Mapping and sequencing of the non-dormant evg mutant in peach [Prunus persica (L.) Batsch] identified six tandem-arrayed DAM (dormancy-associated MADS-box) genes as candidates for regulating growth cessation and terminal bud formation. To narrow the list of candidate genes, an attempt was made to associate bud phenology with the seasonal and environmental patterns of expression of the candidates in wild-type trees. The expression of the six peach DAM genes at the EVG locus of peach was characterized throughout an annual growing cycle in the field, and under controlled conditions in response to a long day–short day photoperiod transition. DAM1, 2, 4, 5, and 6 were responsive to a reduction in photoperiod in controlled conditions and the direction of response correlated with the seasonal timing of expression in field-grown trees. DAM3 did not respond to photoperiod and may be regulated by chilling temperatures. The DAM genes in peach appear to have at least four distinct patterns of expression. DAM1, 2, and 4 are temporally associated with seasonal elongation cessation and bud formation and are the most likely candidates for control of the evg phenotype.
Annual; bud; chilling; perennial; photoperiod
In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points.
We identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure.
The use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.
Background and Aims
In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy.
Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements.
Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development.
Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes.
Apricot; cambium activation; carbohydrate partitioning; chilling requirement; dormancy; meiosis; pollen development; Prunus armeniaca; stamen development; vascular differentiation
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.
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.
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.
Endo-dormant flower buds must pass through a period of chilling to reinitiate growth and subsequent flowering, which is a major obstacle to the forcing culture of tree peony in winter. Customized cDNA microarray (8×15 K element) was used to investigate gene expression profiling in tree peony ‘Feng Dan Bai’ buds during 24 d chilling treatment at 0–4°C. According to the morphological changes after the whole plants were transferred to green house, endo-dormancy was released after 18 d chilling treatment, and prolonged chilling treatment increased bud break rate. Pearson correlation hierarchical clustering of sample groups was highly consistent with the dormancy transitions revealed by morphological changes. Totally 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release process, of which the number of up-regulated (1,611) and that of down-regulated (1,563) was almost the same. Functional annotation of differentially-expressed genes revealed that cellular process, metabolic process, response to stimulus, regulation of biological process and development process were well-represented. Hierarchical clustering indicated that activation of genes involved in carbohydrate metabolism (Glycolysis, Citrate cycle and Pentose phosphate pathway), energy metabolism and cell growth. Based on the results of GO analysis, totally 51 probes presented in the microarray were associated with GA response and GA signaling pathway, and 22 of them were differently expressed. The expression profiles also revealed that the genes of GA biosynthesis, signaling and response involved in endo-dormancy release. We hypothesized that activation of GA pathway played a central role in the regulation of dormancy release in tree peony.
MicroRNAs (miRNAs) are small RNAs (sRNAs) approximately 21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. Within this context, miRNAs and siRNAs are coming to the forefront as molecular mediators of gene regulation in plant responses to annual temperature cycling and cold stress. For this reason, we chose to identify and characterize the conserved and non-conserved miRNA component of peach (Prunus persica (L.) Batsch) focusing our efforts on both the recently released whole genome sequence of peach and sRNA transcriptome sequences from two tissues representing non-dormant leaves and dormant leaf buds. Conserved and non-conserved miRNAs, and their targets were identified. These sRNA resources were used to identify cold-responsive miRNAs whose gene targets co-localize with previously described QTLs for chilling requirement (CR).
Analysis of 21 million peach sRNA reads allowed us to identify 157 and 230 conserved and non-conserved miRNA sequences. Among the non-conserved miRNAs, we identified 205 that seem to be specific to peach. Comparative genome analysis between peach and Arabidopsis showed that conserved miRNA families, with the exception of miR5021, are similar in size. Sixteen of these conserved miRNA families are deeply rooted in land plant phylogeny as they are present in mosses and/or lycophytes. Within the other conserved miRNA families, five families (miR1446, miR473, miR479, miR3629, and miR3627) were reported only in tree species (Populustrichocarpa, Citrus trifolia, and Prunus persica). Expression analysis identified several up-regulated or down-regulated miRNAs in winter buds versus young leaves. A search of the peach proteome allowed the prediction of target genes for most of the conserved miRNAs and a large fraction of non-conserved miRNAs. A fraction of predicted targets in peach have not been previously reported in other species. Several conserved and non-conserved miRNAs and miRNA-regulated genes co-localize with Quantitative Trait Loci (QTLs) for chilling requirement (CR-QTL) and bloom date (BD-QTL).
In this work, we identified a large set of conserved and non-conserved miRNAs and describe their evolutionary footprint in angiosperm lineages. Several of these miRNAs were induced in winter buds and co-localized with QTLs for chilling requirement and bloom date thus making their gene targets potential candidates for mediating plant responses to cold stress. Several peach homologs of genes participating in the regulation of vernalization in Arabidopsis were identified as differentially expressed miRNAs targets, potentially linking these gene activities to cold responses in peach dormant buds. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to peach and/or other tree species.
microRNAs; Distribution; Expression; Cold stress; Chilling requirement; Bud development
American chestnut (Castanea dentata) was devastated by an exotic pathogen in the beginning of the twentieth century. This chestnut blight is caused by Cryphonectria parasitica, a fungus that infects stem tissues and kills the trees by girdling them. Because of the great economic and ecological value of this species, significant efforts have been made over the century to combat this disease, but it wasn't until recently that a focused genomics approach was initiated. Prior to the Genomic Tool Development for the Fagaceae project, genomic resources available in public databases for this species were limited to a few hundred ESTs. To identify genes involved in resistance to C. parasitica, we have sequenced the transcriptome from fungal infected and healthy stem tissues collected from blight-sensitive American chestnut and blight-resistant Chinese chestnut (Castanea mollissima) trees using ultra high throughput pyrosequencing.
We produced over a million 454 reads, totaling over 250 million bp, from which we generated 40,039 and 28,890 unigenes in total from C. mollissima and C. dentata respectively.
The functions of the unigenes, from GO annotation, cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. In silico expression analyses showed that many of the stress response unigenes were expressed more in canker tissues versus healthy stem tissues in both American and Chinese chestnut. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either American or Chinese chestnut canker tissues.
Our study resulted in the identification of a large set of cDNA unigenes from American chestnut and Chinese chestnut. The ESTs and unigenes from this study constitute an important resource to the scientific community interested in the discovery of genes involved in various biological processes in Chestnut and other species. The identification of many defense-related genes differentially expressed in canker vs. healthy stem in chestnuts provides many new candidate genes for developing resistance to the chestnut blight and for studying pathways involved in responses of trees to necrotrophic pathogens. We also identified several candidate genes that may underline the difference in resistance to Cryphonectria parasitica between American chestnut and Chinese chestnut.
• Background and Aims The control of dormancy in yam (Disocorea spp.) tubers is poorly understood and attempts to shorten the long dormant period (i.e. cause tubers to sprout or germinate much earlier) have been unsuccessful. The aim of this study was to identify and define the phases of dormancy in Dioscorea rotundata tubers, and to produce a framework within which dormancy can be more effectively studied.
• Methods Plants of ‘TDr 131’ derived from tissue culture were grown in a glasshouse simulating temperature and photoperiod at Ibadan (7°N), Nigeria to produce tubers. Tubers were sampled on four occasions: 30 d before shoot senescence (149 days after planting, DAP), at shoot senescence (179 DAP), and twice during storage at a constant 25 °C (269 and 326 DAP). The development of the apical shoot bud was described from tissue sections. In addition, the responsiveness of shoot apical bud development to plant growth regulators (gibberellic acid, 2-chloroethanol and thiourea) applied to excised tuber sections was also examined 6 and 12 d after treatment.
• Key Results and Conclusions Three phases of tuber dormancy are proposed: Phase I, from tuber initiation to the appearance of the tuber germinating meristem; Phase II, from the tuber germinating meristem to initiation of foliar primordium; and Phase III, from foliar primordium to appearance of the shoot bud on the surface of the tuber. Phase I is the longest phase (approx. 220 d in ‘TDr 131’), is not affected by PGRs and is proposed to be an endo-dormant phase. Phases II and III are shorter (<70 d in total), are influenced by PGRs and environmental conditions, and are therefore endo-/eco-dormant phases. To manipulate dormancy to allow off-season planting and more than one generation per year requires that the duration of Phase I is shortened.
Dioscorea rotundata; yam; dormancy; phases of dormancy; plant growth regulator; primary thickening meristem; tuber germinating meristem; shoot apical meristem
The detrimental effects of mild winter temperatures on the consistency of cropping of blackcurrant (Ribes nigrum L.) in parts of Europe have led to increasing interest in the genetic control of dormancy release in this species. This study examined patterns of gene expression in leaf buds of blackcurrant to identify key differential changes in these profiles around the time of budbreak.
Using leaf bud tissue of blackcurrant, a cDNA library was generated as a source of blackcurrant ESTs for construction of a custom microarray, which was used to identify differential gene expression during dormancy release. Gene activity was lowest in early stages of dormancy, increasing to reach a maximum around the time of budbreak. Genes with significantly changing expression profiles were clustered and evidence is provided for the transient activity of genes previously associated with dormancy processes in other species. Expression profiling identified candidate genes which were mapped onto a blackcurrant genetic linkage map containing budbreak-related QTL. Three genes, which putatively encode calmodulin-binding protein, beta tubulin and acetyl CoA carboxylase respectively, were found to co-localise with budbreak QTL.
This study provides insight into the genetic control of dormancy transition in blackcurrant, identifying key changes in gene expression around budbreak. Genetic mapping of ESTs enabled the identification of genes which co-localise with previously-characterised blackcurrant QTL, and it is concluded that these genes have probable roles in release of dormancy and can therefore provide a basis for the development of genetic markers for future breeding deployment.
Bud dormancy is a critical developmental process that allows perennial plants to survive unfavorable environmental conditions. Pear is one of the most important deciduous fruit trees in the world, but the mechanisms regulating bud dormancy in this species are unknown. Because genomic information for pear is currently unavailable, transcriptome and digital gene expression data for this species would be valuable resources to better understand the molecular and biological mechanisms regulating its bud dormancy.
We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of ‘Suli’ pear (Pyrus pyrifolia white pear group) using the Illumina RNA-seq system. RNA-Seq generated approximately 100 M high-quality reads that were assembled into 69,393 unigenes (mean length = 853 bp), including 14,531 clusters and 34,194 singletons. A total of 51,448 (74.1%) unigenes were annotated using public protein databases with a cut-off E-value above 10-5. We mainly compared gene expression levels at four time-points during bud dormancy. Between Nov. 15 and Dec. 15, Dec. 15 and Jan. 15, and Jan. 15 and Feb. 15, 1,978, 1,024, and 3,468 genes were differentially expressed, respectively. Hierarchical clustering analysis arranged 190 significantly differentially-expressed genes into seven groups. Seven genes were randomly selected to confirm their expression levels using quantitative real-time PCR.
The new transcriptomes offer comprehensive sequence and DGE profiling data for a dynamic view of transcriptomic variation during bud dormancy in pear. These data provided a basis for future studies of metabolism during bud dormancy in non-model but economically-important perennial species.
‘Suli’ pear (Pyrus pyrifolia white pear group); Transcriptome; Bud dormancy; RNA-Seq
Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).
A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.
We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.
• Background and Aims Asarum caudigerum (Aristolochiaceae) is an important species of paleoherb in relation to understanding the origin and evolution of angiosperm flowers, due to its basal position in the angiosperms. The aim of this study was to isolate floral-related genes from A. caudigerum, and to infer evolutionary relationships among florally expression-related genes, to further illustrate the origin and diversification of flowers in angiosperms.
• Methods A subtracted floral cDNA library was constructed from floral buds using suppression subtractive hybridization (SSH). The cDNA of floral buds and leaves at the seedling stage were used as a tester and a driver, respectively. To further identify the function of putative MADS-box transcription factors, phylogenetic trees were reconstructed in order to infer evolutionary relationships within the MADS-box gene family.
• Key Results In the forward-subtracted floral cDNA library, 1920 clones were randomly sequenced, from which 567 unique expressed sequence tags (ESTs) were obtained. Among them, 127 genes failed to show significant similarity to any published sequences in GenBank and thus are putatively novel genes.
• Conclusions Phylogenetic analysis indicated that a total of 29 MADS-box transcription factors were members of the APETALA3(AP3) subfamily, while nine others were putative MADS-box transcription factors that formed a cluster with MADS-box genes isolated from Amborella, the basal-most angiosperm, and those from the gymnosperms. This suggests that the origin of A. caudigerum is intermediate between the angiosperms and gymnosperms.
Expressed sequence tags (ESTs); Asarum caudigerum; floral genes; suppression subtractive hybridization (SSH); MADS-box gene; phylogenetic reconstruction
Vegetative buds provide plants in temperate environments the possibility for growth and reproduction when environmental conditions are favorable. In grapevine, crucial developmental events take place within buds during two growing seasons in consecutive years. The first season, the shoot apical meristem within the bud differentiates all the basic elements of the shoot including flowering transition in lateral primordia and development of inflorescence primordia. These events practically end with bud dormancy. The second season, buds resume shoot growth associated to flower formation and development. Gene expression has been previously monitored at specific stages of bud development but has never been followed along the two growing seasons.
Gene expression changes were analyzed along the bud annual cycle at eight different time points. Principal Components Analysis (PCA) revealed that the main factors explaining the global gene expression differences were the processes of bud dormancy and active growth as well as stress responses. Accordingly, non dormant buds showed an enrichment in functional categories typical of actively proliferating and growing cells together with the over abundance of transcripts belonging to stress response pathways. Differential expression analyses performed between consecutive time points indicated that major transcriptional changes were associated to para/endodormancy, endo/ecodormancy and ecodormancy/bud break transitions. Transcripts encoding key regulators of reproductive development were grouped in three major expression clusters corresponding to: (i) transcripts associated to flowering induction, (ii) transcripts associated to flower meristem specification and initiation and (iii) transcripts putatively involved in dormancy. Within this cluster, a MADS-box gene (VvFLC2) and other transcripts with similar expression patterns could participate in dormancy regulation.
This work provides a global view of major transcriptional changes taking place along bud development in grapevine, highlighting those molecular and biological functions involved in the main events of bud development. As reported in other woody species, the results suggest that genes regulating flowering could also be involved in dormancy regulatory pathways in grapevine.
Grapevine; Bud development; Dormancy; Flowering; Flower development; Transcriptomics
The present study investigated the expressional regulation of PpDAM5 and PpDAM6, two of the six peach (Prunus persica) dormancy-associated MADS-box genes, in relation to lateral bud endodormancy. PpDAM5 and PpDAM6 were originally identified as homologues of Arabidopsis SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 identified in the EVERGROWING locus of peach. Furthermore, PpDAM5 and PpDAM6 have recently been suggested to be involved in terminal bud dormancy. In this study, seasonal expression analyses using leaves, stems, and lateral buds of high-chill and low-chill peaches in field conditions indicated that both genes were up-regulated during the endodormancy period and down-regulated with endodormancy release. Controlled environment experiments showed that the expression of both PpDAM5 and PpDAM6 were up-regulated by ambient cool temperatures in autumn, while they were down-regulated by the prolonged period of cold temperatures in winter. A negative correlation between expression levels of PpDAM5 and PpDAM6 and bud burst percentage was found in the prolonged cold temperature treatment. Application of the dormancy-breaking reagent cyanamide to endo/ecodormant lateral buds induced early bud break and down-regulation of PpDAM5 and PpDAM6 expression at the same time. These results collectively suggest that PpDAM5 and PpDAM6 may function in the chilling requirement of peach lateral buds through growth-inhibiting functions for bud break.
bud dormancy; chilling requirement; cyanamide; low-chill peaches; Prunus; StMADS11
Branching patterns are major determinants of plant architecture. They depend both on leaf phillotaxy (branch primordia are formed in the axils of leaves) and on the decision of buds to grow out to give a branch or to remain dormant. In Arabidopsis, several genes involved in the long-distance signalling of the control of branch outgrowth have been identified. However, the genes acting inside the buds to cause growth arrest remained unknown until now. In the February issue of Plant Cell we have described the function of BRANCHED1 (BRC1), an Arabidopsis gene coding for a plant-specific transcription factor of the TCP family that is expressed in the buds and prevents their development. Loss of BRC1 function leads to accelerated AM initiation, precocious progression of bud development and excess of shoot branching. BRC1 transcription is affected by endogenous and environmental signals controlling branching and we have shown that BRC1 function mediates the response to these stimuli. Therefore we have proposed that BRC1 function represents the point at which signals controlling branching are integrated within axillary buds.
Arabidopsis; plant architecture; bud dormancy; branching; TCP genes; transcription factors
Bud mutations often arise in citrus. The selection of mutants is one of the most important breeding channels in citrus. However, the molecular basis of bud mutation has rarely been studied. To identify differentially expressed genes in a spontaneous sweet orange [C. sinensis (L.) Osbeck] bud mutation which causes lycopene accumulation, low citric acid, and high sucrose in fruit, suppression subtractive hybridization and microarray analysis were performed to decipher this bud mutation during fruit development. After sequencing of the differentially expressed clones, a total of 267 non-redundant transcripts were obtained and 182 (68.2%) of them shared homology (E-value ≤1×10−10) with known gene products. Few genes were constitutively up- or down-regulated (fold change ≥2) in the bud mutation during fruit development. Self-organizing tree algorithm analysis results showed that 95.1% of the differentially expressed genes were extensively coordinated with the initiation of lycopene accumulation. Metabolic process, cellular process, establishment of localization, response to stimulus, and biological regulation-related transcripts were among the most regulated genes. These genes were involved in many biological processes such as organic acid metabolism, lipid metabolism, transport, and pyruvate metabolism, etc. Moreover, 13 genes which were differentially regulated at 170 d after flowering shared homology with previously described signal transduction or transcription factors. The information generated in this study provides new clues to aid in the understanding of bud mutation in citrus.
Bud mutation; candidate genes; cDNA microarray; Citrus; real-time RT-PCR; suppression subtractive hybridization (SSH)
Background and Aims
Although a claim has been made that dormancy cycling occurs in seeds of Ipomoea lacunosa (Convolvulaceae) with physical dormancy, this would seem to be impossible since the water gap cannot be closed again after it opens (dormancy break). On the other hand, changes in sensitivity (sensitive ↔ non-sensitive) to dormancy-breaking factors have been reported in seeds of Fabaceae with physical dormancy. The primary aim of the present study was to determine if sensitivity cycling also occurs in physically dormant seeds of I. lacunosa.
Treatments simulating conditions in the natural habitat of I. lacunosa were used to break seed dormancy. Storage of seeds at temperatures simulating those in spring, summer, autumn and winter were tested for their effect on sensitivity change. Seeds made non-dormant were stored dry in different temperature regimes to test for dormancy cycling. In addition, seeds collected on different dates (i.e. matured under different climatic conditions) were used to test for maternal effects on sensitivity to dormancy-breaking factors.
Sensitivity was induced by storing seeds under wet conditions and reversed by storing them under dry conditions at low (≤5 °C) or high (≥30 °C) temperatures, demonstrating that seeds of I. lacunosa can cycle between sensitive and insensitive states. Sensitive seeds required ≥2 h at 35 °C on moist sand for release of dormancy. However, there is no evidence to support dormancy cycling per se. Conceptual models are proposed for sensitivity cycling and germination phenology of I. lacunosa in the field.
Seasonal germination behaviour of physically dormant I. lacunosa seeds can be explained by sensitivity cycling but not by dormancy cycling per se. Convolvulaceae is only the second of 16 families known to contain species with physical dormancy for which sensitivity cycling has been demonstrated.
Convolvulaceae; germination phenology; Ipomoea; physical dormancy; sensitivity cycling
The aerobic fast-growing Mycobacterium smegmatis, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior.
In a library generated by transposon mutagenesis, we searched for M. smegmatis mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the uvrA gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively.
Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.
• Background and Aims Dwarf mistletoes (Arceuthobium; Viscaceae) are highly specialized dioecious angiosperms parasitic on many gymnosperm hosts in the northern hemisphere. Several dwarf mistletoe species are capable of inducing an unusual form of isophasic infection in which the internal (endophytic) system proliferates even into the apical buds of its hosts. Studies of the internal endophytic system have, for the most part, focused on the parasite within secondary host tissues. The present anatomical and ultrastructural study characterizes the growth pattern of the isophasic endophytic system of Arceuthobium douglasii within the dormant apical buds of Pseudotsuga menziesii.
• Methods Semi-thin serial sections from dwarf mistletoe-infected host apical buds were mounted, stained and micrographed. Graphic files were created from the serial micrographs and these files were stacked. These stacked files were utilized to describe the pattern of growth of the endophyte within the host tissue. The interface between cells of the mistletoe and host was also examined at the ultrastructural level by transmission electron microscopy.
• Key Results By utilizing a novel technique of superimposed graphics, the current study reveals an organized pattern of mistletoe distribution that penetrates further into host tissues than previously known. A consistent pattern of growth occurring even into the preformed leaves of the host is documented.
• Conclusions The apparently non-intrusive growth of the parasite appears to be developmentally synchronized with that of the host. No symplastic connections were observed in the ultrastructural examination of the parasite/host interface within the apical buds of Pseudotsuga menziesii parasitized by A. douglasii or of Pinus contorta parasitized by A. americanum.
Anatomy; Arceuthobium; British Columbia; dwarf mistletoe; Douglas fir; endophytic; isophasic; Viscaceae
MADS-box genes similar to Arabidopsis SHORT VEGETATIVE PHASE (SVP) have been implicated in the regulation of flowering in annual species and bud dormancy in perennial species. Kiwifruit (Actinidia spp.) are woody perennial vines where bud dormancy and out-growth affect flower development. To determine the role of SVP-like genes in dormancy and flowering of kiwifruit, four MADS-box genes with homology to Arabidopsis SVP, designated SVP1, SVP2, SVP3, and SVP4, have been identified and analysed in kiwifruit and functionally characterized in Arabidopsis. Phylogenetic analysis indicate that these genes fall into different sub-clades within the SVP-like gene group, suggesting distinct functions. Expression was generally confined to vegetative tissues, and increased transcript accumulation in shoot buds over the winter period suggests a role for these genes in bud dormancy. Down-regulation before flower differentiation indicate possible roles as floral repressors. Over-expression and complementation studies in Arabidopsis resulted in a range of floral reversion phenotypes arising from interactions with Arabidopsis MADS-box proteins, but only SVP1 and SVP3 were able to complement the svp mutant. These results suggest that the kiwifruit SVP-like genes may have distinct roles during bud dormancy and flowering.
Actinidia species; AGL24; Arabidopsis; budbreak; bud dormancy; flowering; hydrogen cyanamide; kiwifruit; SVP
Reproductive meristems and embryos display dormancy mechanisms in specialized structures named respectively buds and seeds that arrest the growth of perennial plants until environmental conditions are optimal for survival. Dormancy shows common physiological features in buds and seeds. A genotype-specific period of chilling is usually required to release dormancy by molecular mechanisms that are still poorly understood. In order to find common transcriptional pathways associated to dormancy release, we analyzed the chilling-dependent expression in embryos of certain genes that were previously found related to dormancy in flower buds of peach. We propose the presence of short and long-term dormancy events affecting respectively the germination rate and seedling development by independent mechanisms. Short periods of chilling seem to improve germination in an abscisic acid-dependent manner, whereas the positive effect of longer cold treatments on physiological dwarfing coincides with the accumulation of phenylpropanoids in the seed.
Background and Aims
Pathogen–seed interactions may involve a race for seed resources, so that seeds that germinate more quickly, mobilizing reserves, will be more likely to escape seed death than slow-germinating seeds. This race-for-survival hypothesis was tested for the North American seed pathogen Pyrenophora semeniperda on seeds of the annual grass Bromus tectorum, an invasive plant in North America. In this species, the seed germination rate varies as a function of dormancy status; dormant seeds germinate slowly if at all, whereas non-dormant seeds germinate quickly.
Three experimental approaches were utilized: (a) artificial inoculations of mature seeds that varied in primary dormancy status and wounding treatment; (b) naturally inoculated undispersed seeds that varied in primary dormancy status; and (c) naturally inoculated seeds from the carry-over seed bank that varied in degree of secondary dormancy, habitat of origin and seed age.
In all three approaches, seeds that germinated slowly were usually killed by the pathogen, whereas seeds that germinated quickly frequently escaped. Pyrenophora semeniperda reduced B. tectorum seed banks. Populations in drier habitats sustained 50 times more seed mortality than a population in a mesic habitat. Older carry-over seeds experienced 30 % more mortality than younger seeds.
Given the dramatic levels of seed death and the ability of this pathogen to reduce seed carry-over, it is intriguing to consider whether P. semeniperda could be used to control B. tectorum through direct reduction of its seed bank.
Biocontrol; biotic resistance; cheatgrass; Drechslera campanulata; invasive species; mycoherbicide; pathogen; seed bank; seed-borne
Background and Aims
The water gap is an important morphoanatomical structure in seeds with physical dormancy (PY). It is an environmental signal detector for dormancy break and the route of water into the non-dormant seed. The Convolvulaceae, which consists of subfamilies Convolvuloideae (11 tribes) and Humbertoideae (one tribe, monotypic Humberteae), is the only family in the asterid clade known to produce seeds with PY. The primary aim of this study was to compare the morphoanatomical characteristics of the water gap in seeds of species in the 11 tribes of the Convolvuloideae and to use this information, and that on seed dormancy and storage behaviour, to construct a phylogenetic tree of seed dormancy for the subfamily.
Scanning electron microscopy (SEM) was used to define morphological changes in the hilum area during dormancy break; hand and vibratome sections were taken to describe the anatomy of the water gap, hilum and seed coat; and dye tracking was used to identify the initial route of water entry into the non-dormant seed. Results were compared with a recent cladogram of the family.
Species in nine tribes have (a) layer(s) of palisade cells in the seed coat, a water gap and orthodox storage behaviour. Erycibe (Erycibeae) and Maripa (Maripeae) do not have a palisade layer in the seed coat or a water gap, and are recalcitrant. The hilar fissure is the water gap in relatively basal Cuscuteae, and bulges adjacent to the micropyle serve as the water gap in the Convolvuloideae, Dicranostyloideae (except Maripeae) and the Cardiochlamyeae clades. Seeds from the Convolvuloideae have morphologically prominent bulges demarcated by cell shape in the sclereid layer, whereas the Dicranostyloideae and Cardiochlamyeae have non-prominent bulges demarcated by the number of sub-cell layers. The anatomy and morphology of the hilar pad follow the same pattern.
PY in the subfamily Convolvuloideae probably evolved in the aseasonal tropics from an ancestor with recalcitrant non-dormant seeds, and it may have arisen as Convolvulaceae radiated to occupy the seasonal tropics. Combinational dormancy may have developed in seeds of some Cuscuta spp. as this genus moved into temperate habitats.
Convolvulaceae; evolution; hilar fissure; physical dormancy; water gap
Spore germination of the yeast Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation.
Employing detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors.
Resumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores.
Sporulation; Germination; Saccharomyces cerevisiae
Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear.
We used mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar (Populus simonii × P. nigra). There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues.
This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants.