Deoxyuridine triphosphatase expression defines the transition from dormant to sprouting potato tuber buds

Identification of molecular markers defining the end of tuber dormancy prior to visible sprouting is of agronomic interest for potato growers and the potato processing industry. In potato tubers, breakage of dormancy is associated with the reactivation of meristem function. In dormant meristems, cells are arrested in the G₁/G₀ phase of the cell cycle and re-entry into the G₁ phase followed by DNA replication during the S phase enables bud outgrowth. Deoxyuridine triphosphatase (dUTPase) is essential for DNA replication and was therefore tested as a potential marker for meristem reactivation in tuber buds. The corresponding cDNA clone was isolated from potato by PCR. The deduced amino acid sequence showed 94% similarity to the tomato homologue. By employing different potato cultivars, a positive correlation between dUTPase expression and onset of tuber sprouting could be confirmed. Moreover, gene expression analysis of tuber buds during storage time revealed an up-regulation of the dUTPase 1 week before visible sprouting occurred. Further analysis using an in vitro sprout assay supported the assumption that dUTPase is a good molecular marker to define the transition from dormant to active potato tuber meristems.     

Involvement of endogenous gibberellins in potato tuber dormancy and early sprout growth: a critical assessment

The role of endogenous gibberellins (GAs) in the regulation of potato (Solanum tuberosum) tuber dormancy was examined by determining: 1. changes in endogenous GA levels during natural dormancy progression, 2. the effects of GA biosynthesis inhibitors on tuber dormancy duration and 3. the dormancy status and tuber GA levels in a dwarf mutant of potato. The tubers (cv. Russet Burbank) used in these studies were still completely dormant after 98 days of storage. Between 98 and 134 days of storage, dormancy began to end and tubers exhibited limited (< 2 mm) sprout growth. Tuber dormancy weakened with further storage and tubers exhibited greater rates of sprout growth after 187 days of storage. Tubers stored for 212 days or longer were completely non-dormant and exhibited vigorous sprout growth. Immediately after harvest, the endogenous contents of GA19, GA20, and GA1 were relatively high (0.48-0.62 ng g fresh weight(-1)). The content of these GAs declined between 33 and 93 days of storage. Internal levels of GA19, GA20, and GA, rose slightly between 93 and 135 days of storage reaching levels comparable to those found in highly dormant tubers immediately after harvest. Levels of GA19, GA20, and GA1 continued to increase as sprout growth became more vigorous. Neither GA4 nor GA8 was detected in any tuber sample regardless of dormancy status. Dormant tubers exhibited a time-dependent increase in apparent GA sensitivity. Freshly harvested tubers were completely insensitive to exogenous GAs. As postharvest storage continued, exogenous GAs promoted premature dormancy release with GA1 and GA20 eliciting the greatest response. Injection of up to 5 microg tuber(-1) of kaurene, GA12, GA19 or GA8 had no effect on dormancy release. Sprout growth from non-dormant tubers was also promoted by exogenous GA in the following sequence of activity: GA1 = GA20 > GA19. Kaurene, GA12, and GA8 were inactive. Continuous exposure of developing tubers to inhibitors of GA biosynthesis (AMO-1618, ancymidol, or tetcyclasis) did not extend tuber dormancy but rather hastened dormancy release. Comparison of tuber dormancy and GA1 content in tubers of a wild-type and dwarf mutant of S. tuberosum ssp. andigena revealed a near-identical pattern of dormancy progression in spite of the absence of detectable levels of GA1 in tubers of the dwarf sibling at any time during dormancy progression. Collectively, these results do not support a role for endogenous GA in potato tuber dormancy release but are consistent with a role for GAs in the regulation of subsequent sprout growth.     

Chemically forced dormancy termination mimics natural dormancy progression in potato tuber meristems by reducing ABA content and modifying expression of genes involved in regulating ABA synthesis and metabolism

The length of potato tuber dormancy depends on both the genotype and the environmental conditions during growth and storage. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms regulating ABA content during dormancy, as well as the sites of ABA synthesis, and catabolism are unknown. Recently, a temporal correlation between changes in ABA content and certain ABA biosynthetic and catabolic genes has been reported in stored field tubers during physiological dormancy progression. However, the protracted length of natural dormancy progression complicated interpretation of these data. To address this issue, in this study the synthetic dormancy-terminating agent bromoethane (BE) was used to induce rapid and highly synchronous sprouting of dormant tubers. The endogenous ABA content of tuber meristems increased 2-fold 24 h after BE treatment and then declined dramatically. By 7 d post-treatment, meristem ABA content had declined by >80%. Exogenous [(3)H]ABA was readily metabolized by isolated meristems to phaseic and dihydrophaseic acids. BE treatment resulted in an almost 2-fold increase in the rate of ABA metabolism. A differential expression of both the StNCED and StCYP707A gene family members in meristems of BE-treated tubers is consistent with a regulatory role for StNCED2 and the StCYP707A1 and StCYP707A2 genes. The present results show that the changes in ABA content observed during tuber dormancy progression are the result of a dynamic equilibrium of ABA biosynthesis and degradation that increasingly favours catabolism as dormancy progresses.     

Starch-related enzymes during potato tuber dormancy and sprouting

Activities of enzymes presumably involved in starch biosynthesis (ADP-glucose pyrophosphorylase, AGPase) and/or breakdown (starch phosphorylase, STP; amylases) were determined during potato (Solanum tuberosum L.) tuber dormancy and sprouting. Overall activities of all these enzymes decreased during the first stage of tuber dormancy. No clear changes were detected at the time of dormancy breaking and sprouting. However, when AGPase activity was monitored by in situ staining during the entire dormancy period, a clear decrease during the dormant period and a large increase before visible sprouting could be observed. This increase was especially evident near the vascular tissue and at the apical bud, which showed a very intensive staining. In situ staining of STP activity in sprouting tubers showed that the tissue distribution of STP was the same as for AGPase. As a possible explanation, direct starch cycling is suggested: STP produces glucose-1-phosphate during starch breakdown, which can be directly used as a substrate by AGPase for starch synthesis. Gene expression studies with the AGPaseS promoter coupled to the firefly luciferase reporter gene also clearly showed a higher activity in sprouting tubers as compared to dormant tubers, with the highest expression levels observed around the apical buds. The presence of amylase activity at dormancy initiation and AGPase activity persistent at the sprouting stage suggest that starch was cycling throughout the entire dormancy period. According to the in situ studies, the AGPase activity increased well before visible sprout growth and could therefore be one of the first physiological determinants of dormancy breakage.     

Effect of growth regulators on proton transport through cytoplasmic membrane of potato tuber cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H+ through the membranes of plasma membrane vesicles of potato (Solanum tuberosum L.) tuber cells were studied. The ATP-dependent accumulation of H+ in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H+ in the vesicles. The data suggest that the plasma membrane H+ ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

Regulation of potato tuber dormancy and sprouting

Dormancy is the final stage of tuber life serving to preserve tubers as organs of vegetative reproduction under unfavorable growth conditions. Since the duration of potato tuber dormancy and their sprouting time have significant economic importance, much attention is given to the study of the regulation of these processes. This review considers metabolite, genetic, and hormonal aspects of regulation of potato (Solanum tuberosum L.) tuber dormancy and sprouting. Particular attention is paid to the relationship between processes occurring in different parts of the tuber: its storage tissues and buds. The interaction of hormonal and metabolite (carbohydrate) regulation of dormancy and sprouting is discussed.     

Ethanol breaks dormancy of the potato tuber apical bud

Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Changes in GA 20-oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants

Gene StGA20ox1 encoding potato GA 20-oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over-expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20-oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.     

Effects of Growth Regulators on H+Translocation across the Membranes of Plasma Membrane Vesicles from Potato Tuber Cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H⁺through the membranes of plasma membrane vesicles of potato (Solanum tuberosumL.) tuber cells were studied. The ATP-dependent accumulation of H⁺in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H⁺in the vesicles. The data suggest that the plasma membrane H⁺ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

Detection of Potato Viruses Y and A in Tubers by Enzyme-Linked Immunosorbent Assay after Natural and Artificial Break of Dormancy

The effects of natural and artificial break of dormancy on the detection of potato viruses Y (PVY) and A (PVA) in tubers of field-grown potato cultivars with primary and secondary infections were studied by enzyme-linked immunosorbent assay (ELISA). Both viruses were at low concentration and unevenly distributed in dormant -tubers. Four to six weeks after treatment with Rindite, maximum concentrations of PVY and PVA were detected at both tuber ends with generally higher values at the rose end than at the heel end. Slower and smaller virus increase and a retarded and/or limited virus translocation were observed in treated tubers of resistant cultivars. Consequently, detectability of PVY and PVA was higher in tubers of susceptible cultivars than in those of the resistant ones. Detection of both viruses was very difficult and unreliable in tubers after natural break of dormancy. The very low concentration of PVY and PVA in dormant tubers and their drastic multiplication in Rindite- treated tubers appear to be characteristic of potyviruses and are unique among the major potato viruses.     

Reduction of abscisic acid content and induction of sprouting in potato,Solanum tuberosum L., by thidiazuron

The effect of [(N-phenyl-N-1,2,3-thidiazol-5-ylurea)] (thidazuron) on sprouting of potato (Solanum tuberosum L.) tubers and the role of ABA in bud break and subsequent bud growth were studied. Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) from the peel of potato tubers. The ELISA results were also validated by gas chromatography-electron capture detector and confirmed by gas chromatography-mass spectrometry and by a lettuce hypocotyl bioassay. The degree of rest in the tubers was associated with ABA content in the peel. Basal portion (where tuber was attached to mother plant) contained the highest amount of ABA. Thidiazuron reduced ABA content and induced potato tuber sprouting. Exogenously applied ABA stimulated growth of buds that had emerged from dormancy.On leave from the Department of Horticulture, South China Agricultural University, Guangzhou, People's Republic of China.     

Can loss of apical dominance in potato tuber serve as a marker of physiological age?

The potato tuber constitutes a model system for the study of dormancy release and sprouting, suggested to be regulated by endogenous plant hormones and their balance inside the tuber. During dormancy, potato tubers cannot be induced to sprout without some form of stress or exogenous hormone treatment. When dormancy is released, sprouting of the apical bud may be inhibited by sprout control agents or cold temperature. Dominance of the growing apical bud over other lateral buds decreases during storage and is one of the earliest morphophysiological indicators of the tuber's physiological age. Three main types of loss of apical dominance (AD) affect sprouting shape. Hallmarks of programmed cell death (PCD) have been identified in the tuber apical bud meristem (TAB-meristem) during normal growth, and are more extensive when AD is lost following extended cold storage or chemical stress. Nevertheless, the role of hormonal regulation in TAB-meristem PCD remains unclear.     

The control of bud dormancy in potato tubers

Potato (Solanum tuberosum L.) tuber buds normally remain dormant through the growing season until several weeks after harvest. In the cultivar Majestic, this innate dormancy persisted for 9 to 12 weeks in storage at 10° C, but only 3 to 4 weeks when the tubers were stored at 2° C. At certain stages, supplying cytokinins to tubers with innately dormant buds induced sprout growth within 2 d. The growth rate was comparable to that of buds whose innate dormancy had been lost naturally. Cytokinin-treatment did not accelerate the rates of cell division and cell expansion in buds whose innate dormancy had already broken naturally. Gibberellic acid did not induce sprout growth in buds with innate dormancy. We conclude that cytokinins may well be the primary factor in the switch from innate dormancy to the non-dormant state in potato tuber buds, but probably do not control the subsequent sprout growth.Abbreviations tio ⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)purine, zeatin - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)-9--D-ribofuranosyl purine, zeatin riboside     

Changes in cis-zeatin and cis-zeatin riboside levels and biological activity during potato tuber dormancy

The effects of postharvest storage duration and temperature on endogenous cis -zeatin ( cis -Z) and cis -zeatin riboside ( cis -ZR) levels in potato ( Solanum tuberosum L.) tubers were determined in relation to tuber bud dormancy. The tubers used in these studies were completely dormant for at least 81 days of storage. Thereafter, tuber bud dormancy diminished gradually and after 165 days of postharvest storage, the tubers were completely non-dormant. Immediately after harvest, endogenous levels of cis- Z and cis -ZR were approximately 25 pmol (g fresh weight)⁻¹ and 8 pmol (g fresh weight)⁻¹, respectively. In tubers exiting dormancy but stored at a growth-inhibiting temperature (3°C), endogenous levels of cis -Z rose over threefold after 25 days of storage and remained elevated for the duration of the study. Levels of cis -ZR remained essentially constant during this same period. In tubers transferred to a growth permissive temperature (20°C) prior to use, the rise in endogenous cis -Z was less dramatic and more protracted; increasing twofold after 53 days of storage. No change in cis -Z riboside content was observed in these tubers during this period. Dose-response studies using either cis -Z or trans -Z demonstrated a time-dependent increase in cytokinin sensitivity during postharvest storage. Immediately after harvest, dormant tubers were insensitive to both zeatin isomers. Thereafter, tubers exhibited a dose-dependent increase in premature sprouting following injection with either cytokinin isomer. After injection into dormant tubers, cis -[8-¹⁴C]-zeatin was metabolized primarily to adenine/adenosine and cis -Z riboside. Seven days after injection, less than 10% of the recovered radioactivity was associated with trans -ZR. These results are consistent with a role for endogenous cis -Z (and its derivatives) in the regulation of potato tuber dormancy.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

Combining conventional QTL analysis and whole-exome capture-based bulk-segregant analysis provides new genetic insights into tuber sprout elongation and dormancy release in a diploid potato population

Tuber dormancy and sprouting are commercially important potato traits as long-term tuber storage is necessary to ensure year-round availability. Premature dormancy release and sprout growth in tubers during storage can result in a significant deterioration in product quality. In addition, the main chemical sprout suppressant chlorpropham has been withdrawn in Europe, necessitating alternative approaches for controlling sprouting. Breeding potato cultivars with longer dormancy and slower sprout growth is a desirable goal, although this must be tempered by the needs of the seed potato industry, where dormancy break and sprout vigour are required for rapid emergence. We have performed a detailed genetic analysis of tuber sprout growth using a diploid potato population derived from two highly heterozygous parents. A dual approach employing conventional QTL analysis allied to a combined bulk-segregant analysis (BSA) using a novel potato whole-exome capture (WEC) platform was evaluated. Tubers were assessed for sprout growth in storage at six time-points over two consecutive growing seasons. Genetic analysis revealed the presence of main QTL on five chromosomes, several of which were consistent across two growing seasons. In addition, phenotypic bulks displaying extreme sprout growth phenotypes were subjected to WEC sequencing for performing BSA. The combined BSA and WEC approach corroborated QTL locations and served to narrow the associated genomic regions, while also identifying new QTL for further investigation. Overall, our findings reveal a very complex genetic architecture for tuber sprouting and sprout growth, which has implications both for potato and other root, bulb and tuber crops where long-term storage is essential.Subject terms: Genetic markers, Next-generation sequencing, Plant breeding, Agricultural genetics, Genetic mapping