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.     

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.     

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.     

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.     

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties,activity, and expression during tuber dormancy progression

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [³H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.     

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+)].     

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.     

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     

A review of the physiology of potato tuber dormancy

A review of the scientific literature relating to the physiology of potato (Solanum tuberosum) tuber dormancy is presented. Effort has been concentrated on an up-to-date overview of the current state of understanding, rather than comprehensively covering the very extensive literature going back over many decades. The format chosen follows the fate of the crop. After defining tuberisation and dormancy, the physiological activity of the dormant tuber is reviewed and the storage environment is considered from both a physical and chemical standpoint. Advances in chemical control and the potential for molecular biology are highlighted.     

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.     

Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.     

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.     

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8′-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

In vitro micro-tuber initiation and dormancy in yam

Dormancy is a mechanism that regulates the timing of sprouting (germination) of affected plant parts as well as ensures that the food quality of edible parts is maintained in storage until the following growing season. In yam, however, little is known about the control of tuber initiation or tuber dormancy. The objective of this study was to determine the effects of selected plant growth regulators (PGRs) on tuber initiation and dormancy, using an in vitro system. In two replicated experiments, 2-chloroethylphosphonic acid (ethephon, an ethylene source), abscisic acid (ABA) and gibberellin (GA₃) – and their inhibitors silver nitrate, fluridone and 2-chloroethyl-trimethylammonium chloride, respectively – were added at two concentrations to the culture medium prior to explant culture. Dates of micro-tuber initiation and sprouting (end of dormancy) and tuber number were recorded. In the control (no PGR) in Experiment 1, micro-tubers were initiated at the base of the stem after 176 days and sprouted 235 days later, that is 411 days after culturing. Most PGR treatments had only small effects (±30 days) on the duration of dormancy and the time of micro-tuber initiation. However, in GA₃ micro-tuber initiation occurred after 76 days, about 100 days earlier than in the control, whereas fluridone affected the position of micro-tubers and duration of dormancy. With fluridone treatments, tubers were found at the base of the stem (normal position) and on lower and upper nodes. Lower node tubers sprouted within 225 days of culturing compared with about 420 days after culturing at other nodal positions and in other PGR treatments. These data suggest an important role for ABA and gibberellic acid in yam micro-tuber initiation and the induction of dormancy.     

Polyamines bound to nucleic acids during dormancy and activation of tuber cells of Helianthus tuberosus

Tuber tissue of Helianthus tuberosus L. (cv. OB1) contains a low amount of polyamines during dormancy but they are rapidly synthesized when tuber cells are activated in a growth medium and enter a new cell cycle. It was assumed that one of the reasons for this synthesis is that polyamines are necessary for the active conformation and correct functioning of nucleic acids. Complexes were found between spermine, spermidine and putrescine and rRNA, tRNA and an RNA fraction which contains poly(A) RNA and proteins. The amount of RNA-bound polyamines in the parenchyma cells of dormant tubers is dependent on the stage of dormancy and clearly increases (especially putrescine) when cells are activated. There are both tightly-bound and non-tightlybound polyamines. The significance of these bound polyamines is discussed in relation to their stabilizing role on nucleic acids.     

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.     

Changes in histone H3 and H4 multi-acetylation during natural and forced dormancy break in potato tubers

The effects of post-harvest storage and dormancy progression on histone acetylation patterns were examined in potato ( Solanum tuberosum L. cv. Russet Burbank) tubers. Storage of field-grown tubers at 3°C in the dark resulted in the progressive loss of tuber meristem dormancy, defined as measurable growth after transfer to 20°C for 7 days. Dormancy emergence was concomitant with sustained increases in histone H3.1 and H3.2 multi-acetylation, and with transient increases in H4 multi-acetylation that peaked 4–5 months post-harvest. Treatment of dormant tubers with bromoethane (BE) resulted in rapid loss of dormancy over 9 days. Similar to cold-stored field-grown tubers, dormancy break in BE-treated tubers occurred at the same time as transient rises in H4 and H3.1/3.2 multi-acetylation, peaking at days 1 and 4, respectively. BE treatment also resulted in small increases in RNA synthesis at day 6, and a three-fold, sustained activation of DNA synthesis thereafter. A defined sequence of epigenetic events, beginning with previously characterized transient cytosine demethylation, followed by increased H3 and H4 histone acetylation and ultimately, tuber meristem re-activation, may thus exist in potatoes during dormancy exit and resumption of rapid growth.     

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