Characterization, expression and function of DORMANCY ASSOCIATED MADS-BOX genes from leafy spurge

DORMANCY ASSOCIATED MADS-BOX (DAM) genes are related to AGAMOUS-LIKE 24 and SHORT VEGETATIVE PHASE genes of arabidopsis and are differentially regulated coordinately with endodormancy induction and release in buds of several perennial plant species. DAM genes were first shown to directly impact endodormancy in peach where a deletion of a series of DAM resulted in loss of endodormancy induction. We have cloned and characterized several MADS box genes from the model perennial weed leafy spurge. Leafy spurge DAM genes are preferentially expressed in shoot tips and buds in response to cold temperatures and day length in a manner that is relative to the level of endodormancy induced by various environmental conditions. Over-expression of one DAM gene in arabidopsis delays flowering. Additionally, we show that at least one DAM gene is differentially regulated by chromatin remodeling. Comparisons of the DAM gene promoters between poplar and leafy spurge have identified several conserved sequences that may be important for their expression patterns in response to dormancy-inducing stimuli.     

Seasonal shifts in dormancy status, carbohydrate metabolism, and related gene expression in crown buds of leafy spurge

Crown buds of field-grown leafy spurge (Euphorbia esula L.) were examined to determine relationships between carbohydrate metabolism and gene expression throughout para-, endo-, and eco-dormancy during the transition from summer, autumn, and winter, respectively. The data indicates that endo-dormancy plays a role in preventing new shoot growth during the transition from autumn to winter. Cold temperature was involved in breaking endo-dormancy, inducing flowering competence, and inhibiting shoot growth. An inverse relationship developed between starch and soluble sugar (mainly sucrose) content in buds during the shift from para- to endo-dormancy, which continued through eco-dormancy. Unlike starch content, soluble sugars were lowest in crown buds during para-dormancy but increased over two- to three-fold during the transition to endo-dormancy. Several genes (AGPase, HK, SPS, SuSy, and UGPase) coding for proteins involved in sugar metabolism were differentially regulated in conjunction with well-defined phases of dormancy in crown buds. Marker genes for S-phase progression, cell wall biochemistry, or responsive to auxin were also differentially regulated during transition from para-, endo-, and eco-dormancy. The results were used to develop a model showing potential signalling pathways involved in regulating seasonal dormancy status in leafy spurge crown buds.     

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge

Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula β-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this β-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.     

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.     

Effect of Leafy Spurge (Euphorbia esula) Genotype on Feeding Damage and Reproduction of Aphthona spp.: Implications for Biological Weed Control

North American leafy spurge (Euphorbia esula L.) is genetically diverse and composed of multiple genotypes introduced from several areas of Europe and Asia. Five species of leafy spurge flea beetle (Aphthona spp.) have been introduced as biological control agents for leafy spurge, but were collected in a relatively small region of Europe. Greenhouse and field experiments were conducted to determine if observed variation in feeding preference and reproduction of Aphthona spp. on North American leafy spurge may be due in part to leafy spurge genotype. Leafy spurge genotypes were collected from Austria; Manitoba in Canada; and the states of Montana, Nebraska, North Dakota, South Dakota, and Wyoming in the United States. Leafy spurge genotype affected feeding but not egg laying by Aphthona spp. adults. Aphthona czwalinae/lacertosa fed slightly less in a free-choice test on a genotype from Manitoba (7%) compared to genotypes from Nebraska and North Dakota (14%). Aphthona flava tended to feed less on the Nebraska genotype than any other genotype evaluated. Reproduction of Aphthona spp. was greatly affected by leafy spurge genotype. For instance, A. czwalinae/lacertosa produced 72 adults per plant from a Nebraska genotype compared to 11 to 32 adults per plant from all other genotypes evaluated. Thus, some observed variation in establishment and reproduction of Aphthona spp. can be attributed to leafy spurge genotype.     

Molecular analysis of signals controlling dormancy and growth in underground adventitious buds of leafy spurge

Dormancy and subsequent regrowth of adventitious buds is a critical physiological process for many perennial plants. We have used the expression of hormone and cell cycle-responsive genes as markers to follow this process in leafy spurge (Euphorbia esula). In conjunction with earlier studies, we show that loss of mature leaves results in decreased sugar levels and increased gibberellin perception in underground adventitious buds. Gibberellin is sufficient for induction of S phase-specific but not M phase-specific gene expression. Loss of both apical and axillary buds or inhibition of polar auxin transport did not result in induction of S phase- or M phase-specific gene expression. Loss of polar auxin transport was necessary for continuation of the cell cycle and further bud development if the S phase was previously initiated.