Jeffrey C. Suttle

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

BackgroundDormancy 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.ResultsNearly 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.ConclusionGene 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.

Role of Endogenous Abscisic Acid in Potato Microtuber Dormancy

Potato (Solanum tuberosum L. cv Russet Burbank) microtubers generated in vitro from single-node explants contained substantial amounts (approximately 250 pmol/g fresh weight) of free abscisic acid (ABA) and were completely dormant for a minimum of 12 weeks. Microtubers that developed in the presence of 10 [mu]M fluridone (FLD) contained considerably reduced amounts (approximately 5–25 pmol/g fresh weight) of free ABA and exhibited a precocious loss of dormancy. Inclusion of exogenous racemic ABA in the FLD-containing medium suppressed the premature sprouting of these microtubers in a dose-dependent manner. At a concentration of 50 [mu]M, exogenous ABA restored internal ABA levels to control values and completely inhibited FLD-induced precocious sprouting. Exogenous jasmonic acid was ineffective in suppressing FLD-induced sprouting. Application of FLD to preformed, fully dormant microtubers also resulted in a reduction in internal ABA content and precocious sprouting. These results indicate that endogenous ABA is essential for the induction and maintenance of potato microtuber dormancy.

Physiological regulation of potato tuber dormancy

At harvest, potato (Solanum tuberosum L.) tubers are dormant and will not sprout. As the period of postharvest storage is extended, tuber dormancy is broken and sprout growth commences. The loss of tuber dormancy and onset of sprout growth is accompanied by numerous biochemical changes, many of which are detrimental to the nutritional and processing qualities of potatoes. Endogenous hormones have been proposed to play a significant role in tuber dormancy regulation. The involvement of all major classes of endogenous hormones in tuber dormancy is reviewed. Based on available evidence, it is concluded that both ABA and ethylene are required for dormancy induction, but only ABA is needed to maintain bud dormancy. An increase in cytokinin sensitivity and content appear to be the principal factors leading to the loss of dormancy. Changes in endogenous IAA and GA content appear to be more closely related to the regulation of subsequent sprout growth.ResumenLos tubérculos de papa (Solanum tuberosum L.), al momento de la cosecha se encuentran en estado latente y no tienen capacidad de germinación. A medida que transcurre el periodo de almacenamiento, se rompe la latencia y comienza el crecimiento del brote. La supresión de la latencia del tubérculo y el inicio del crecimiento del brote son acompañados por numerosos cambios bioquímicos, muchos de los cuales son perjudiciales para la calidad nutricional y el procesamiento de la papa. Se señala que las hormonas endégenas juegan un rol significativo en la regulación de la latencia. Se hace una revisión sobre la forma en que intervienen las principales clases de hormonas endógenas en la latencia del tubérculo. En base a la evidencia disponible se concluye que tanto el ABA como el etileno son requeridos para la inducción de la latencia, pero sólo el ABA es necesario para mantener la latencia de la yema. Un incremento en la sensibilidad a la citoquinina y su contenido, parecen ser los factores principales que conducen a la pérdida de la latencia. Los cambios en el contenido de IAA y AG parece que están mas estrechamente relacionados a la regulación del crecimiento ulterior del brote.

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.

Effect of polyamines on ethylene production

Abstract The polyamines putrescine, cadaverine, spermidine and spermine reduced the amount of ethylene produced by senescing petals of Tradescantia but they did not prevent anthocyanin leakage from these same petals. These polyamines also inhibited auxin-mediated ethylene production by etiolated soybean hypocotyls to less than 7 % of the control. The basic amino acids lysine and histidine reduced the amount of auxin-induced ethylene produced by soybean hypocotyls by ca 50 %. In the hypocotyls, methionine was unable to overcome the inhibition caused by the polyamines. The polyamines spermidine and spermine inhibited ethylene production induced by the application of 1-aminocyclopropane-1-carboxylic acid and they also reduced the endogenous content of this amino acid in the treated tissues.

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

Dormancy in potato tuber meristems: chemically induced cessation in dormancy matches the natural process based on transcript profiles

Meristem dormancy in perennial plants is a developmental process that results in repression of metabolism and growth. The cessation of dormancy results in rapid growth and should be associated with the production of nascent transcripts that encode for gene products controlling for cell division and growth. Dormancy cessation was allowed to progress normally or was chemically induced using bromoethane (BE), and microarray analysis was used to demonstrate changes in specific transcripts in response to dormancy cessation before a significant increase in cell division. Comparison of normal dormancy cessation to BE-induced dormancy cessation revealed a commonality in both up and downregulated transcripts. Many transcripts that decrease as dormancy terminates are inducible by abscisic acid particularly in the conserved BURP domain proteins, which include the RD22 class of proteins and in the storage protein patatin. Transcripts that are associated with an increase in expression encoded for proteins in the oxoglutarate-dependent oxygenase family. We conclude that BE-induced cessation of dormancy initiates transcript profiles similar to the natural processes that control dormancy.

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.

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.

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

Calcium/calmodulin (Ca2+/CaM) has long been considered a crucial component in wound signaling pathway. However, very few Ca2+/CaM-binding proteins have been identified which regulate plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca2+/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 Ca2+/CaM-binding to AtSR1 is indispensable for the suppression of salicylic acid (SA) accumulation and disease resistance. Here we report that Ca2+/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 Ca2+/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 Ca2+/CaM-mediated signaling regulates plant response to herbivore attack/wounding by modulating the SA-JA crosstalk through AtSR1.