Floral Bud Development Research Articles

Genome-Wide Identification MIKC-Type MADS-Box Gene Family and Their Roles during Development of Floral Buds in Wheel Wingnut (Cyclocarya paliurus).

MADS-box transcription factors (TFs) have fundamental roles in regulating floral organ formation and flowering time in flowering plants. In order to understand the function of MIKC-type MADS-box family genes in Cyclocarya paliurus (Batal.) Iljinskaja, we first implemented a genome-wide analysis of MIKC-type MADS-box genes in C. paliurus. Here, the phylogenetic relationships, chromosome location, conserved motif, gene structure, promoter region, and gene expression profile were analyzed. The results showed that 45 MIKC-type MADS-box were divided into 14 subfamilies: BS (3), AGL12 (1), AP3-PI (3), MIKC* (3), AGL15 (3), SVP (5), AGL17 (2), AG (3), TM8 (1), AGL6 (2), SEP (5), AP1-FUL (6), SOC1 (7), and FLC (1). The 43 MIKC-type MADS-box genes were distributed unevenly in 14 chromosomes, but two members were mapped on unanchored scaffolds. Gene structures were varied in the same gene family or subfamily, but conserved motifs shared similar distributions and sequences. The element analysis in promoters’ regions revealed that MIKC-type MADS-box family genes were associated with light, phytohormone, and temperature responsiveness, which may play important roles in floral development and differentiation. The expression profile showed that most MIKC-type MADS-box genes were differentially expressed in six tissues (specifically expressed in floral buds), and the expression patterns were also visibly varied in the same subfamily. CpaF1st24796 and CpaF1st23405, belonging to AP3-PI and SEP subfamilies, exhibited the high expression levels in PA-M and PG-F, respectively, indicating their functions in presenting heterodichogamy. We further verified the MIKC-type MADS-box gene expression levels on the basis of transcriptome and qRT-PCR analysis. This study would provide a theoretical basis for classification, cloning, and regulation of flowering mechanism of MIKC-type MADS-box genes in C. paliurus.

Preanthesis changes in freeze resistance, relative water content, and ovary growth preempt bud phenology and signify dormancy release of sour cherry floral buds.

Growing degree hours (GDH) predicted floral bud development of 'Montmorency' sour cherry and explained changes in lethal temperatures (LT50) that preempted any visible changes in bud phenology. The gradual warming during late winter and early spring promotes floral bud development and, concomitantly, the de-acclimation of Prunus sp. flowers. In fact, once ecodormancy releases, an approximate 20°C loss of hardiness occurs prior to any distinguishable changes in external bud phenology. The aim of the following work was to characterize the physiological changes of 'Montmorency' sour cherry floral buds as they transition from endo- and ecodormancy and resume growth, and to determine whether physiological and anatomical characteristics within the buds preempt or signify dormancy release to enable a better prediction of freeze susceptibility. Here, we present a developmental timeline of the preanthesis changes of 'Montmorency' floral buds, ovaries and anthers over 2 years following their completion of chilling and relate these changes to growing degree hours (GDH) and the lethal temperature (LT50) of flowers. Changes in bud dry weight (DW), fresh weight (FW), volume, and external phenology stage including the percentage of green color development of bud scales were predicted by heat accumulation but were not early predictors of the increasing freeze susceptibility of pistils. Between endodormancy and green tip stage, ovary volume increased nearly threefold and relative water content (RWC) increased from ~ 45 to 70% in both years. A linear mixed regression model indicated that RWC and the interaction between RWC and ovary growth were significant predictors of LT50. Importantly, the loss of ~ 20°C of freeze resistance occurred between 45 and 57% RWC and preceded any detectable changes in bud phenology. Microsporogenesis was observed after dormancy release when measurable changes in the ovary and bud RWC had already occurred. A GDH model estimated freeze sensitivity of pistils and explained 93% of the variation in LT50 during preanthesis development. A simple GDH model to predict critical freeze temperature of pistils should aid producers to manage frost protection.

ITRAQ-based proteomic analysis of apple buds provides new insights into regulatory mechanisms of flowering in response to shoot bending

Flowering is an important life cycle stage in apple trees, and young ‘Nagafu No. 2’ produce limited inferior flower buds. Shoot bending, an effective agronomic measure, is widely used to promote flowering but little is known regarding the underlying molecular mechanisms. Using isobaric tags for relative and absolute quantification LC−MS/MS, 8,585 proteins expressed in buds during flower bud differentiation were identified. The 1,060 up- and 1,079 down-regulated proteins after shoot bending were divided into 7 pattern-based groups: I (22/5), II (30/57), III (34/21), IV (77/61), V (381/452), VI (218/170), and VII (298/313) types. Number of proteins were associated with carbohydrate, fatty acid, and lipid metabolism, as well as hormone, transcription factor, circadian rhythm, and photoperiod and flowering pathways. The differentially expressed proteins involved in fatty acid and lipid pathways were up-regulated by shoot bending, while other proteins that were involved in starch synthesis were down-regulated. In addition, circadian rhythm- and flowering-related genes were up-regulated by shoot bending. This research provides a valuable inventory of proteins involved in floral bud development and contributes to the understanding of the regulatory mechanisms of flower bud differentiation caused by shoot bending in apple trees.

Is seasonal flowering time of Paphiopedilum species caused by differences in initial time of floral bud differentiation?

Members of the genus Paphiopedilum are world-famous for their large, colourful flowers, unique floral morphology and long floral lifespan. Most Paphiopedilum species bloom in spring or autumn. The control of flowering time is of great significance to the commercial production of floral crops, because it affects the sales and prices of flowers. However, the mechanism that regulates when Paphiopedilum species bloom is unclear. In the present study, floral bud initiation and development of P. micranthum (spring-flowering species with one flower per stalk), P. dianthum (autumn-flowering species with multiple flowers per stalk) and P. henryanum (autumn-flowering species with one flower per stalk) were investigated by morphological and anatomical methods. We divided Paphiopedilum floral bud differentiation into six phases: the initiation of differentiation, inflorescence primordium differentiation, flower primordium differentiation, sepal primordium differentiation, petal primordium differentiation and column primordium differentiation. We found that the timing of floral bud differentiation for the three species was synchronized when experiencing the same environment, while the period from initiation to flowering largely differed. In addition, initiation of floral bud differentiation in P. dianthum was earlier at a warmer environment. The difference in flowering time of three species was mainly caused by the duration of floral bud development, rather than the initiation time. The findings were of great significance for the cultivation and flowering regulation of Paphiopedilum species.

Characterization of Japanese Apricot (Prunus mume) Floral Bud Development Using a Modified BBCH Scale and Analysis of the Relationship between BBCH Stages and Floral Primordium Development and the Dormancy Phase Transition

Bud dormancy is an important developmental stage that ensures that trees can tolerate environmental stresses in winter and bloom uniformly in the following spring. Regarding Rosaceae floral buds, exposure to chilling conditions promotes floral primordium development and the transition from endodormancy to ecodormancy. A subsequent period of warm conditions induces blooming. In Japanese apricot (Prunus mume), dormancy progression is accompanied by morphological changes that alter the bud appearance and internal structures. We used a modified BBCH scale and conducted microscopy analyses to elucidate the bud developmental stage of three cultivars with contrasting chilling requirements. The floral bud developmental period corresponding to BBCH stages 51–53 includes the transition from endodormancy to ecodormancy in all three cultivars. Male meiosis and microspore development occurred during this transition in high-chill cultivars, but were detected considerably later than the transition in the low-chill cultivar. A slow or suspended developmental phase was observed only for the high-chill cultivars upon completion of floral primordium organ differentiation, suggesting that chilling may be required to induce floral bud maturation and dormancy release only in high-chill cultivars. Possible relationships among BBCH stages, flowering-related morphological characteristics, and the dormancy phase transition in Japanese apricot are discussed.

Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic floral buds of andromonoecious Diospyros kaki Thunb

BackgroundPersimmon (Diospyros kaki Thunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluated OGI and MeGI expression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon ‘Longyanyeshi 1’.ResultsStages 2 and 4 were identified as the critical morphological periods for sex differentiation of ‘Longyanyeshi 1’ by histological and cytological observation. At both stages, OGI was differentially expressed in male and hermaphroditic buds, but MeGI was not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA3, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (including Aux/IAA, ARFs, DELLA, AHP, A-ARR, B-ARR, CYP735A, CRE1, PP2C, JAZ, MYC2, COI1, CTR1, SIMKK, ACO, and MPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition, MYB, FAR1, bHLH, WRKY, and MADS transcription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation.ConclusionThe present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of ‘Longyanyeshi 1’ during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

Genome-wide identification, characterization, and expression analysis of the NAC transcription factor family in orchardgrass (Dactylis glomerata L.)

BackgroundOrchardgrass (Dactylis glomerata L.) is one of the most important cool-season perennial forage grasses that is widely cultivated in the world and is highly tolerant to stressful conditions. However, little is known about the mechanisms underlying this tolerance. The NAC (NAM, ATAF1/2, and CUC2) transcription factor family is a large plant-specific gene family that actively participates in plant growth, development, and response to abiotic stress. At present, owing to the absence of genomic information, NAC genes have not been systematically studied in orchardgrass. The recent release of the complete genome sequence of orchardgrass provided a basic platform for the investigation of DgNAC proteins.ResultsUsing the recently released orchardgrass genome database, a total of 108 NAC (DgNAC) genes were identified in the orchardgrass genome database and named based on their chromosomal location. Phylogenetic analysis showed that the DgNAC proteins were distributed in 14 subgroups based on homology with NAC proteins in Arabidopsis, including the orchardgrass-specific subgroup Dg_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 15, and multitudinous DgNAC genes contained three exons. Chromosomal mapping analysis found that the DgNAC genes were unevenly distributed on seven orchardgrass chromosomes. For the gene expression analysis, the expression levels of DgNAC genes in different tissues and floral bud developmental stages were quite different. Quantitative real-time PCR analysis showed distinct expression patterns of 12 DgNAC genes in response to different abiotic stresses. The results from the RNA-seq data revealed that orchardgrass-specific NAC exhibited expression preference or specificity in diverse abiotic stress responses, and the results indicated that these genes may play an important role in the adaptation of orchardgrass under different environments.ConclusionsIn the current study, a comprehensive and systematic genome-wide analysis of the NAC gene family in orchardgrass was first performed. A total of 108 NAC genes were identified in orchardgrass, and the expression of NAC genes during plant growth and floral bud development and response to various abiotic stresses were investigated. These results will be helpful for further functional characteristic descriptions of DgNAC genes and the improvement of orchardgrass in breeding programs.

A gene expression atlas for kiwifruit (Actinidia chinensis) and network analysis of transcription factors

BackgroundTranscriptomic studies combined with a well annotated genome have laid the foundations for new understanding of molecular processes. Tools which visualise gene expression patterns have further added to these resources. The manual annotation of the Actinidia chinensis (kiwifruit) genome has resulted in a high quality set of 33,044 genes. Here we investigate gene expression patterns in diverse tissues, visualised in an Electronic Fluorescent Pictograph (eFP) browser, to study the relationship of transcription factor (TF) expression using network analysis.ResultsSixty-one samples covering diverse tissues at different developmental time points were selected for RNA-seq analysis and an eFP browser was generated to visualise this dataset. 2839 TFs representing 57 different classes were identified and named. Network analysis of the TF expression patterns separated TFs into 14 different modules. Two modules consisting of 237 TFs were correlated with floral bud and flower development, a further two modules containing 160 TFs were associated with fruit development and maturation. A single module of 480 TFs was associated with ethylene-induced fruit ripening. Three “hub” genes correlated with flower and fruit development consisted of a HAF-like gene central to gynoecium development, an ERF and a DOF gene. Maturing and ripening hub genes included a KNOX gene that was associated with seed maturation, and a GRAS-like TF.ConclusionsThis study provides an insight into the complexity of the transcriptional control of flower and fruit development, as well as providing a new resource to the plant community. The Actinidia eFP browser is provided in an accessible format that allows researchers to download and work internally.

Genome-wide comparative transcriptome analysis of the A4-CMS line ICPA 2043 and its maintainer ICPB 2043 during the floral bud development of pigeonpea.

Cytoplasmic male sterility (CMS) offers a unique system to understand cytoplasmic nuclear crosstalk, and is also employed for exploitation of hybrid vigor in various crops. Pigeonpea A4-CMS, a predominant source of male sterility, is being used for efficient hybrid seed production. The molecular mechanisms of CMS trait remain poorly studied in pigeonpea. We performed genome-wide transcriptome profiling of A4-CMS line ICPA 2043 and its isogenic maintainer ICPB 2043 at two different stages of floral bud development (stage S1 and stage S2). Consistent with the evidences from some other crops, we also observed significant difference in the expression levels of genes in the later stage, i.e., stage S2. Differential expression was observed for 143 and 55 genes within the two stages of ICPA 2043 and ICPB 2043, respectively. We obtained only 10 differentially expressed genes (DEGs) between the stage S1 of the two genotypes, whereas expression change was significant for 582 genes in the case of stage S2. The qRT-PCR assay of randomly selected six genes supported the differential expression of genes between ICPA 2043 and ICPB 2043. Further, GO and KEGG pathway mapping suggested a possible compromise in key bioprocesses during flower and pollen development. Besides providing novel insights into the functional genomics of CMS trait, our results were in strong agreement with the gene expression atlas of pigeonpea that implicated various candidate genes like sucrose-proton symporter 2 and an uncharacterized protein along with pectate lyase, pectinesterase inhibitors, L-ascorbate oxidase homolog, ATPase, β-galactosidase, polygalacturonase, and aldose 1-epimerase for pollen development of pigeonpea. The dataset presented here provides a rich genomic resource to improve understanding of CMS trait and its deployment in heterosis breeding in pigeonpea.

Genome-wide characterization of PEBP family genes in nine Rosaceae tree species and their expression analysis in P. mume

BackgroundPhosphatidylethanolamine-binding proteins (PEBPs) constitute a common gene family found among animals, plants and microbes. Plant PEBP proteins play an important role in regulating flowering time, plant architecture as well as seed dormancy. Though PEBP family genes have been well studied in Arabidopsis and other model species, less is known about these genes in perennial trees.ResultsTo understand the evolution of PEBP genes and their functional roles in flowering control, we identified 56 PEBP members belonging to three gene clades (MFT-like, FT-like, and TFL1-like) and five lineages (FT, BFT, CEN, TFL1, and MFT) across nine Rosaceae perennial species. Structural analysis revealed highly conserved gene structure and protein motifs among Rosaceae PEBP proteins. Codon usage analysis showed slightly biased codon usage across five gene lineages. With selection pressure analysis, we detected strong purifying selection constraining divergence within most lineages, while positive selection driving the divergence of FT-like and TFL1-like genes from the MFT-like gene clade. Spatial and temporal expression analyses revealed the essential role of FT in regulating floral bud breaking and blooming in P. mume. By employing a weighted gene co-expression network approach, we inferred a putative FT regulatory module required for dormancy release and blooming in P. mume.ConclusionsWe have characterized the PEBP family genes in nine Rosaceae species and examined their phylogeny, genomic syntenic relationship, duplication pattern, and expression profiles during flowering process. These results revealed the evolutionary history of PEBP genes and their functions in regulating floral bud development and blooming among Rosaceae tree species.

Gain-of-function of the 1-aminocyclopropane-1-carboxylate synthase gene ACS1G induces female flower development in cucumber gynoecy.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

Positive Correlation between Color and Scent in Rose Petals with Floral Bud Development

Positive Correlation between Color and Scent in Rose Petals with Floral Bud Development

High Temperature Suppresses Fruit/Seed Set and Weight, and Cladode Regreening in Red-fleshed ‘Da Hong’ Pitaya (Hylocereus polyrhizus) under Controlled Conditions

Following high summer temperatures in Taiwan, erratic fruit production and yellowed cladode have been observed in red-fleshed ‘Da Hong’ pitaya (Hylocereus polyrhizus). However, the specific environmental influences that result in the yield loss and cladode damage are unknown. The aim of this study was to evaluate how high temperature affects fruit production and cladode yellowing of ‘Da Hong’ pitaya under controlled conditions. One-year-old field-grown potted plants with moderately yellow-colored cladodes were placed in the phytotron at either 40/30 °C ± 1 °C [day/night, high-temperature treatment (HT)] or 30/20 °C ± 1 °C [day/night, control (CK)] during the natural long-day reproductive period. Floral bud development duration, flower opening behavior, fruit set and development, as well as fruit characteristics and seed setting (which was expressed as the estimated number of seeds), and the weight per fruit at harvest were investigated. In addition, the percentage of dry matter and color change (regreening) of cladodes were examined. We found that floral bud development was completed 8 days earlier than the control, but the time of blooming was 2 to 3 hours later within a day; and fruit set, fruit size, seed weight, and peel color were strongly suppressed in HT-treated plants compared with the control. Furthermore, both the estimated seed number and seed weight were positively correlated with fruit weight, suggesting that reduced seed setting and weight arising from incomplete fertilization in the HT plants may have resulted in fruit drop and smaller fruit. Although the color on the sunny (sun-exposed) side of the cladode remained yellow, the percentage of dry matter in the HT cladodes was not significantly different from the control, indicating that the yellow-colored cladodes did not reduce their carbon supply potential. The results indicate that HT during bloom led to poorer fruit set and lower fruit weight, presumably due to lower seed setting/weight per fruit arising from incomplete fertilization. The HT treatment also caused less regreening of cladodes, but this did not seem to impact fruit production. Further study is required to ascertain whether disrupted stamens or pistils resulting from HT treatment lead to incomplete fertilization.

EMBRYONIC FLOWER2B, coming from a stable QTL, represses the floral transition in cotton

The EMBRYONIC FLOWER 2 (EMF2) gene encodes a VEFS (VRN2–EMF2–FIS2–Su(z)12) domain protein involved in plant growth and development. Herein, genome-wide characterization of the VEFS-box gene family in Gossypium raimondii, G. arboreum, G. barbadense, and G. hirsutum was performed with a total of 3, 3, 6, and 6 homologous sequences respectively identified in the four species. The gene structure, protein motifs, and gene expression were further investigated. Based on our previous research on multiple stable quantitative trait loci for early maturity, GhEMF2B on chromosome D03 was selected as a candidate gene for further study. Quantitative real-time PCR analysis indicated that GhEMF2B was upregulated in the apical buds of late-maturing cultivars at the fourth and fifth true-leaf stages compared to that of early-maturing cultivars. Virus-induced gene silencing of GhEMF2B in cotton seedlings repressed expression by 50%–70%, which led to earlier floral bud development, young curled leaves, and abnormal petal formation. Further analysis demonstrated that the silencing of GhEMF2B enhanced the expression levels of the positive floral regulators AGAMOUS-LIKE 6 (GhAGL6), FLOWERING LOCUS T (GhFT), and APETALA 1 (GhAP1). Thus, it can be inferred that GhEMF2B plays important roles in the floral transition and development of cotton.

Regulation of floral bud development and emergence by ambient temperature under a long-day photoperiod in white-fleshed pitaya (Hylocereus undatus)

Night-breaking is used for the off-season production of pitaya (Hylocereus spp.) fruit in subtropical regions but is less successful at higher latitudes with colder winters. Consequently, ambient temperature has been proposed as a crucial factor controlling flowering in pitaya. However, the effects of temperature on reproductive bud development and emergence remain unclear. Therefore, here, we performed two phytotron experiments to clarify these effects in 2–3-year-old field-grown, potted white-fleshed pitaya (Hylocereus undatus “VN White”) under an inductive long-day photoperiod (14/10 h) in Taiwan. Experiment I was conducted in late March 2016 and used day/night temperatures of 32/22 °C (control) and 23/13 °C (low temperature), while experiment II was conducted in late March 2017 and used temperatures of 32/22 °C (optimal temperature), 29/19 °C (required temperature), and 25/15 °C (low temperature). In experiment I, the temperature of the low temperature treatment was increased by 2 °C every 4 weeks from the emergence of reproductive buds in the 32/22 °C treatment until reproductive bud formation, whereas in experiment II, the temperature of the low temperature treatment was increased to 27/17°C 2 weeks after the first reproductive buds emerged in the 29/19°C treatment. The development (stages 0–3) and the emergence of reproductive and vegetative buds were recorded weekly in all treatment groups. Experiment I showed that temperature had no effect on vegetative bud emergence. However, warm temperatures (32/22 °C) enhanced bud development (to stage 3) and induced reproductive bud emergence 3–4 weeks after treatment, whereas most buds remained in a pre-reproductive stage (stage 2) under the low temperature treatment (23/13 °C). More buds reached stages 2 and 3 after raising the temperature to 25/15 °C, but reproductive buds did not emerge until the temperature reached 29/19 °C. Experiment II further showed that plants needed to be kept at 29/19 °C for 9 weeks for reproductive bud emergence. Thus, 29/19 °C may be the minimum required temperature for reproductive bud emergence, while 32/22 °C is the optimal temperature for reproductive bud development and emergence in white-fleshed pitaya. These findings suggest that the induction of reproductive buds in pitaya is first initiated under a long-day photoperiod but warmer temperatures are required for their subsequent emergence.

A Methodological Approach to Determine Flower Bud Vulnerability to Low Temperatures for Deciduous Crops in Early Spring Using Degree Days

A common problem for decision makers in selecting frost control options is uncertainty about the level of injury that can be caused by low temperatures. During the past few years, the concept of lethal temperature (LT) at which 10% of the bud population dies (LT10) has been used as an index for evaluating the vulnerability of flower buds to low temperature conditions. This concept has shown to be a useful tool for frost control decision-making. However, the current methods used to obtain LT values assume no spatial or temporal variability, which results in a high level of uncertainty. The goal of this study was to develop an approach that decreases the uncertainty based on the known effects of temperature on bud vulnerability. A growth chamber experiment was conducted to determine flower bud vulnerability to low temperature as a function of temperature. The results from this study showed that thermal time expressed in degree days could explain changes in floral bud development and vulnerability to frost injury. According to our findings, LT10 is a fully acceptable index for determining flower bud vulnerability to low temperatures in orchard crops. Based on this information, we found that among the five apple and cherry cultivars analyzed, ‘Gala’ is the least vulnerable to low temperature because it starts at the beginning of spring with a high level of hardiness and increases its vulnerability at a low rate. The approach described in this article may enhance decision-making certainty associated with the timing and methods to increase air temperature in orchards during low-temperature events to avoid frost damage.

Comparative transcriptomic analysis reveals novel roles of transcription factors and hormones during the flowering induction and floral bud differentiation in sweet cherry trees (Prunus avium L. cv. Bing).

In sweet cherry trees, flowering is commercially important because the flowers, after fertilization, will generate the fruits. In P. avium, the flowering induction and flower organogensis are the first developmental steps towards flower formation and they occur within specialized organs known as floral buds during the summer, nine months before blooming. During this period the number of floral buds per tree and the bud fruitfulness (number of flowers per bud) are stablished affecting the potential yield of orchards and the plant architecture. The floral bud development is sensitive to any type of stress and the hotter and drier summers will interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular and hormonal mechanisms would be of help, but unlike the model plant Arabidopsis, very little is known about floral induction in sweet cherry. To explore the molecular mechanism of floral bud differentiation, high-throughput RNA sequencing was used to detect differences in the gene expression of P. avium floral buds at five differentiation stages. We found 2,982 differentially expressed genes during floral bud development. We identified genes associated with floral initiation or floral organ identity that appear to be useful biomarkers of floral development and several transcription factor families (ERF, MYB, bHLH, MADS-box and NAC gene family) with novel potential roles during floral transition in this species. We analyzed in deep the MADS-box gene family and we shed light about their key role during floral bud and organs development in P. avium. Furthermore, the hormonal-related signatures in the gene regulatory networks and the dynamic changes of absicic acid, zeatin and indolacetic acid contents in buds suggest an important role for these hormones during floral bud differentiation in sweet cherry. These data provide a rich source of novel informacion for functional and evolutionary studies about floral bud development in sweet cherry and new tools for biotechnology and breeding.

Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana

MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

Comparative Transcriptome Analysis between Cytoplasmic Male-sterile Line and Its Maintainer During the Floral Bud Development of Pepper

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704A and its maintainer 9704B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes (DEGs) were identified in the stage S1, S2 and S3, respectively, and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility.

Genome-wide identification and functional analysis of JmjC domain-containing genes in flower development of Rosa chinensis.

We genome-wide identified 28 JmjC domain-containing genes, further spatio-temporal expression profiling and genetic analysis defined them as epigenetic regulators in flowering initiation of Rosa chinensis. The JmjC domain-containing histone demethylases play critical roles in maintaining homeostasis of histone methylations, thus are vital for plant growth and development. Genome-wide identification of the JmjC domain-containing genes have been reported in several species, however, no systematic study has been performed in rose plants. In this paper, we identified 28 JmjC domain-containing genes from the newly published genome database of Rosa chinensis. The JmjC domain-containing proteins in R. chinensis were divided into seven groups, KDM3 was the largest group with 13 members, and JmjC domain-only A and KDM5B were the smallest clades both with only one member. Although all the JmjC domain proteins having a conserved JmjC domain, the gene and protein structure experienced differentiation and specification during the evolution, especially in KDM3 clade, one gene (RcJMJ40) was found carrying site deletions for cofactors Fe (II) and α-KG binding which were crucial for demethylase activities, three genes (RcJMJ41, RcJMJ43 and RcJMJ44) had no intron while two of them had tandem JmjC domains. Spatial expression pattern analysis of these JmjC domain-containing genes in different tissues showed most of them were highly expressed in reproductive tissues such as floral meristem and closed flowers than vegetative tissues, demonstrating their important functions in developmental switch from vegetative to reproductive growth of roses. Temporal expression profiling indicated majority of JmjC domain-containing genes from R. chinensis fluctuated along with floral bud differentiation and development, further proving their essential roles in flower organogenesis. VIGS induced silencing of RcJMJ12 led to delayed flowering time, and decreased the expression levels of flowering integrator such as RcFT, RcSOC1, RcFUL, RcLFY and RcAP1, therefore providing the genetic evidence of RcJMJ12 in flowering initiation. Collectively, spatio-temporal expression profiling and genetic analysis defined the JmjC domain-containing genes as important epigenetic regulators in flower development of R. chinensis.