Anther Development Research Articles

Constitutive expression of cucumber CsACS2 in Arabidopsis Thaliana disrupts anther dehiscence through ethylene signaling and DNA methylation pathways.

Constitutive expression of cucumber CsACS2 in Arabidopsis disrupts anther dehiscence and male fertility via ethylene signaling and DNA methylation, revealing new avenues for enhancing crop reproductive traits. The cucumber gene CsACS2, encoding ACC (1-aminocyclopropane-1-carboxylic acid) synthase, plays a pivotal role in ethylene biosynthesis and sex determination. This study investigates the effects of constitutive CsACS2 expression in Arabidopsis thaliana on anther development and male fertility. Transgenic Arabidopsis plants overexpressing CsACS2 exhibited male sterility due to inhibited anther dehiscence, which was linked to suppressed secondary cell wall thickening. RNA-Seq analysis revealed upregulation of ethylene signaling pathway genes and downregulation of secondary cell wall biosynthesis genes, with gene set enrichment analysis indicating the involvement of DNA methylation. Rescue experiments demonstrated that silver nitrate (AgNO₃) effectively restored fertility, while 5-azacytidine (5-az) partially restored it, highlighting the roles of ethylene signaling and DNA methylation in this process. Constitutive CsACS2 expression in Arabidopsis disrupts anther development through ethylene signaling and DNA methylation pathways, providing new insights into the role of ethylene in plant reproductive development and potential applications in crop improvement.

Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris)

Pollen coat proteins (PCPs) are cysteine-rich small-molecule proteins, which exhibit high levels of polymorphism and are expressed in gametocytes. Previous investigations have revealed that PCP genes are involved in pollen wall synthesis, pollen-stigma recognition, and pollen development and germination. However, gene expression and function of PCP family in pollen development is not well understood in Wucai (Brassica campestris L.). In this study, genome-wide identification and expression analysis of the BcPCP gene family members were conducted, including their physical and chemical properties, chromosome localization, phylogenetic relationships, gene structure, and tertiary structure. A total of 20 BcPCP genes were identified and classified into three subfamilies showing high homology to Arabidopsis thaliana. Expression pattern analysis indicated that the BcPCP gene family exhibits higher expression levels in reproductive organs, suggesting their potential involvement in the reproductive development. Notably, BraA02g002400.3 C, potentially associated with male sterility, was identified through multiple transcriptomic and proteomic datasets. Subsequently, sequence analysis revealed its homology with the Arabidopsis GRP20 gene, and thus it was named BcGRP20. Functional analysis of this gene showed that overexpression of BcGRP20 gene in the Arabidopsis grp20 mutant could restore anther fertility. Overall, our findings indicate that BcGRP20 plays a critical role in pollen development and may be the causative gene for male sterility in Wucai. This study provides candidate genes for further functional identification of BcPCP genes in Wucai, which are crucial for anther development.

Duplicate MADS-box genes with split roles and a genetic regulatory network of floral development in long-homostyle common buckwheat

The classic ABC model postulates how three classes of floral homeotic genes (A, B and C) work in a combinational way to confer organ identity to each whorl that make up a perfect flower in core eudicot plants. Fagopyrum esculentum (Polygonaceae) produces dimorphic flowers with single whorl showy tepals, representing a considerable difference with most core eudicots flowers. Here, we explain in detail the function of a duplicated pair of floral homeotic genes involved in the formation of tepals and stamens in the LH F. esculentum. FaesAP1_1 and FaesAP1_2 work together to specify tepal identity. FaesAP3_1/2 or FaesPI_1/2 have redundant function in specifying filament identity, while FaesAP3_2 and FaesPI_2 also retain a conserved role in specifying anther development and gain novel function in style length determination. However, FaesPI_1 gain novel function in floral color formation. In addition, FaesAG can directly regulate stamen and pistil development or binds to the CArG-box of pFaesPI_1 to indirectly regulate stamen and pistil development by a gene regulatory pathway involving FaesAP1_1/2, FaesAP3_1/2 and FaesPI_1/2. Moreover, FaesAP1_1/2 can directly or indirectly regulate B-class gene (FaesAP3_1/2 and FaesPI_1/2) expression to be involved in floral development. Our work has led to detailed insights into the MADS-box gene regulatory networks that control floral developmental process in LH F. esculentum.

Cell Wall Invertases from Maternal Tissues Modulate Sucrose Flux in Apoplastic Pathways During Rice Anther and Seed Development.

Efficient sucrose transport and metabolism are vital for seed and pollen development in plants. Cell wall invertases (CINs) hydrolyze sucrose into glucose and fructose, maintaining a sucrose gradient in the apoplast of sink tissues. In rice, two CIN isoforms, OsCIN1 and OsCIN2, were identified as being specifically expressed in the anthers but not in pollen. Functional analyses through genetic crosses and mutant characterization showed that oscin1/2 double mutants exhibit a sporophytic male-sterile phenotype and produce shrunken seeds. This suggests that CIN activity is essential for proper pollen development and seed formation in rice. Observation of the progeny genotypes and phenotypes from various genetic crosses revealed that the phenotype of oscin1/2 seeds is determined by the genotype of the maternal tissue, indicating the critical role of CIN function in the apoplast between maternal and filial tissues for sucrose transport and metabolism. The CIN activity in the anthers and seeds of wild-type rice was found to be significantly higher-over 500-fold in the anthers and 5-fold in the seeds-than in the leaves, highlighting the importance of CIN in facilitating the efficient unloading of sucrose. These findings suggest that the fine-tuning of CIN activity in the apoplast, achieved through tissue-specific expression and CIN isoform regulation, plays a key role in determining the carbohydrate distribution across different tissues. Understanding this regulatory mechanism could provide opportunities to manipulate carbohydrate allocation to sink organs, potentially enhancing crop yields.

Ribosome binding of phasiRNA precursors accelerates the 24-nt phasiRNA burst in meiotic maize anthers.

Reproductive phasiRNAs (phased, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different maize inbred lines, with ribosomes enriched upstream of miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers.

Transcriptome Remodeling in Arabidopsis: A Response to Heterologous Poplar MSL-lncRNAs Overexpression.

Stamens are vital reproductive organs in angiosperms, essential for plant growth, reproduction, and development. The genetic regulation and molecular mechanisms underlying stamen development are, however, complex and varied among different plant species. MSL-lncRNAs, a gene specific to the Y chromosome of Populus deltoides, is predominantly expressed in male flower buds. Heterologous expression of MSL-lncRNAs in Arabidopsis thaliana resulted in an increase in both stamen and anther count, without affecting pistil development or seed set. To reveal the molecular regulatory network influenced by MSL-lncRNAs on stamen development, we conducted transcriptome sequencing of flowers from both wild-type and MSL-lncRNAs-overexpressing Arabidopsis. A total of 678 differentially expressed genes were identified between wild-type and transgenic Arabidopsis. Among these, 20 were classified as transcription factors, suggesting a role for these regulatory proteins in stamen development. GO enrichment analysis revealed that the differentially expressed genes were significantly associated with processes such as pollen formation, polysaccharide catabolic processes, and secondary metabolism. KEGG pathway analysis indicated that MSL-lncRNAs might promote stamen development by upregulating genes involved in the phenylpropanoid biosynthesis pathway. The top three upregulated genes, all featuring the DUF295 domain, were found to harbor an F-box motif at their N-termini, which is implicated in stamen development. Additionally, in transgenic Arabidopsis flowers, genes implicated in tapetum formation and anther development were also observed to be upregulated, implying a potential role for MSL-lncRNAs in modulating pollen development through the positive regulation of these genes. The findings from this study establish a theoretical framework for elucidating the genetic control exerted by MSL-lncRNAs over stamen and pollen development.

Gene expression profiles and metabolic pathways responsible for male sterility in cybrid pummelo.

Abnormal expression of genes regulating anther and pollen development and insufficient accumulation of male sterility (MS)- related metabolites lead to MS in cybrid pummelo Male sterility (MS) is a major cause of seedlessness in citrus, which is an important trait for fresh fruit. Understanding the mechanism of MS is important for breeding seedless citrus cultivars. In this study, we dissected the transcriptional, metabolic and physiological mechanisms of MS in somatic cybrid of pummelo (G1 + HBP). G1 + HBP exhibited severe male sterility, manifesting as retarded anther differentiation, abnormal anther wall development (especially tapetum and endothecium), and deficient pollen wall formation. In the anthers of G1 + HBP, the expression of genes regulating anther differentiation and tapetum development was abnormal, and the expression of genes regulating endothecium secondary lignification thickening and pollen wall formation was down-regulated. The transcription of genes involved in MS-related biological processes, such as jasmonic acid (JA) signaling pathway, primary metabolism, flavonoid metabolism, and programmed cell death, was altered in G1 + HBP anthers, and the accumulation of MS-associated metabolites, including fatty acids, amino acids, sugars, ATP, flavonols and reactive oxygen species (ROS), was down-regulated in G1 + HBP anthers. In summary, abnormal expression of key genes regulating anther and pollen development, altered transcription of key genes involved in MS-related metabolic pathways, and insufficient accumulation of MS-related metabolites together lead to MS in G1 + HBP. The critical genes and the metabolism pathways identified herein provide new insights into the formation mechanism of MS in citrus and candidate genes for breeding seedless citrus.

RNA sequencing and functional analysis uncover key long non-coding RNAs involved in regulating pollen fertility during the process of gametocidal action in wheat.

Gametocidal (Gc) chromosomes have been widely utilized in genetic breeding due to their ability to induce chromosomal breakage and eliminate gametes that lack them. Long noncoding RNAs (lncRNAs) have various functional mechanisms in regulating pollen and anther development; however, their regulatory contributions to Gc action are still unknown. Here, we identified 2824 differentially expressed lncRNAs (DE-lncRNAs) from the anther tissues of Triticum aestivum cv. Chinese Spring (CS) and Chinese Spring-Gc 3C chromosome monosomic addition line (CS-3C) through sequencing. In this study, we predicted 161 target mRNAs for 145 DE-lncRNAs, including 104 cis-regulatory, 60 trans-regulatory, and three both cis-regulatory and trans-regulatory manner. Combined with our previous miRNA sequencing data, 241 DE-lncRNAs functioned as potential endogenous target mimics (eTMs) for 84 differentially expressed microRNAs (DE-miRNAs, including 12 novel miRNAs). The results of transient transformation in tobacco leaves indicated that L006278 could bind to MTCONS_00006277, which encoded a calcineurin CBL-interacting protein kinase 19-like, and suppress its expression. Furthermore, L117735 could function as an eTM for tae-miR9657b-3p, and L056972 could function as an eTM for gc-m2240-5p. To explore the function of lncRNAs in the process of Gc action, we transformed L006278, an up-regulated lncRNA in CS-3C, into rice to analyze its effect on pollen fertility. Overexpression of L006278 led to a reduction in rice pollen fertility. Overall, our findings indicate that lncRNAs can contribute to the regulation of pollen fertility during the process of Gc action by regulating the expression levels of target mRNAs and acting as eTMs for certain key miRNAs.

Cryptic cytoplasmic male sterility-causing gene in the mitochondrial genome of common japonica rice.

Cytoplasmic male sterility (CMS) is an agronomically significant trait that causes dysfunction in pollen and anther development. It is often observed during successive backcrossing between distantly related species. Here, we show that Asian japonica cultivars (Oryza sativa) exhibit CMS when the nucleus is replaced with that of the African rice Oryza glaberrima. The CMS line produced stunted anthers and did not set any seeds. Mitochondrial orf288 RNA was detected in the anthers of CMS lines but not in fertility restorer lines. The mitochondrial genome-edited japonica rice that was depleted of orf288 did not exhibit male sterility when backcrossed with O. glaberrima. These results demonstrate that orf288 is a CMS-causing gene. As orf288 commonly occurs in the mitochondrial genomes of japonica rice, these results indicate that common japonica rice cultivars possess a cryptic CMS-causing gene hidden in their mitochondrial genomes.

Comparative Analysis of the Mitochondrial Genome of Eggplant (Solanum melongena L.) to Identify Cytoplasmic Male Sterility Candidate Genes.

Cytoplasmic male sterility (CMS) is important for commercial hybrid seed production. However, it is still not used in eggplant (Solanum melongena L.), and corresponding regulatory genes and mechanisms of action have not been reported. We report CMS line 327A, which was derived from the hybridization between cultivated and wild eggplants. By looking at different stages of anther development under a microscope, we saw that the 327A anther's tapetum layer vacuolized during meiosis, which caused abortion. To investigate the 327A CMS regulatory genes, the mitochondrial genomes of 327A and its maintainer line 327B were assembled de novo. It was found that 15 unique ORFs (Open Reading Frame) were identified in 327A. RT-PCR and RT-QPCAR tests confirmed that orf312a and orf172a, 327A-specific ORFs with a transmembrane domain, were strongly expressed in sterile anthers of 327A. In addition, orf312a has a chimeric structure with the ribosomal protein subunit rpl16. Therefore, orf312a and orf172a can be considered strong candidate genes for CMS. Concurrently, we analyzed the characteristics of CMS to develop a functional molecular marker, CMS312, targeting a future theoretical basis for eggplant CMS three-line molecular breeding.

Genome-Wide Analysis of the Histone Modification Gene (HM) Family and Expression Investigation during Anther Development in Rice (Oryza sativa L.).

Histone modification plays a crucial role in chromatin remodeling and regulating gene expression, and participates in various biological processes, including plant development and responses to stress. Several gene families related to histone modification have been reported in various plant species. However, the identification of members and their functions in the rice (Oryza sativa L.) histone modification gene family (OsHM) at the whole-genome level remains unclear. In this study, a total of 130 OsHMs were identified through a genome-wide analysis. The OsHM gene family can be classified into 11 subfamilies based on a phylogenetic analysis. An analysis of the genes structures and conserved motifs indicates that members of each subfamily share specific conserved protein structures, suggesting their potential conserved functions. Molecular evolutionary analysis reveals that a significant number of OsHMs proteins originated from gene duplication events, particularly segmental duplications. Additionally, transcriptome analysis demonstrates that OsHMs are widely expressed in various tissues of rice and are responsive to multiple abiotic stresses. Fourteen OsHMs exhibit high expression in rice anthers and peaked at different pollen developmental stages. RT-qPCR results further elucidate the expression patterns of these 14 OsHMs during different developmental stages of anthers, highlighting their high expression during the meiosis and tetrad stages, as well as in the late stage of pollen development. Remarkably, OsSDG713 and OsSDG727 were further identified to be nucleus-localized. This study provides a fundamental framework for further exploring the gene functions of HMs in plants, particularly for researching their functions and potential applications in rice anthers' development and male sterility.

An epigenetically mediated double negative cascade from EFD to HB21 regulates anther development

Epigenetic modifications are crucial for plant development. EFD (ExineFormationDefect) encodes a SAM-dependent methyltransferase that is essential for the pollen wall pattern formation and male fertility in Arabidopsis. In this study, we find that the expression of DRM2, a de novo DNA methyltransferase in plants, complements for the defects in efd, suggesting its potential de novo DNA methyltransferase activity. Genetic analysis indicates that EFD functions through HB21, as the knockout of HB21 fully restores fertility in efd mutants. DNA methylation and histone modification analyses reveal that EFD represses the transcription of HB21 through epigenetic mechanisms. Additionally, we demonstrate that HB21 directly represses the expression of genes crucial for pollen formation and anther dehiscence, including CalS5, RPG1/SWEET8, CYP703A2 and NST2. Collectively, our findings unveil a double negative regulatory cascade mediated by epigenetic modifications that coordinates anther development, offering insights into the epigenetic regulation of this process.

Changes in anatomical structure and cell wall composition during male inflorescence development of Castanea mollissima

The Chinese chestnut ( Castanea mollissima Blume) is a dioecious plant in which the number of male inflorescences far exceeds that of female inflorescences. Therefore, it is of interest to study the differentiation of male flowers in C. mollissima. In this study, the male inflorescences of “Yanshanhongli” C. mollissima were observed. Then, the main components of the cell wall were stained by fluorescence and immunofluorescence labeling techniques to examine the changes in cell wall components. The results showed that the development of the male inflorescence of C. mollissima had different transverse elongation and longitudinal elongation. In addition, the development of male inflorescences and anthers were divided into five and four major stages, respectively, according to the microstructural changes. Immunostaining indicated that the fluorescence intensity of cellulose increased with the development of male inflorescences of C. mollissima. The fluorescence intensity of low-esterified homogalacturonan (HG) was stronger in the early stage, while the fluorescence intensity of high-esterified HG was stronger thereafter. This research provides new insights into the changes in cell wall components during the growth and development of male inflorescences of C. mollissima and important clues for exploring the growth and development mechanism of male inflorescences of C. mollissima.

Temperature change regulates pollen fertility of a PTGMS rice line PA64S by modulating the ROS homeostasis and PCD within the tapetum.

Photoperiod and temperature-sensitive male sterility rice is an important line for two-line hybrid rice, and the changes in the cultivation temperature strictly control its pollen fertility. However, the mechanism by which temperature variation regulates pollen fertility is still unclear. This study obtained stable fertile PA64S(F) and sterile PA64S(S) rice from PA64S by controlling temperature changes. PA64S(F) shows a normal anther development and fertile pollen under low temperature (21°C), and PA64S(S) shows delayed degradation of the tapetum cells, leading to abnormal pollen wall formation and ubisch development under normal temperature (28°C). The accumulation of reactive oxygen species (ROS) positively correlates with the programmed cell death (PCD) process of tapetum cells. The delayed accumulation of ROS in the PA64S(S) tapetum at early stages leads to a delayed initiation of the PCD process. Importantly, we localized ascorbic acid (ASA) accumulation in the tapetum cells and determined that ASA is a major antioxidant for ROS homeostasis. ROS-inhibited accumulation plants (PA64S-ASA) demonstrated pollen sterility, higher ASA and lower ROS accumulation in the tapetum, and the absence of PCD processes in the tapetum cell. Abnormal changes in the tapetum of PA64S(S) rice disrupted metabolic pathways such as lipid metabolism, cutin and wax synthesis, sugar accumulation, and phenylpropane, affecting pollen wall formation and substance accumulation, suggesting that the timely accumulation of ROS is critical for male fertility. This study highlights the central role of ROS homeostasis in fertility alteration and also provides an avenue to address the effect of environmental temperature changes on pollen fertility in rice.

Multi-Omics analysis reveals the important role of indole-3-acetic acid homeostasis in male fertility of cotton

Auxin (Indole-3-acetic acid, IAA) is essential for anther development and male fertility, but little is known about its homeostasis in developing anthers due to its complex and diverse synthesis pathways. Here, we employed transcriptomic, targeted metabolomic, and amino acid analyses on a wild type (WT) cotton (Gossypium hirsutum) line SD98–6 and its nearly-isogenic male sterile (MS) line SD98–6A, to delve into the relationship between IAA and anther fertility. In WT anthers, IAA homeostasis was achieved mainly through self-synthesis, conjugate hydrolysis, and polar transport, and accumulation of IAA exhibited an inverted "U" pattern with the highest content observed at the tetrad stage. In MS anthers, no IAA accumulation was observed after the meiosis stage and the IAA content remained constantly low, mainly due to reduction of the substrates (phenylalanine and tryptophan) for IAA synthesis, and significantly decreased expression of the genes involved in IAA synthesis. Meanwhile, carbohydrate metabolism pathways directly contributing to IAA synthesis and synthesis of amino acids were malfunctional in MS anthers based on the expression profile of the metabolic genes and content measurement. These results provide valuable insights into the IAA homeostasis in cotton anthers and imply that genes related to IAA homeostasis are potential targets for development of IAA-sensitive male sterile cotton lines.

R2R3-MYB Gene Family in Coptis teeta Wall.: Genome-Wide Identification, Phylogeny, Evolutionary Expansion, and Expression Analyses during Floral Development.

The R2R3-MYB gene family represents a widely distributed class of plant transcription factors. This gene family plays an important role in many aspects of plant growth and development. However, the characterization of R2R3-MYB genes present in the genome of Coptis teeta has not been reported. Here, we describe the bioinformatic identification and characterization of 88 R2R3-MYB genes in this species, and the identification of members of the R2R3-MYB gene family in species within the order Ranales most closely related to Coptis teeta. The CteR2R3-MYB genes were shown to exhibit a higher degree of conservation compared to those of A. thaliana, as evidenced by phylogeny, conserved motifs, gene structure, and replication event analyses. Cis-acting element analysis confirmed the involvement of CteR2R3-MYB genes in a variety of developmental processes, including growth, cell differentiation, and reproduction mediated by hormone synthesis. In addition, through homology comparisons with the equivalent gene family in A. thaliana, protein regulatory network prediction and transcriptome data analysis of floral organs across three time periods of flower development, 17 candidate genes were shown to exhibit biased expression in two floral phenotypes of C. teeta. This suggests their potential involvement in floral development (anther development) in this species.

Decoding the role of OsPRX83 in enhancing osmotic stress tolerance in rice through ABA-dependent pathways and ROS scavenging

ABSTRACT Plant Class III peroxidases have diverse roles in controlling root hair growth, anther development, and abiotic and biotic stress responses. However, their abiotic stress response mechanism in rice remains elusive. Here, we identified a peroxidase precursor gene, OsPRX83, and investigated its role in enhancing osmotic stress tolerance in rice. We used OsPRX83 overexpression and CRISPR-Cas9-generated mutant lines to elucidate OsPRX83‘s function and expression patterns under stress conditions. The expression of OsPRX83 was induced by H2O2, PEG, NaCl, and ABA treatments. Using qRT-PCR, RNA sequencing, and physiological assays, we demonstrated that overexpression of OsPRX83 enhanced the osmotic and oxidative stress tolerance as compared to the wild-type and mutant seedlings, as evident from the higher survival rates, enhanced peroxidase (POD) and ascorbate peroxidase (APX) activities, and increased ABA sensitivity compared with mutants and wild-type plants. Transcriptome analysis further supported the involvement of OsPRX83 in the ROS scavenging, by modulating the expression of OsDREB1B, OsDREB1E, OsDREB1F, OsDREB1G in response to osmotic treatment. In summary, our study suggests that OsPRX83 plays a pivotal role in enhancing stress tolerance in rice through ABA-dependent pathways and ROS scavenging. Therefore, this study elucidates the function of a novel abiotic stress response gene in rice, thereby may contribute to a new genetic engineering resource for engineering drought-resistant rice varieties.

The non-canonically organized members of MIR395 gene family in Brassica juncea are associated with developmentally regulated, sulfate-stress responsive bidirectional promoters that exhibit orientation-dependent differential transcriptional activity

Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom.In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5’end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.

The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.)

BackgroundThe male sterile lines are an important foundation for heterosis utilization in wheat (Triticum aestivum L.). Thereinto, pollen development is one of the indispensable processes of wheat reproductive development, and its fertility plays an important role in wheat heterosis utilization, and are usually influencing by genes. However, these key genes and their regulatory networks during pollen abortion are poorly understood in wheat.ResultsDEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) is a member of the R2R3-MYB family and has been shown to be essential for early tapetal layer development and pollen grain fertility in rice (Oryza sativa L.) and Arabidopsis thaliana. In order to clarify the function of TDF1 in wheat anthers development, we used OsTDF1 gene as a reference sequence and homologous cloned wheat TaTDF1 gene. TaTDF1 is localized in the nucleus. The average bolting time of Arabidopsis thaliana overexpressed strain (TaTDF1-OE) was 33 d, and its anther could be colored normally by Alexander staining solution, showing red. The dominant Mosaic suppression silence-line (TaTDF1-EAR) was blue-green in color, and the anthers were shrimpy and thin. The TaTDF1 interacting protein (TaMAP65) was confirmed using Yeast Two-Hybrid Assay (Y2H) and Bimolecular-Fluorescence Complementation (BiFC) experiments. The results showed that downregulated expression of TaTDF1 and TaMAP65 could cause anthers to be smaller and shrunken, leading to pollen abortion in TaTDF1 wheat plants induced by virus-induced gene-silencing technology. The expression pattern of TaTDF1 was influenced by TaMAP65.ConclusionsThus, systematically revealing the regulatory mechanism of wheat TaTDF1 during anther and pollen grain development may provide new information on the molecular mechanism of pollen abortion in wheat.

Overexpression of sesame O-acetylserine(thiol)lyase induces male sterility by delaying tapetum programmed cell death

Male sterile lines are key resources for hybrid seed production and for ensuring high varietal purity. However, the genes and mechanisms underlying sesame male sterility remain largely unknown. Hence, this study identified an O-acetylserine(thiol)lyase gene SiOASTL1 and functionally characterized its roles in inducing defective anther development. Spatiotemporal expression analysis revealed that SiOASTL1 is significantly (2.7 fold) up-regulated in sterile sesame anthers at the microspore stage compared with fertile ones. Sequence and phylogenetic analyses showed that SiOASTL1 is homologous to Arabidopsis OAS-TL plastid isoforms. We thus overexpressed SiOASTL1 in Arabidopsis to unravel its regulatory roles. Cytological observation revealed that SiOASTL1 overexpression transformed transgenic plants into male sterile lines arising at the microspore development stage. SiOASTL1 overexpression decreased cysteine biosynthesis and down-regulated the expression of the sporopollenin synthesis-related genes, including AtTKPR1, AtTKPR2, AtPKSA, and AtPKSB in transgenic Arabidopsis. Consequently, the tapetum programmed cell death (PCD) was delayed, resulting in the formation of defective pollen grains with irregular walls and empty cytoplasm. Our findings prove that the induction of SiOASTL1 expression disrupts pollen development and contributes to sesame male sterility. Moreover, these results suggest that genetic manipulation of SiOASTL1 expression may facilitate the development of new hybrid varieties in sesame and other crops.