679 PW 375 R Novy et al. 2016 J.R. Simplot Innate (W8) Transformed Russet Burbank R Clark et al. 2014 Co.

aReference is to LTS phenotype

Table 2. Effects of duration of high soil temperature during bulking (80 -120 DAP) and maturation (120-160 DAP) on yield, average tuber weight, specific gravity and tuber dry matter. Data are averaged across Ranger Russet, POR06V12-3, A02138-2 and Payette Russet (see supplementary table for clone specific data). Soil temperatures averaged 8 oC above ambient (19 oC) for 20 or 40 days during the indicated growth stages before return to ambient temperature. Tubers were harvested 169 DAP. Letters indicate mean separation within a column (LSD, P<0.05). ANOVA results ( P levels) for the effects of growth stage, duration of elevated temperature, and their interaction are summarized beneath the table.

Yield (MT/ha) Duration Soil Temp Tubers Specific % Dry Heat On (days) (oC) plant -1 Total Mkt #1 g tuber -1 gravity Matter

N/A - 19 6.9 b 64.9 a 63.9 a 228 a 1.088 a 24.2 a 80 DAP 20 27 8.2 a 56.8 b 52.6 b 157 c 1.073 c 20.1 c 40 27 7.1 b 42.7 c 39.6 c 136 d 1.063 d 18.8 d 120 DAP 20 27 5.4 c 41.6 c 40.8 c 185 b 1.079 b 22.8 b 40 27 4.4 d 30.5 d 26.1 d 143 cd 1.081 b 22.6 b

LSD 0.05 0.78 6.7 6.10 20.1 0.004 1.0 19 oC (ambient) vs Heat 0.05 0.001 0.001 0.001 0.001 0.001 Stage (80 vs 120 DAP) 0.001 0.001 0.001 0.05 0.001 0.001 Duration (20 d vs 40 d) 0.001 0.001 0.001 0.001 0.01 0.05 Stage x Duration NS NS NS NS 0.001 NS

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Table 4 . Significance levels ( P-values) for planned comparisons among LTS- susceptible and resistant cultivars/clones in response to postharvest heat stress (PHHS) treatments. Ranger Russet (RR), Russet Burbank (RB), POR06V12-3 (V12), A02138-2 (138), Payette Russet (PR) and Innate Russet Burbank (W8) tubers were either stored at 9 oC (control), heat-primed for 21 days at 32 oC (HS), cold sweetened for 32 days at 4 oC (CS) or subjected to the combination treatment (HS+CS). Effects of HS and CS treatments on changes in process quality (French fry color, basal photovolt), tuber sugar concentrations and invertase activities relative to control tubers were then compared. See Figs. 6, 7 and 8 for process quality, sugar concentrations and invertase data.

Basal Reducing Invertase b Comparisons Photovolt Sugars Sucrose (+inh) (-inh) Cultivar (CV) (9 oC) RR RB/V12 138 0.001 a 0.001 0.05 0.01 0.001 RR RB/PR W8 0.001 0.001 0.05 0.01 0.001 V12 138/PR W8 0.05 NS 0.001 NS NS PR/W8 NS NS 0.001 NS NS 9oC vs HS 0.05 NS 0.001 NS NS 9oC vs CS 0.001 0.001 0.01 0.001 0.001 9oC vs HS+CS 0.001 0.001 0.001 0.01 0.001 CV x 9 oC vs HS RR RB/V12 138 0.001 0.01 NS NS NS RR RB/PR W8 0.001 0.05 NS NS NS V12 138/PR W8 NS NS NS NS NS PR/W8 NS NS NS NS NS CV x 9 oC vs CS RR RB/V12 138 NS NS NS 0.001 0.001 RR RB/PR W8 0.05 0.01 NS 0.001 0.001 V12 138/PR W8 NS NS 0.05 NS 0.01 PR/W8 NS NS 0.05 NS NS CV x 9 oC vs HS+CS RR RB/V12 138 0.001 NS NS NS 0.01 RR RB/PR W8 NS 0.001 0.001 NS NS V12 138/PR W8 0.001 0.001 0.001 0.01 0.001 PR/W8 NS NS 0.05 NS NS aLevels of significance (P values) for the indicated comparisons; NS , not significant. bInvertase activities were assessed for RR, RB, 138, PR and W8 only (V12 excluded).

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Fig. 1. (a) Two soil warming cables (61 m in length) were installed in furrow to increase soil temperature during the bulking and maturation phases of tuber development. Seed pieces were spaced 25.4 cm apart and 20.3 cm deep after hilling. Hill cross-sectional temperature profiles for control (ambient, 19 oC) (b) and high temperature (27oC) rows (c) during tuber bulking were measured on July 23, 2014.

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tuberization early bulkinglate bulking maturation harvest a 44 Early HS o 40 Ambient 19 C (80 DAP) 20 days 27 oC 36 Heat off Heat off 40 days 27 oC 32 C) o 28 24 20 16 Temperature( 12 Heat on 8 4

0 Jun 15 Jul 3 Jul 23 Aug 12 Sep 1 Sep 21 60 70 80 90 100 110 120 130 140 150 160 b 44 Late HS 40 (120 DAP) o 36 Ambient 19 C Heat off Heat off 20 days 27 oC 32 o 40 days at 27 C C) o 28 24 20 16 Temperature( 12 Heat on 8 4 0 Jun 15 Jul 3 Jul 23 12 Aug Sep 1 Sep 21 60 70 80 90 100 110 120 130 140 150 160 Days After Planting

Fig. 2. Soil temperature profiles of control (ambient, 19 oC) and heated rows from 57-165 DAP in Othello, WA (2014 growing season). Average soil temperatures during the bulking (a) (80-

100 and 80-120 DAP) and maturation phases (b) (120-140 and 120-160 DAP) of tuber development were 27oC.

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Fig. 3. Polygonal plots depicting the shift in tuber size distribution induced by a +8 oC increase in soil temperature (19 vs. 27 oC) for 0, 20 or 40 days during the bulking (a) and maturation (b) phases of tuber development (average of 4 cultivars/clones). The yields of <113-g, 113-170-g,

170-284-g, 284-340-g, 340-397-g and >397-g U.S. No. 1 tubers are plotted as percent marketable yield on each axis. Marketable yields (Mkt yld), tuber numbers and average tuber fresh weights are compared in the inset table (letters indicate mean separation by LSD, P<0.05). Yields with factorial ANOVA results are presented in Table 2.

142

< 113 g a Bulking 40 (80 -120 DAP) 35 % Yield 30 25 >397 g 113-170 g 20 15 10 HS (days at 27 oC) 0 20 40 5 Mkt Yield (MT ha -1) 63.9 a 52.6 b 39.6 c 0 Tubers plant -1 6.9 b 8.2 a 7.1 b g tuber -1 228 a 157 c 136 d

340-397 g 170-284 g

284-340 g < 113 g b Maturation 40 (120 -160 DAP) 35 % Yield 30 25 >397 g 113-170 g 20 15 10 HS (days at 27 oC) 5 0 20 40 Mkt Yield (MT ha -1) 63.9 a 40.8 c 26.1 d 0 Tubers plant -1 6.9 b 5.4 c 4.4 d g tuber -1 228 a 185 b 143 cd

340-397 g 170-284 g

284-340 g

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Fig. 4. (a) Changes in process quality (basal end French fry color), (b) concentrations of reducing sugars (glc + fru) and (c) sucrose during storage of Ranger Russet, POR06V12-3,

A02138-2 and Payette Russet tubers at 4 oC as affected by 20 or 40 days of heat stress (+8oC soil temperature) during the bulking (80-100 and 80-120 DAP) or maturation (120-140 and 120-160

DAP) phases of tuber development. Data are plotted ±SE. Color shading indicates the USDA color ratings (0-4) of French fries. ANOVA results comparing the effects of soil treatments on fry color, sucrose and reducing sugar concentrations at zero and 30 days of LTS are given in

Table 3.

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Ranger Russet POR06V12-3 A02138-2 Payette Russet 55 55 55 55

50 19 oC Ambient 50 50 50 80 DAP-20 d 80 DAP-40 d 45 27 oC 45 45 45 120 DAP-20 d 0 40 120 DAP-40 d 40 40 40

35 35 35 35 30 30 30 30 1 25 25 25 25 USDA color rating 20 20 20 20 2 3 Stem end fry color (Photovolt ref) (Photovolt color fry end Stem 15 15 15 15 4 10 10 10 10 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 50 50 50 50 45 45 45 45 40 40 40 40 35 35 35 35 DW) -1 14 30 30 30 30

5 25 25 25

25 20 20 20 20 15 15 15 15 Glc + Fru (mgg Fru + Glc 10 10 10 10 5 5 5 5 0 0 0 0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 40 40 40 40

35 35 35 35

30 30 30 30 DW)

-1 25 25 25 25

20 20 20 20

15 15 15 15

Sucrose (mg g (mg Sucrose 10 10 10 10

5 5 5 5

0 0 0 0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 Days at 4 oC Days at 4 oC Days at 4 oC Days at 4 oC

a 45 b 45 Ranger Russet Payette Russet 40 40 35 19 oC Ambient 35 80 DAP-20 d 30 27 oC 80 DAP-40 d 30 120 DAP-20 d 25 120 DAP-40 d 25 20 20 15 15 10 10 Longest Sprout (mm) LongestSprout (mm) LongestSprout 5 5 0 0

0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 100 120 140 160 o Days After Harvest (9 oC) Days After Harvest (9 C) c 4.0 d 4.0 Ranger Russet a Payette Russet o o 3.5 (87 DAH 9 C) (165 DAH 9 C) 3.5 ) ) -1 3.0 3.0 -1 a 2.5 2.5 b 2.0 2.0 bc 1.5 bc b 1.5 b 1.0 c b 1.0

Sprout WeightSprout tuber (g b Weight (g Sprout tuber 0.5 0.5

0.0 0.0 C d d 9C 0 d 1 -40 d -4 -20 -40 d -20 nt P P P P P A A A A A D D D D mbie 0 DAP-20 d 0 A 8 80 20 D 80 8 20 120 DAP-201 d Ambient 19 1 120 DAP-40 d

Fig. 5. Effects of duration (20 vs. 40 d) of elevated soil temperature (+8oC above ambient) during tuber bulking (80-100 and 80-120 DAP) and maturation (120-140 and 120-160 DAP) on emergence from dormancy and sprout growth from Ranger Russet (a, c) and Payette Russet (b, d) tubers stored at 9oC (95% RH). Each point represents the average of 20 tubers ±SE. Final sprout fresh weights (c, d) were determined at 87 and 165 DAH for Ranger Russet and Payette

Russet, respectively. Letters indicate mean separation by LSD (P<0.05).

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Fig. 6. (a) Changes in process quality (color) of French fry planks from LTS-susceptible and resistant cultivars/clones as affected by storage for 32 d at 4 oC (CS) or the combination of HS

(21 days at 32 oC) plus CS. Control tubers were stored at 9 oC. Fry planks are oriented with the stem (basal) end down. The four fry planks for each treatment are from different tubers and represent the average color observed from a 12-tuber sample. Numbers on fries depict USDA color ratings for the average basal Photovolt reflectance value. (b) Changes in basal end

Photovolt reflectance values (fry color) as affected by the PHHS treatments (n=12, ±SE). Letters indicate LSD (P<0.05) for comparison across PHHS treatments and cultivars.

147

148

a b 35 a a o 0.05 70 a 0.05 9 C 9oC HS a HS 30 CS 60 a HS+CS CS HS+CS dry wt) dry a -1 25 50 a dry wt) dry a 20 a -1 40 a b a b b 15 a 30 b b b b a a 10 b 20 b a a

cc b g (mg Sucrose

c c ab c 149 c b ab a b a a 5 10 c a c Reducing Sugars (mg (mg g Sugars Reducing c a c c b b d b b b b b b b b c bc c c b c c c c 0 0 t t t t t t k 3 2 2 8 k 3 2 2 8 n e e - - e - n e e - - e - s s s s s s a 2 8 2 W a 2 8 2 W s s 1 3 s 0 s s 1 3 s 0 b u u u e b u u u e r V 1 7 t r V 1 7 t u R R 2 R 9 u R R 2 R 9 6 a 6 a 0 0 0 0 B r e 0 e n B r e 0 e n t t t e g R A t A n t e g R A t A n e g I e g I a O e G a O e G s n y s n y s S E s S E a P a a P a u u R P R P R R

Fig. 7. Changes in (a) reducing sugar (glc + fru) and (b) sucrose concentrations of LTS-susceptible and resistant cultivars/clones as

affected by PHHS treatments (see Fig. 6). Each point represents the average of 12 tubers (n=4, ±SE). Letters indicate LSD (P<0.05)

for comparison of PHHS treatments within a cultivar.

25 a a 0.05 o Inhibitor Present 9 C 20 HS CS

a HS+CS protein) -1 15 mg -1

10 a

b Invertase Activity Invertase a ab

(nmol sucrose h 5 bc b b b a c c a a a c a a a 0 a

b 25 a 0.05 a 9oC Inhibitor Absent 20 HS CS HS+CS protein) -1 15 mg

-1 a b

10 ab a b b Invertase Activity Invertase bc b 5 c (nmol h sucrose

c a c ab ab a a a 0 b a t t 2 k 8 e - e n s s 8 a W s 3 s u u b e 1 r t R 2 R u a r 0 B e n e t A t t n g I e e n y s s a a u R P R

Fig. 8. Changes in basal (endogenous inhibitor present) and total (endogenous inhibitor removed) acid invertase activities of LTS-susceptible and resistant cultivars/clones in response to

CS (32 d at 4 oC ), HS (21 days at 32 oC) and the combined CS+HS treatment. Data represents the average of 12 tubers (n=4, ±SE). Letters indicate LSD (P<0.05) for comparing PHHS treatments within a cultivar/clone.

150

Fig. 9. Effects of PHHS treatments (see Fig. 8) on activities of the plastidic isoform of a -1,4- glucan phosphorylase from Russet Burbank, Innate W8 and Payette Russet tubers. The plastidic isoform was separated on glycogen-containing native gels, which were subsequently incubated with glc-1-phosphate and starch to initiate starch synthesis. Each lane was loaded with a sample extract pooled from four replicates of three tubers per replicate, representing 12 tubers.

151

Fig. 10. Changes in expression (qPCR) of (a) acid invertase and (b) apoplastic (Inh1) and (c) vacuolar (Inh2) invertase inhibitors in Russet Burbank, A02138-2, Innate Russet Burbank (W8) and Payette Russet tubers in response to 32 d at 4 oC (CS) or the combination of HS (21 days at

o o 32 C) plus CS. Control tubers were held at 9 C. Data ±SE were normalized (DDC T) to expression of EF1-α. Letters indicate LSD (P<0.05) for comparing PHHS treatments within a cultivar/clone.

152

8 a Acid Invertase )

-2 7 9oC CS a 6 a HS+CS 5 b b 4 ab

b 2 a 1 Relative mRNA (x10 expression Relative a a a a a 0 b 28 Inh1 a ) -2 9oC 24 CS HS+CS b 20 b 16

12 a 8

a b 4 b b Relative mRNA expression (x10 expression mRNA Relative c a a b 0 10 c Inh2 ) 9 -4 9oC 8 CS HS+CS 7 a 6 5 b 4 a 3 a a 2 a a a c a b Relative mRNA expression (x10 expression mRNA Relative 1 b 0 Russet Burbank A02138-2 Innate W8 Payette Russet

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Appendix

Supplementary Table. Effects of duration of high soil temperature during bulking and maturation on yield, average tuber weight and specific gravity of ‘Ranger Russet’, ‘POR06V12-3’, ‘A02138-2’ and ‘Payette Russet’ tubers. Soil temperatures averaged 8 oC above ambient (19 oC) for 20 or 40 days during the indicated growth stages before return to ambient temperature. Tubers were harvested 169 DAP. Letters indicate mean separation within a column and within each clone (LSD, P<0.05). ANOVA results ( P levels) for the effects of growth stage, duration of elevated temperature, and their interaction are summarized for each cultivar.

Yield (MT/ha) Tubers Specific % Dry Duration Soil Temp -1 Clone Heat On (days) (oC) plant -1 Total Mkt #1 g tuber gravity Matter

Ranger N/A - 19 7.6 abc 67.3 a 65.0 a 208 a 1.084 a 23.5 a 80 DAP 20 27 8.7 a 65.0 ab 57.6 ab 162 bc 1.067 c 18.6 b 40 27 7.8 ab 48.7 bc 42.8 bc 133 c 1.053 d 17.6 b 120 DAP 20 27 5.9 bc 47.2 bc 46.0 bc 194 ab 1.078 ab 21.7 a 40 27 5.3 c 41.6 c 34.3 c 157 bc 1.077 b 21.6 a

LSD 0.05 2.4 17.9 16.4 43 0.006 3.3 19 oC (ambient) vs Heat NS 0.05 0.01 0.05 0.01 0.01 Stage (80 vs 120 DAP) 0.01 NS NS NS 0.01 0.01 Duration (20 d vs 40 d) NS NS 0.05 0.05 0.01 NS Stage x Duration NS NS NS NS 0.05 NS V12-3 N/A - 19 7.0 b 65.6 a 65.0 a 228 a 1.092 a 24.7 a 80 DAP 20 27 8.8 a 53.5 ab 50.1 ab 139 b 1.075 c 21.2 b 40 27 7.3 ab 39.2 bc 37.8 bc 126 b 1.071 c 18.8 c 120 DAP 20 27 5.5 c 38.6 bc 37.8 bc 168 b 1.084 b 24.4 a 40 27 4.4 c 29.1 c 24.9 c 133 b 1.089 ab 24.5 a

LSD 0.05 1.5 17.0 16.6 54 0.007 3.2 19 oC (ambient) vs Heat NS 0.01 0.01 0.01 0.01 0.05 Stage (80 vs 120 DAP) 0.01 0.05 0.05 NS 0.01 0.01 Duration (20 d vs 40 d) 0.05 0.05 0.05 NS NS NS Stage x Duration NS NS NS NS NS NS 138-2 N/A - 19 6.3 b 58.4 a 57.9 a 227 a 1.081 a 22.5 a 80 DAP 20 27 8.3 a 51.6 b 49.0 b 145 b 1.068 c 18.5 b 40 27 6.1 b 33.8 c 33.1 c 132 b 1.055 d 18. 4b 120 DAP 20 27 5.7 bc 37.5 c 37.2 c 163 b 1.075 b 21.4 a 40 27 4.2 c 25.9 d 22.8 d 133 b 1.078 ab 20.6 a

LSD 0.05 1.7 5.8 6.3 40 0.005 2.8 19 oC (ambient) vs Heat NS 0.01 0.01 0.01 0.01 0.01 Stage (80 vs 120 DAP) 0.01 0.01 0.01 NS 0.01 0.01 Durat ion (20 d vs 40 d) 0.01 0.01 0.01 NS 0.05 NS Stage x Duration NS NS NS NS 0.01 NS

Payette N/A - 19 6.7 a 68.2 a 67.6 a 248 a 1.094 a 26.2 a 80 DAP 20 27 7.1 a 57.4 ab 53.6 ab 183 bc 1.081 b 22.2 bc 40 27 7.1 a 48.9 ab 44.7 b 153 c 1.074 b 20.6 c 120 DAP 20 27 4.8 b 43.2 bc 42.4 b 214 ab 1.080 b 23.8 ab 40 27 3.5 b 25.2 c 22.6 c 148 c 1.078 b 23.5 b

LSD 0.05 1.9 20.9 19.1 57 0.009 3.6 19 oC (ambient) vs Hea t NS 0.01 0.01 0.01 0.01 0.01 Stage (80 vs 120 DAP) 0.01 0.05 0.05 NS NS 0.05 Duration (20 d vs 40 d) NS NS 0.05 0.05 NS NS Stage x Duration NS NS NS NS NS NS

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