Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Increase in ACC oxidase levels and activities during paradormancy release of leafy spurge (Euphorbia esula) buds.

The plant hormone ethylene is known to affect various developmental processes including dormancy and growth. Yet, little information is available about the role of ethylene during paradormancy release in underground adventitious buds of leafy spurge. In this study, we examined changes in ethylene evolution and the ethylene biosynthetic enzyme ACC oxidase following paradormancy release (growth induction). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1 mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of a Euphorbia esula ACC oxidase (Ee-ACO) increased up to threefold following growth induction. In addition, a 2.5- to 4-fold increase in ACO activity was observed 4 days after decapitation, and the Ee-ACO accounted for 40 % of the total ACO activity. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. This protein was highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization of Ee-ACO on growing bud sections revealed strong labeling of the nucleus and cytoplasm in cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred in cells undergoing mitosis, where labeling of Ee-ACO was negligible. Taken together, our results indicated an increase in the levels of Ee-ACO during paradormancy release of leafy spurge that was not correlated with an increase in ethylene synthesis.

Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response through calmodulin-binding transcription factor AtSR1/CAMTA3.

Calcium/calmodulin (Ca(2+)/CaM) has long been considered a crucial component in wound signaling pathway. However, very few Ca(2+)/CaM-binding proteins have been identified which regulate plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca(2+)/CaM-binding transcription factors designated as AtSRs (also known as AtCAMTAs) can respond differentially to wounding stress. Further studies revealed that AtSR1/CAMTA3 is a negative regulator of plant defense, and Ca(2+)/CaM-binding to AtSR1 is indispensable for the suppression of salicylic acid (SA) accumulation and disease resistance. Here we report that Ca(2+)/CaM-binding is also critical for AtSR1-mediated herbivore-induced wound response. Interestingly, atsr1 mutant plants are more susceptible to herbivore attack than wild-type plants. Complementation of atsr1 mutant plants by overexpressing wild-type AtSR1 protein can effectively restore plant resistance to herbivore attack. However, when mutants of AtSR1 with impaired CaM-binding ability were overexpressed in atsr1 mutant plants, plant resistance to herbivore attack was not restored, suggesting a key role for Ca(2+)/CaM-binding in wound signaling. Furthermore, it was observed that elevated SA levels in atsr1 mutant plants have a negative impact on both basal and induced biosynthesis of jasmonates (JA). These results revealed that Ca(2+)/CaM-mediated signaling regulates plant response to herbivore attack/wounding by modulating the SA-JA crosstalk through AtSR1.

Auxin and ABA act as central regulators of developmental networks associated with paradormancy in Canada thistle (Cirsium arvense).

Dormancy in underground vegetative buds of Canada thistle, an herbaceous perennial weed, allows escape from current control methods and contributes to its invasive nature. In this study, ~65 % of root sections obtained from greenhouse propagated Canada thistle produced new vegetative shoots by 14 days post-sectioning. RNA samples obtained from sectioned roots incubated 0, 24, 48, and 72 h at 25°C under 16:8 h light-dark conditions were used to construct four MID-tagged cDNA libraries. Analysis of in silico data obtained using Roche 454 GS-FLX pyrosequencing technologies identified molecular networks associated with paradormancy release in underground vegetative buds of Canada thistle. Sequencing of two replicate plates produced ~2.5 million ESTs with an average read length of 362 bases. These ESTs assembled into 67358 unique sequences (21777 contigs and 45581 singlets) and annotation against the Arabidopsis database identified 15232 unigenes. Among the 15232 unigenes, we identified processes enriched with transcripts involved in plant hormone signaling networks. To follow-up on these results, we examined hormone profiles in roots, which identified changes in abscisic acid (ABA) and ABA metabolites, auxins, and cytokinins post-sectioning. Transcriptome and hormone profiling data suggest that interaction between auxin- and ABA-signaling regulate paradormancy maintenance and release in underground adventitious buds of Canada thistle. Our proposed model shows that sectioning-induced changes in polar auxin transport alters ABA metabolism and signaling, which further impacts gibberellic acid signaling involving interactions between ABA and FUSCA3. Here we report that reduced auxin and ABA-signaling, in conjunction with increased cytokinin biosynthesis post-sectioning supports a model where interactions among hormones drives molecular networks leading to cell division, differentiation, and vegetative outgrowth.

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.

The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato.

The factors that regulate storage organ carotenoid content remain to be fully elucidated, despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined, at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether Carotenoid Cleavage Dioxygenase4 (CCD4) activity affects potato (Solanum tuberosum) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNA interference (RNAi) approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, 2- to 5-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems, and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers, and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses.

Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar.

BACKGROUND: Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development. RESULTS: We report that poplar (Populus spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast. CONCLUSION: These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.

The qSD12 underlying gene promotes abscisic acid accumulation in early developing seeds to induce primary dormancy in rice.

Seeds acquire primary dormancy during their development and the phytohormone abscisic acid (ABA) is known to play a role in inducing the dormancy. qSD12 is a major seed dormancy quantitative trait locus (QTL) identified from weedy rice. This research was conducted to identify qSD12 candidate genes, isolate the candidates from weedy rice, and determine the relation of the dormancy gene to ABA. A fine mapping experiment, followed by marker-assisted progeny testing for selected recombinants, narrowed down qSD12 to a genomic region of <75 kb, where there are nine predicted genes including a cluster of six transposon/retrotransposon protein genes and three putative (a PIL5, a hypothetic protein, and a bHLH transcription factor) genes based on the annotated Nipponbare genome sequence. The PIL5 and bHLH genes are more likely to be the QTL candidate genes. A bacterial artificial chromosome (BAC) library equivalent to 8-9 times of the haploid genome size was constructed for the weedy rice. One of the two BAC contigs developed from the library covers the PIL5 to bHLH interval. A pair of lines different only in the QTL-containing region of <200 kb was developed as isogenic lines for the qSD12 dormancy and non-dormancy alleles. The dormant line accumulated much higher ABA in 10-day developing seeds than the non-dormant line. In the QTL-containing region there is no predicted gene that has been assigned to ABA biosynthetic or metabolic pathways. Thus, it is concluded that the qSD12 underlying gene promotes ABA accumulation in early developing seeds to induce primary seed dormancy.

Age-induced loss of wound-healing ability in potato tubers is partly regulated by ABA.

Wounding of potato (Solanum tuberosum L.) tubers induces the development of a suberized closing layer and wound periderm that resists desiccation and microbial invasion. Wound-healing ability declines with tuber age (storage period). The mechanism of loss in healing capacity with age is not known; however, upregulation of superoxide production, increased ABA biosynthesis and phenylalanine ammonia lyase (PAL) activity in response to wounding are processes critical to the development of a suberized closing layer and wound periderm. Therefore, the role of ABA in modulating the age-induced loss of wound-healing ability of tubers was examined. Non-wounded older tubers had 86% less ABA (dry matter basis) than younger tubers. PAL transcript increased in younger tubers within 24 h of wounding, but transcription was delayed by 5 days in older tubers. Wound-induced PAL activity increased more rapidly in younger than older tubers. ABA treatment increased PAL expression and activity in tissue from both ages of tubers and restored the 24 h transcription time line in older tubers. Moreover, ABA treatment of wounded older tubers enhanced their resistance to water vapor loss following a 6-day wound-healing period. Wound-induced accumulation of suberin poly(phenolic(s)) (SPP) and suberin poly(aliphatic(s)) (SPA) was measurably slower in older versus younger tubers. ABA treatment hastened SPP accumulation in older tubers to match that in younger tubers, but only enhanced SPA accumulations over the initial 4 days of healing. Age-induced loss of wound-healing ability is thus partly due to reduced ability to accumulate ABA and modulate the production of SPP through PAL in response to wounding and to dysfunction in the downstream signaling events that couple SPA biosynthesis and/or deposition to ABA. ABA treatment partly restored the healing ability of older tubers by enhancing the accumulation of SPP without restoring wound-induced superoxide forming ability to the level of younger tubers. The coupling of phenolic monomers into the poly(phenolic) domain of suberin was therefore not limited by the diminished wound-induced superoxide production of older tubers.

Transcriptome analysis identifies novel responses and potential regulatory genes involved in seasonal dormancy transitions of leafy spurge (Euphorbia esula L.).

BACKGROUND: Dormancy of buds is a critical developmental process that allows perennial plants to survive extreme seasonal variations in climate. Dormancy transitions in underground crown buds of the model herbaceous perennial weed leafy spurge were investigated using a 23 K element cDNA microarray. These data represent the first large-scale transcriptome analysis of dormancy in underground buds of an herbaceous perennial species. Crown buds collected monthly from August through December, over a five year period, were used to monitor the changes in the transcriptome during dormancy transitions. RESULTS: Nearly 1,000 genes were differentially-expressed through seasonal dormancy transitions. Expected patterns of gene expression were observed for previously characterized genes and physiological processes indicated that resolution in our analysis was sufficient for identifying shifts in global gene expression. CONCLUSION: Gene ontology of differentially-expressed genes suggests dormancy transitions require specific alterations in transport functions (including induction of a series of mitochondrial substrate carriers, and sugar transporters), ethylene, jasmonic acid, auxin, gibberellic acid, and abscisic acid responses, and responses to stress (primarily oxidative and cold/drought). Comparison to other dormancy microarray studies indicated that nearly half of the genes identified in our study were also differentially expressed in at least two other plant species during dormancy transitions. This comparison allowed us to identify a particular MADS-box transcription factor related to the DORMANCY ASSOCIATED MADS-BOX genes from peach and hypothesize that it may play a direct role in dormancy induction and maintenance through regulation of FLOWERING LOCUS T.

Regulatory involvement of abscisic acid in potato tuber wound-healing.

Rapid wound-healing is crucial in protecting potato tubers from infection and dehydration. Wound-induced suberization and the accumulation of hydrophobic barriers to reduce water vapour conductance/loss are principal protective wound-healing processes. However, little is known about the cognate mechanisms that effect or regulate these processes. The objective of this research was to determine the involvement of abscisic acid (ABA) in the regulation of wound-induced suberization and tuber water vapour loss (dehydration). Analysis by liquid chromatography-mass spectrometry showed that ABA concentrations varied little throughout the tuber, but were slightly higher near the periderm and lowest in the pith. ABA concentrations increase then decrease during tuber storage. Tuber wounding induced changes in ABA content. ABA content in wound-healing tuber discs decreased after wounding, reached a minimum by 24 h, and then increased from the 3rd to the 7th day after wounding. Wound-induced ABA accumulations were reduced by fluridone (FLD); an inhibitor of de novo ABA biosynthesis. Wound-induced phenylalanine ammonia lyase activity was slightly reduced and the accumulation of suberin poly(phenolics) and poly(aliphatics) noticeably reduced in FLD-treated tissues. Addition of ABA to the FLD treatment restored phenylalanine ammonia lyase activity and suberization, unequivocally indicating that endogenous ABA is involved in the regulation of these wound-healing processes. Similar experiments showed that endogenous ABA is involved in the regulation of water vapour loss, a process linked to wax accumulation in wound-healing tubers. Rapid reduction of water vapour loss across the wound surface is essential in preventing desiccation and death of cells at the wound site; live cells are required for suberization. These results unequivocally show that endogenous ABA is involved in the regulation of wound-induced suberization and the processes that protect surface cells from water vapour loss and death by dehydration.

Wounding induces changes in cytokinin and auxin content in potato tuber, but does not induce formation of gibberellins.

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0-5/6 days), and (II) initiation of phellogen and wound periderm formation (∼ 7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c-ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t-ZR and c-ZR content at 0.5 days and large increases in IPA and c-ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5-7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes.

Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions.

Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato (Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 33% decrease in genome-wide 5-methylcytosine (5mC) content and a transient threefold increase in transcription rates that were most evident at 6 and 9 days after subculture, respectively. In contrast to the global reduction in 5mC content, subculture resulted in a transient twofold increase in 5mC levels within 5'-CCGG-3' sequences and no detectable change in 5'-CG-3' methylation. Multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by approximately 25% at day 9, then decreased 5'-mCmCGG-3' by 30-50% and increased H3 and H4 multi-acetylation by 30-60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was approximately twofold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension cultures, rapid, reversible changes in 5mC levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells.

Mechanism-based irreversible inhibitors of cytokinin dehydrogenase.

The effects of three N(6)-substituted aminopurine derivatives containing either allenic or acetylenic side-chains on in vitro and in vivo cytokinin dehydrogenase (CKX; EC 1.5.99.12) activities were determined. At concentrations < or = 100 microM, the acetylenic derivative (HA-2) had no effect on in vitro CKX activity. In contrast, the two allenic derivatives (HA-1, HA-8) inhibited in vitro CKX activity in a dose-dependent manner with 50% inhibition occurring at HA-1 and HA-8 concentrations of 9.0 and 0.4 microM (respectively). HA-8 inhibited the degradation of both the free bases and ribosides of N6-(2-isopentenyl)adenine and zeatin. Pretreatment with HA-8 inhibited CKX activity in both a time- and concentration-dependent manner. In contrast to the reversible phenylurea inhibitor N-(chloro-4-pyridyl)-N'-phenylurea, inhibition of CKX activity by HA-8 was not relieved by 24 h of dialysis. Both HA-1 and HA-8 (but not HA-2) inhibited the metabolism of exogenous [3H]-N(6)-(2-isopentenyl)adenosine in excised aseptic potato (Solanum tuberosum) leaves. These results demonstrate that HA-8 is a mechanism-based irreversible (suicide) inhibitor of CKX and indicate that it may be useful in determining the role of CKX in cytokinin homeostasis in planta.

Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation.

Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing.

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 degrees C in the dark resulted in the progressive loss of tuber meristem dormancy, defined as measurable growth after transfer to 20 degrees 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.

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.

Kinetics and localization of wound-induced DNA biosynthesis in potato tuber.

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive "Click-iT EdU Assay" employing incorporation of the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), in conjunction with 4',6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12h, rapidly reached a maximum at about 18h and then declined to near zero at 48h. About 28% of the nuclei were EdU labeled at 18h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7-8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.

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. [(3)H]-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.

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes.

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48h after wounding and remained elevated through 96h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8'-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3h of wounding and remained elevated through 96h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.