Wun S. Chao

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.

Review: A current review on the regulation of dormancy in vegetative buds

Abstract In this review, we examine current techniques and recent advances directed toward understanding cellular mechanisms involved in controlling dormancy in vegetative propagules. Vegetative propagules (including stems, rhizomes, tubers, bulbs, stolons, creeping roots, etc.) contain axillary and adventitious buds capable of producing new stems/branches under permissive environments. Axillary and adventitious buds are distinct in that axillary buds are formed in the axil of leaves and are responsible for production of lateral shoots (branches). Adventitious buds refer to buds that arise on the plant at places (stems, roots, or leaves) other than leaf axils. Both axillary and adventitious buds generally undergo periods of dormancy. Dormancy has been described as a temporary suspension of visible growth of any plant structure containing a meristem (Lang et al. 1987). Dormancy can be subdivided into three categories: (1) ecodormancy-arrest is under the control of external environmental factors; (2) paradormancy-arrest is under the control of external physiological factors within the plant; and (3) endodormancy-arrest is under the control of internal physiological factors. One common feature in all of these processes is prevention of growth under conditions where growth should otherwise continue. There is growing evidence that lack of growth is due to blockage of cell division resulting from interactions between the signaling pathways controlling dormancy and those controlling the cell cycle. Nomenclature: Abscisic acid, ABA; CDK-activating kinase, CAK; cyclin-dependent protein kinase, CDK; extracellular-signal-regulated kinase, ERK; gibberellic acid, GA; growth factor receptor, GFR; mitogen-activated protein kinase, MAPK; underground adventitious buds, UABs; nuclear export signal, NES; nuclear localization signal, NLS; retinoblastoma, RB; virus-induced gene silencing, VIGS.

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.

Potential model weeds to study genomics, ecology, and physiology in the 21st century

Abstract Plant model systems have contributed greatly to the dramatic progress in understanding the fundamental aspects of plant biology. Using model weeds will also help facilitate focused funding and research in the weed science community. Criteria for developing model weeds require attention to weedy characteristics that impart economic losses and a wide geographic distribution, attributes that present the potential for political and scientific support. Expressed sequence tag (EST) databases for model weeds are the most practical approach to identifying new genes and obtaining data on the gene expression underlying weedy characteristics. Weeds such as Canada thistle, eastern black nightshade, johnsongrass, jointed goatgrass, leafy spurge, waterhemp, and weedy rice are proposed as model systems. Nomenclature: Canada thistle, Cirsium arvense (L.) Scop CIRAR; common waterhemp, Amaranthus rudis Sauer AMATA; eastern black nightshade, Solanum ptycanthum Dun. SOLPT; johnsongrass, Sorghum halepense (L.) Pers SORHA; jointed goatgrass, Aegilops cylindrica Host. AEGCY; leafy spurge, Euphorbia esula L. EUPES; red rice (weedy rice), Oryza sativa L. ORYSA; tall waterhemp, Amaranthus tuberculatus (Moq.) J. D. Sauer AMATU.

Characterization of an EST Database for the Perennial Weed Leafy Spurge: An Important Resource for Weed Biology Research

Abstract Genomics programs in the weed science community have not developed as rapidly as that of other crop, horticultural, forestry, and model plant systems. Development of genomic resources for selected model weeds are expected to enhance our understanding of weed biology, just as they have in other plant systems. In this report, we describe the development, characteristics, and information gained from an expressed sequence tag (EST) database for the perennial weed leafy spurge. ESTs were obtained using a normalized cDNA library prepared from a comprehensive collection of tissues. During the EST characterization process, redundancy was minimized by periodic subtractions of the normalized cDNA library. A sequencing success rate of 88% yielded 45,314 ESTs with an average read length of 671 nucleotides. Using bioinformatic analysis, the leafy spurge EST database was assembled into 23,472 unique sequences representing 19,015 unigenes (10,293 clusters and 8,722 singletons). Blast similarity searches to the GenBank nonredundant protein database identified 18,186 total matches, of which 14,205 were nonredundant. These data indicate that 77.4% of the 23,472 unique sequences and 74.7% of the 19,015 unigenes are similar to other known proteins. Further bioinformatics analysis indicated that 2,950, or 15.5%, of the unigenes have previously not been identified suggesting that some may be novel to leafy spurge. Functional classifications assigned to leafy spurge unique sequences using Munich Information Center for Protein or Gene Ontology were proportional to functional classifications for genes of arabidopsis, with the exception of unclassified or unknowns and transposable elements which were significantly reduced in leafy spurge. Although these EST resources have been developed for the purpose of constructing high-density leafy spurge microarrays, they are already providing valuable information related to sugar metabolism, cell cycle regulation, dormancy, terpenoid secondary metabolism, and flowering. Nomenclature: Leafy spurge, Euphorbia esula L. EPHES, arabidopsis, Arabidopsis thaliana (L.) Heynh

Sugars, hormones, and environment affect the dormancy status in underground adventitious buds of leafy spurge (Euphorbia esula)

Abstract Signals from both leaves and apical or axillary meristems of leafy spurge are known to inhibit root bud growth. To test the hypothesis that carbohydrates and growth regulators affect root bud growth, decapitated leafy spurge plants were hydroponically treated with glucose, sucrose, gibberellic acid (GA), abscisic acid (ABA), 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA), and a GA biosynthesis inhibitor, paclobutrazol. Both glucose and sucrose caused suppression of root bud growth at concentrations of 30 mM. The inhibitory effect of sucrose was counteracted by GA at 15 μM. In contrast, BA, ABA, NAA, and paclobutrazol inhibited root bud growth at concentrations as low as 1, 2, 1, and 16 μM, respectively. Sugar and starch levels were also determined in root buds at various times after decapitation. Buds of intact plants contained the highest level of sucrose compared with buds harvested 1, 3, and 5 d after decapitation. To determine how seasonal changes affect root bud dormancy, growth from root buds of field-grown plants was monitored for several years. Root buds of field-grown leafy spurge had the highest level of innate dormancy from October to November, which persisted until a prolonged period of freezing occurred in November or early December. Our data support the hypothesis that carbohydrates may be involved in regulating dormancy status in root buds of leafy spurge. Nomenclature: Leafy spurge, Euphorbia esula L. EPHES.

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula).

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis “general purpose” traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species.

Bud Dormancy in Perennial Plants: A Mechanism for Survival

Dormancy in vegetative buds of perennial plants plays an important role for surviving harsh environmental conditions. Identifying the genetic and physiological mechanisms regulating dormancy in these vegetative structures will allow manipulation of plant growth and development in both crops and weeds. Model plants have been used to study the physiological effects that photoperiod and temperature impart on dormancy regulation in perennial buds. At the molecular level, models derived through analysis of the transcriptome have shed new light on multiple cellular pathways and physiological processes associated with dormancy transitions and, in some cases, have revealed overlap with pathways regulating flowering and cold acclimation. In this chapter, we discuss proposed models based on advances to our understanding of physiological and molecular factors affecting dormancy regulation in vegetative buds of perennials.

Transcriptome analysis of leafy spurge (Euphorbia esula) crown buds during shifts in well-defined phases of dormancy

Abstract Microarray analysis was used to follow changes in gene expression coinciding with seasonal changes in the dormancy status of crown buds of field-grown leafy spurge. Known cold-regulated genes were induced, and numerous gibberellic acid–responsive genes were down-regulated during the transition from paradormancy to endodormancy. Genes involved in photomorphogenesis were induced during endodormancy. Also, ethylene signaling responses were observed during ecodormancy rather than endodormancy. These results provide additional insights into the signals regulating expression of several genes previously associated with transition from paradormancy to growth in root buds. Nomenclature: Leafy spurge, Euphorbia esula L. EPHES.