Stamen Development Research Articles

OsMADS58 Stabilizes Gene Regulatory Circuits during Rice Stamen Development.

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cells, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

Ethylene Activates the EIN2-EIN3/EIL1 Signaling Pathway in Tapetum and Disturbs Anther Development in Arabidopsis.

Ethylene was previously reported to repress stamen development in both cucumber and Arabidopsis. Here, we performed a detailed analysis of the effect of ethylene on anther development. After ethylene treatment, stamens but not pistils display obvious developmental defects which lead to sterility. Both tapetum and microspores (or microsporocytes) degenerated after ethylene treatment. In ein2-1 and ein3-1 eil1-1 mutants, ethylene treatment did not affect their fertility, indicating the effects of ethylene on anther development are mediated by EIN2 and EIN3/EIL1 in vivo. The transcription of EIN2 and EIN3 are activated by ethylene in the tapetum layer. However, ectopic expression of EIN3 in tapetum did not induce significant anther defects, implying that the expression of EIN3 are regulated post transcriptional level. Consistently, ethylene treatment induced the accumulation of EIN3 in the tapetal cells. Thus, ethylene not only activates the transcription of EIN2 and EIN3, but also stabilizes of EIN3 in the tapetum to disturb its development. The expression of several ethylene related genes was significantly increased, and the expression of the five key transcription factors required for tapetum development was decreased after ethylene treatment. Our results thus point out that ethylene inhibits anther development through the EIN2-EIN3/EIL1 signaling pathway. The activation of this signaling pathway in anther wall, especially in the tapetum, induces the degeneration of the tapetum and leads to pollen abortion.

Stamen and pollen development in Menispermaceae with contrasting androecium structure

Stamen and pollen development in Menispermaceae with contrasting androecium structure

Co-expression network analysis of genes and networks associated with wheat pistillody.

Crop male sterility has great value in theoretical research and breeding application. HTS-1, whose stamens transformed into pistils or pistil-like structures, is an important male sterility material selecting from Chinese Spring three-pistil (CSTP) wheat. However the molecular mechanism of pistillody development in HTS-1 remains a mystery. RNA-seq data of 11 wheat tissues were obtained from the National Center for Biotechnology Information (NCBI), including the stamens of CSTP and the pistils and pistillodic stamen of HTS-1. The Salmon program was utilized to quantify the gene expression levels of the 11 wheat tissues; and gene quantification results were normalized by transcripts per million (TPM). In total, 58,576 genes were used to construct block-wise network by co-expression networks analysis (WGCNA) R package. We obtained all of modules significantly associated with the 11 wheat tissues. AgriGO V2.0 was used to do Gene Ontology (GO) enrichment analysis; and genes and transcription factors (TFs) in these significant modules about wheat pistillody development were identified from GO enrichment results. Basic local alignment search tool (BLAST) was used to align HTS-1 proteins with the published pistillody-related proteins and TFs. Genes about wheat pistillody development were analyzed and validated by qRT-PCR. The MEturquoise, MEsaddlebrown, MEplum, MEcoral1, MElightsteelblue1, and MEdarkslateblue modules were significantly corelated to pistillodic stamen (correlation p < 0.05). Moreover, 206 genes related to carpel development (GO:0048440) or gynoecium development (GO:0048467) were identified only in the MEturquoise module by Gene Ontology (GO) analysis, and 42 of 206 genes were hub genes in MEturquoise module. qRT-PCR results showed that 38 of the 42 hub genes had highly expressed in pistils and pistillodic stamens than in stamens. A total of 15 pistillody development-related proteins were validated by BLAST. Transcription factors (TFs) were also analyzed in the MEturquoise module, and 618 TFs were identified. In total, 56 TFs from 11 families were considered to regulate the development of pistillodic stamen. The co-expression network showed that six of HB and three of BES1 genes were identified in 42 hub genes. This indicated that TFs played important roles in wheat pistillody development. In addition, there were 11 of ethylene-related genes connected with TFs or hub genes, suggesting the important roles of ethylene-related genes in pistillody development. These results provide important insights into the molecular interactions underlying pistillody development.

Disruption of transcription factor RhMYB123 causes the transformation of stamen to malformed petal in rose (Rosa hybrida)

We find that the R2R3 MYB transcription factor RhMYB123 has a novel function to regulate stamen-petal organ specification in rose. Rose is one of the ornamental plants with economic importance worldwide. Malformed flower seriously affects the ornamental value and fertility of rose. However, the regulatory mechanism is largely unknown. In this work, we identified a R2R3 MYB transcription factor RhMYB123 from rose, the expression of which significantly decreased from flower differentiation stage to floral organ development stage. Phylogenetic analysis indicated that it belongs to the same subgroup as MYB123 of Arabidopsis and located in nucleus. In addition, RhMYB123 was confirmed to have transcriptional activation function by dual luciferase assay. Silencing RhMYB123 using Virus-Induced Gene Silencing (VIGS) in rose could increase the number of malformed petaloid stamen. Furthermore, we identified 549 differential expressed genes (DEGs) in TRV-RhMYB123 lines compared to TRV controls by RNA-seq of floral buds (flower differentiation stage). Among of those genes, expression of 3 MADS box family genes related to floral organ development reduced in TRV-RhMYB123 lines, including AGAMOUS (RhAG), AGAMOUS LIKE 15 (RhAGL15), and SHATTERPROOF 1 (RhSHP1). Given, previous studies have shown that auxin plays a crucial role in floral meristem initiation and stamen-petal organ specification. We also found 6 DEGs were involved in auxin signal transduction, of which five were reduced expression in TRV-RhMYB123. Taken together, our findings suggested that RhMYB123 may govern the development of malformed petaloid stamen by regulating the expressions of some MADS box family members and auxin signaling pathway elements.

Defect in BrMS1, a PHD-finger transcription factor, induces male sterility in ethyl methane sulfonate-mutagenized Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Male sterility is an ideal character for the female parent in commercial hybrid seed production in Chinese cabbages. We identified three allele male sterile mutants msm2-1/2/3 in progenies of ethyl methane sulfonate mutagenized Chinese cabbage. It was proved that their male sterilities were controlled by a same recessive nuclear gene. Cytological observation showed that the delayed tapetal programmed cell death (PCD) as well as the abnormal pollen exine and intine led to pollen abortion in these mutants. MutMap combined with KASP analyses showed that BraA10g019050.3C, a homologous gene of AtMS1 encoding a PHD-finger transcription factor and regulated pollen development, was the causal gene. A single-nucleotide mutation from G to A occurred at the 2443th base of BrMS1 in msm2-1 which results in premature termination of the PHD-finger protein translation; a single-nucleotide mutation from G to A existed at 1372th base in msm2-2 that makes for frameshift mutation; a single-nucleotide mutation from G to A distributed at 1887th base in msm2-3 which issues in the amino acid changed from Asp to Asn. The three allelic mutations in BrMS1 all led to the male sterile phenotype, which revealed its function in stamen development. Quantitative reverse transcription polymerase chain reaction analysis indicated that BrMS1 specially expressed in the anther at the early stage of pollen development and its expression level was higher in msm2-1/2/3 than that in the wild-type "FT." BrMS1 was located at the nucleus and a length of 12 amino acid residues at the C-terminus had transcriptional activation activity. RNA-seq indicated that the mutation in BrMS1 affected the transcript level of genes related to the tapetum PCD and pollen wall formation, which brought out the pollen abortion. These male sterile mutants we developed provided a novel gene resource for hybrid breeding in Chinese cabbage.

Whole-transcriptome analysis of differentially expressed genes between ray and disc florets and identification of flowering regulatory genes in Chrysanthemum morifolium.

Chrysanthemum morifolium has ornamental and economic values. However, there has been minimal research on the morphology of the chrysanthemum florets and related genes. In this study, we used the leaves as a control to screen for differentially expressed genes between ray and disc florets in chrysanthemum flowers. A total of 8,359 genes were differentially expressed between the ray and disc florets, of which 3,005 were upregulated and 5,354 were downregulated in the disc florets. Important regulatory genes that control flower development and flowering determination were identified. Among them, we identified a TM6 gene (CmTM6-mu) that belongs to the Class B floral homeotic MADS-box transcription factor family, which was specifically expressed in disc florets. We isolated this gene and found it was highly similar to other typical TM6 lineage genes, but a single-base deletion at the 3′ end of the open reading frame caused a frame shift that generated a protein in which the TM6-specific paleoAP3 motif was missing at the C terminus. The CmTM6-mu gene was ectopically expressed in Arabidopsis thaliana. Petal and stamen developmental processes were unaffected in transgenic A. thaliana lines; however, the flowering time was earlier than in the wild-type control. Thus, the C-terminal of paleoAP3 appears to be necessary for the functional performance in regulating the development of petals or stamens and CmTM6-mu may be involved in the regulation of flowering time in chrysanthemum. The results of this study will be useful for future research on flowering molecular mechanisms and for the breeding of novel flower types.

Connective modifications and origin of stamen diversity in Melastomataceae.

The androecium of Melastomataceae presents notable modifications in its merosity, morphology between whorls and in prolonged connectives and appendages. We carried out a comparative study of six Melastomataceae species to shed light on the developmental processes that originate such stamen diversity. The development of stamens was studied using scanning electron microscopy and histological observations. The stamens of all species studied have a curved shape because they emerge on a plane displaced by the perigynous hypanthium. They are the last flower organs to initiate and therefore their growth is inwards and towards the floral center. Despite the temporal inversion between carpels and stamens in Melastomataceae, the androecium maintains the centripetal pattern of development, the antepetalous stamens emerging after antesepalous stamens. The isomerous androecium can be the result of abortion of the antepetalous stamens, whereas heterostemony seems to be caused by differences in position and the stamen development time. Pedoconnectives and ventral appendages originate from the basal expansion of the anther late in floral development. The delay in stamen development may be a consequence of their dependence on the formation of a previous space so that they can grow. Most of the stamen diversity is explained by the formation of the connectives and their appendages. The formation of a basal-ventral anther prolongation, which culminates in the development of the pedoconnective, does not differ from other types of sectorial growth of the connective, which form shorter structures.

Functional analysis of flower development related gene SvGLOBOSA from Senecio vulgaris.

The unique capitulum of Asteraceae has important ornamental and research value. Few studies have described the complex molecular mechanism of flower development. In this study, SvGLOBOSA(SvGLO), the MADS-box gene of Senecio vulgaris, was identified by screening the transcriptome data, and its function was examined. The gene structure was analyzed and its function was predicted through bioinformatics. The relative expression levels in different tissues of wild-type S. vulgaris were analyzed by qRT-PCR. SvGLO was overexpressed in Solanum nigrum and morphological observations were made. Histological staining was used in analyzing the histological changes in the ovary of transgenic S. nigrum. The results showed that the open reading frame of SvGLO was 591 bp long, encoding 196 amino acids. It has typical MADS-box and K-box domains and contains a PI motif at the C-terminal. SvGLO belongs to the PI/GLO subfamily of class B MADS-box genes. qRT-PCR results showed that SvGLO was highly expressed in inflorescence tissues but not in vegetative organs. In SvGLO-overexpressed S. nigrum, the sepals showed some characteristics of petals, carpels transformed into stamen-like organs, and fruit development was abnormal. Histological staining revealed that the morphology of ovary wall cells of transgenic S. nigrum was similar to that of anther wall cells of the stamen of wild-type S. vulgaris. Therefore, SvGLO may be involved in the regulation of petal and stamen development in S. vulgaris.

The structures of floral organs and reproductive characteristics of an ornamental bamboo species, Pleioblastus pygmaeus

As a woody grass, bamboo has special reproductive habits with an extremely long vegetative phase. Pleioblastus pygmaeus is a kind of ornamental dwarf bamboo species with high ecological and economic value in China. There was no documentary record of the flowering of P. pygmaeus until 2015, which bloomed in the Bamboo Garden of Nanjing Forestry University. Generally, bamboo plants rarely bear fruit or have a low seed setting rate, but this species has a high seed production. This study explores developmental reasons using anatomical methods. The process of flower bud differentiation and a series of important reproductive development processes including anther wall development, microsporogenesis, megasporogenesis, male and female gametophyte development, and embryonic development were investigated sequentially. Each stamen contained three tetrasporangiate anthers and the development of anther wall was of Monocotyledonous type. The microspores finally matured into 3-celled pollen grains by two successive mitoses. The pistil was composed of three carpels, with an anatropous, tenuinucellate and bitegmic ovule in a single ovary. The formation of embryo sac belonged to Polygonum type. With three mitoses, functional megaspore developed into the embryo sac with eight nuclei within seven cells. The development of stamen and pistil was synchronous in the same floret. The development of embryo followed Grass type and the development of endosperm belonged to Nuclear type. The development of endosperm was earlier than that of embryo. The study enriches basic knowledge of reproductive biology of bamboo plants and is of great fundamental significance to furtherly explore flowering mechanism of P. pygmaeus and to cultivate bamboo plants sustainably.

Hydroxyproline-O-Galactosyltransferases Synthesizing Type II Arabinogalactans Are Essential for Male Gametophytic Development in Arabidopsis.

In flowering plants, male reproductive function is determined by successful development and performance of stamens, pollen grains, and pollen tubes. Despite the crucial role of highly glycosylated arabinogalactan-proteins (AGPs) in male gamete formation, pollen grain, and pollen tube cell walls, the underlying mechanisms defining these functions of AGPs have remained elusive. Eight partially redundant Hyp-galactosyltransferases (named GALT2-GALT9) genes/enzymes are known to initiate Hyp-O-galactosylation for Hyp-arabinogalactan (AG) production in Arabidopsis thaliana. To assess the contributions of these Hyp-AGs to male reproductive function, we used a galt2galt5galt7galt8galt9 quintuple Hyp-GALT mutant for this study. Both anther size and pollen viability were compromised in the quintuple mutants. Defects in male gametogenesis were observed in later stages of maturing microspores after meiosis, accompanied by membrane blebbing and numerous lytic vacuoles. Cytological and ultramicroscopic observations revealed that pollen exine reticulate architecture and intine layer development were affected such that non-viable collapsed mature pollen grains were produced, which were devoid of cell content and nuclei, with virtually no intine. AGP immunolabeling demonstrated alterations in cell wall architecture of the anther, pollen grains, and pollen tube. Specifically, the LM2 monoclonal antibody (which recognized β-GlcA epitopes on AGPs) showed a weak signal for the endothecium, microspores, and pollen tube apex. Pollen tube tips also displayed excessive callose deposition. Interestingly, expression patterns of pollen-specific AGPs, namely AGP6, AGP11, AGP23, and AGP40, were determined to be higher in the quintuple mutants. Taken together, our data illustrate the importance of type-II AGs in male reproductive function for successful fertilization.

Effects of Different Chemicals on Sexual Regulation in Persimmon (Diospyros kaki Thunb.) Flowers

Research on crop sexuality is important for establishing systems for germplasm innovation and cultivating improved varieties. In this study, androecious persimmon trees were treated with various concentrations of ethrel (100, 500, and 1,000 mg/L) and zeatin (1, 5, and 10 mg/L) to investigate the morphological, physiological, and molecular characteristics of persimmon. Ethrel at 1,000 mg/L and zeatin at 10 mg/L both significantly reduced the stamen length and pollen grain diameter in androecious trees. Ethrel treatment also led to reduced stamen development with degenerated cellular contents; zeatin treatment promoted the development of arrested pistils via maintaining relatively normal mitochondrial morphology. Both treatments altered carbohydrate, amino acid, and endogenous phytohormone contents, as well as genes associated with hormone production and floral organ development. Thereafter, we explored the combined effects of four chemicals, including ethrel and zeatin, as well as zebularine and 5-azacytidine, both of which are DNA methylation inhibitors, on androecious persimmon flower development. Morphological comparisons showed that stamen length, pollen viability, and pollen grain diameter were significantly inhibited after combined treatment. Large numbers of genes involving in carbohydrate metabolic, mitogen-activated protein kinase (MAPK) signaling, and ribosome pathways, and metabolites including uridine monophosphate (UMP) and cyclamic acid were identified in response to the treatment, indicating complex regulatory mechanisms. An association analysis of transcriptomic and metabolomic data indicated that ribosomal genes have distinct effects on UMP and cyclamic acid metabolites, explaining how male floral buds of androecious persimmon trees respond to these exogenous chemicals. These findings extend the knowledge concerning sexual differentiation in persimmon; they also provide a theoretical basis for molecular breeding, high-yield cultivation, and quality improvement in persimmon.

RhAGL24 Regulating Auxin-Related Gene RhARF18 Affects Stamen Petaloidy in Rose

AGAMOUS-LIKE 24 (AGL24) is a key gene regulating floral transition, but its involvement in flower organ identity remains largely unknown. In this study, we found that RhAGL24 is strongly related to petal and stamen development in rose. Its expression increases rapidly at the petal primordium development stage and maintains a high level until the complete differentiation stage. RhAGL24 silencing increases the number of malformed petals and decreases the number of stamens, indicating that this gene affects stamen petaloidy. RhAG (AGAMOUS), a class C gene associated with petal and stamen development, is downregulated in RhAGL24-silenced plants. Moreover, we found that RhAGL24 could directly bind to the promoter region of RhARF18 (AUXIN RESPONSE FACTORS 18), a regulator of RhAG. Our results suggested that RhAGL24-RhARF18 module regulates stamen petaloidy in rose and provide new insights into the function of AGL24 for plants.

Arabidopsis TWISTED DWARF1 regulates stamen elongation by differential activation of ABCB1,19-mediated auxin transport.

Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development. In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport, and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation, and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1-as a described ABCB chaperone-is a positive regulator of ABCB1- and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional players downstream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB1,19-mediated auxin transport in the stamen.

An ortholog of the MADS-box gene SEPALLATA3 regulates stamen development in the woody plant Jatropha curcas.

Overexpression of JcSEP3 causes defective stamen development in Jatropha curcas, in which brassinosteroid and gibberellin signaling pathways may be involved. SEPALLATAs (SEPs), the class E genes of the ABCE model, are required for floral organ determination. In this study, we investigated the role of the JcSEP3 gene in floral organ development in the woody plant Jatropha curcas. Transgenic Jatropha plants overexpressing JcSEP3 displayed abnormal phenotypes such as deficient anthers and pollen, as well as free stamen filaments, whereas JcSEP3-RNA interference (RNAi) transgenic plants had no obvious phenotypic changes, suggesting that JcSEP3 is redundant with other JcSEP genes in Jatropha. Moreover, we compared the transcriptomes of wild-type plants, JcSEP3-overexpressing, and JcSEP3-RNAi transgenic plants. In the JcSEP3-overexpressing transgenic plants, we discovered 25 upregulated genes involved in anther and pollen development, as well as 12 induced genes in brassinosteroid (BR) and gibberellin (GA) signaling pathways. These results suggest that JcSEP3 directly or indirectly regulates stamen development, concomitant with the regulation of BR and GA signaling pathways. Our findings help to understand the roles of SEP genes in stamen development in perennial woody plants.

Characterization of PISTILLATA-like Genes and Their Promoters from the Distyly Fagopyrum esculentum.

Arabidopsis PISTILLATA (PI) encodes B-class MADS-box transcription factor (TF), and works together with APETALA3 (AP3) to specify petal and stamen identity. However, a small-scale gene duplication event of PI ortholog was observed in common buckwheat and resulted in FaesPI_1 and FaesPI_2. FaesPI_1 and FaesPI_2 were expressed only in the stamen of dimorphic flower (thrum and pin) of Fagopyrum esculentum. Moreover, intense beta-glucuronidase (GUS) staining was found in the entire stamen (filament and anther) in pFaesPI_1::GUS transgenic Arabidopsis, while GUS was expressed only in the filament of pFaesPI_2::GUS transgenic Arabidopsis. In addition, phenotype complementation analysis suggested that pFaesPI_1::FaesPI_1/pFaesPI_2::FaesPI_2 transgenic pi-1 Arabidopsis showed similar a flower structure with stamen-like organs or filament-like organs in the third whorl. This suggested that FaesPI_2 only specified filament development, but FaesPI_1 specified stamen development. Meanwhile, FaesPI_1 and FaesPI_2 were shown to function redundantly in regulating filament development, and both genes work together to require a proper stamen identity. The data also provide a clue to understanding the roles of PI-like genes involved in floral organ development during the early evolution of core eudicots and also suggested that FaesPI_1 and FaesPI_2 hold the potential application in bioengineering to develop a common buckwheat male sterile line.

Functional analysis of a PISTILLATA-like gene CcMADS20 involved in floral organs specification in citrus

PISTILLATA (PI), as a member of MADS-box transcription factor, plays an important role in petal and stamen specification in Arabidopsis. However, little is known about PI-like genes in citrus. To understand the molecular mechanism of PI during the developmental process of citrus flower, a PI-like gene CcMADS20 was isolated from Citrus Clemantina. Sequence alignment and phylogenetic analysis revealed that CcMADS20 had relatively high similarity with PI-like homolog and was classified in the core dicotyledonous group. The temporal and spatial expression analyses showed that CcMADS20 was specifically expressed in petal and stamen of citrus flower, which was consistent with PI expression pattern in Arabidopsis. Protein interaction revealed that CcMADS20 could form heterodimer with AP3-like proteins. Furthermore, ectopic overexpression of CcMADS20 in Arabidopsis resulted in transformation of sepals into petal-like structure, as observed in other plants overexpressing a functional PI-like homolog. Additionally, promoter fragments of CcMADS20 were also cloned in the representative 21 citrus varieties. Interestingly, four types of promoters were discovered in these citrus varieties, resulting from two stable insert/deletion fragments (Locus1 and Locus2). The homo/hetero-zygosity of promoter alleles in each variety was strongly related to the evolutionary origin of citrus. Four promoters activity analysis indicated that Locus1 presence inhibited CcMADS20 transcriptional activity and Locus2 presence promoted its transcriptional activity. These findings suggested that CcMADS20 determines petal and stamen development during the evolutionary process of citrus and four promoters discovered, as effective genetic markers, are valuable for citrus breeding practices.

AtGH3.10 is another jasmonic acid-amido synthetase in Arabidopsis thaliana.

Jasmonoyl-isoleucine (JA-Ile) is a key signaling molecule that activates jasmonate-regulated flower development and the wound stress response. For years, JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl-amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine, and the source of persisting JA-Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate through recombinant enzyme assays and loss-of-function mutant analyses that AtGH3.10 functions as a JA-amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA-Ile accumulation in the gh3.10-2 jar1-11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative 12OH-JA-Ile was undetected in the flower buds and unwounded leaves. Residual levels of JA-Ile, JA-Ala, and JA-Val were nonetheless detected in gh3.10-2 jar1-11, suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA-Ile and 12OH-JA-Ile and the expression of JA-responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM-DOMAIN1 observed in WT, gh3.10-1, and jar1-11 leaves were effectively abolished in gh3.10-2 jar1-11. Additionally, an increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10-2 jar1-11. These findings conclusively show that AtGH3.10 contributes to JA-amino acid biosynthesis and functions partially redundantly with AtJAR1 in sustaining flower development and the wound stress response in Arabidopsis.

ITRAQ and PRM -based proteomics analysis for the identification of differentially abundant proteins related to male sterility in ms-7 mutant tomato (Solanum lycoperscium) plants

Male sterile mutants can be used in breeding or commercial cultivation in tomato, but there are few research reports on their proteomics. In this study, we analyzed the metabolic pathways and biological functions of differentially abundant proteins (DAPs) involved in two stages of stamen development of the tomato flowers by using a high through-put iTRAQ labeled proteomic approach. There was a total of 1476 DAPs which should associated with the occurrence of pollen abortion in tomato. Moreover, there were more DAPs in the four membrane systems. It shows that membrane systems are very important for tomato pollen development. According to KEGG analysis, these signaling pathways including starch and sucrose metabolism (map00500), tropane, piperidine and pyridine alkaloids biosynthesis (map00960), amino sugar and nucleotide sugar metabolism (map00520) have important effects on pollen development. These results were verified by using mass spectrometry PRM. Finally, two candidate genes (Solyc11g065770 and Solyc11g065530) were found that may be related to pollen development and cause pollen abortion by comparison of protein-protein interaction networks and on the basis of previous studies on ms-7 gene. This data and model will provide a new insight into tomato genetic male sterility 7 and contribute to the improvement of tomato hybrid breeding.Biological significance: Artificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes. SignificanceArtificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes.

Flower Bud Differentiation, Mega-micro Spore Production, and Male-female Gametophyte Development in Camellia weiningensis

Camellia weiningensis is a typical woody edible oil tree species in the northwest alpine area of Guizhou Province, China, but its embryological development is not fully elucidated. Here, we assessed flower bud differentiation, microsporogenesis, and male-female gametophyte development in this species. We performed cytological observations of flower bud development in C. weiningensis through conventional paraffin sectioning, scanning electron microscopy, and stereomicroscopy to establish the corresponding relationships between the external morphology and internal structure. The flowers were hermaphroditic and exhibited a short flower bud differentiation time. Although pistil development occurred later than stamen development, both organs matured synchronously before flowering. The anther contained four sacs that exhibited a butterfly shape in transverse sections. The anther wall comprised the epidermis, anther chamber inner wall, two middle layers, and a glandular tapetum (from outside to inside). Microspore mother cells formed a tetrahedral tetrad through meiosis, mature pollen was two-celled with three germination pores, and the ovary comprised three to five chambers (three chambers predominated). Multiple ovules were invertedly attached to the axial placentation and exhibited double integuments and a thin nucellus. The embryo sac exhibited Allium-type development, and the mature embryo sac was seven-celled and eight-nucleated. In C. weiningensis, embryonic development does not exhibit abnormalities, and stamen development occurs earlier than pistil development. During flower bud development, the inner development process of male and female cells can be judged according to their external morphological characteristics. Our results may provide a theoretical basis for regulating flowering in and the cross-breeding of C. weiningensis.