Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

Convergent evolutionary patterns of heterostyly across angiosperms support the pollination-precision hypothesis.

Since the insights by Charles Darwin, heterostyly, a floral polymorphism with morphs bearing stigmas and anthers at reciprocal heights, has become a model system for the study of natural selection. Based on his archetypal heterostylous flower, including regular symmetry, few stamens and a tube, Darwin hypothesised that heterostyly evolved to promote outcrossing through efficient pollen transfer between morphs involving different areas of a pollinator's body, thus proposing his seminal pollination-precision hypothesis. Here we update the number of heterostylous and other style-length polymorphic taxa to 247 genera belonging to 34 families, notably expanding known cases by 20%. Using phylogenetic and comparative analyses across the angiosperms, we show numerous independent origins of style-length polymorphism associated with actinomorphic, tubular flowers with a low number of sex organs, stamens fused to the corolla, and pollination by long-tongued insects. These associations provide support for the Darwinian pollination-precision hypothesis as a basis for convergent evolution of heterostyly across angiosperms.

Comprehensive analysis of WOX transcription factors provide insight into genes related to the regulation of unisexual flowers development in Akebia trifoliata.

Akebia trifoliata is a fascinating economic and medicinal plant that produces functionally unisexual flowers due to stamen/pistil abortion during flower development, and the genetic regulation pathway of this process remain completely unknown. Here, 10 AktWOXs were identified for the first time, all contained a highly conserved homeodomain. AktWOXs were divided into three clades, each with the same or similar intron, exon, and motifs distribution. Many cis-elements related to stress response, growth and development, and hormone response were found in the AktWOXs promoter region. In addition, four candidate genes AktWOX8, AktWOX11, AktWOX13.2 and AktWUS that might be involved in unisexual flowers development were screened, all of which were located in the nucleus and showed transcriptional activation activity. Yeast one-hybrid showed that both AktKNU and AktAG1, the potential core transcription factors in the activity termination pathway of flower meristem stem cells, could bind to the promoter region of AktWUS. Dual-luciferase assay further confirmed that only AktKNU inhibited the expression of AktWUS. Collectively, this study revealed the mechanism of AktWUS that might affect the formation of unisexual flowers by regulating the timely termination of flower meristem in A. trifoliata.

[Exploration of cross-cultivar group characteristics of a new cultivar of Prunus mume 'Zhizhang Guhong Chongcui'].

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.

Unlocking a 'lock-key' mechanism governing pollen-pistil interactions.

Pollen-pistil interactions ensure genetic diversity and shape the reproductive success of plants. Lan et al. recently revealed that the interaction among various receptor-like kinases, cell-wall proteins, and stigmatic RALF peptides (sRALFs) or pollen RALF peptides (pRALFs) on the stigma surface govern the penetration of pollen tubes in members of the Brassicaceae.

Phenolic Acids and Flavonoids Play Important Roles in Flower Bud Differentiation in Mikania micrantha: Transcriptomics and Metabolomics.

Mikania micrantha is a highly invasive vine, and its ability to sexually reproduce is a major obstacle to its eradication. The long-distance dissemination of M. micrantha depends on the distribution of seeds; therefore, inhibiting M. micrantha flowering and seed production is an effective control strategy. The number of blooms of M. micrantha differs at different altitudes (200, 900, and 1300 m). In this study, we used a combination of metabolomics and transcriptomics methods to study the patterns of metabolite accumulation in the flower buds of M. micrantha. Using LC-MS/MS, 658 metabolites were found in the flower buds of M. micrantha at three different altitudes (200, 900, and 1300 m). Flavonoids and phenolic acids were found to be the main differential metabolites, and their concentrations were lower at 900 m than at 200 m and 1300 m, with the concentrations of benzoic acid, ferulic acid, and caffeic acid being the lowest. The biosynthesis pathways for flavonoids and phenolic compounds were significantly enriched for differentially expressed genes (DEGs), according to the results of transcriptome analysis. The production of flavonoid and phenolic acids was strongly linked with the expressions of phenylalanine ammonia-lyase (PAL), caffeoyl-CoA O-methyltransferase (COMT), and 4-coumarate-CoA ligase (4CL), according to the results of the combined transcriptome and metabolome analysis. These genes' roles in the regulation of distinct phenolic acids and flavonoids during M. micrantha bud differentiation are still unknown. This study adds to our understanding of how phenolic acids and flavonoids are regulated in M. micrantha flower buds at various altitudes and identifies regulatory networks that may be involved in this phenomenon, offering a new approach for the prevention and management of M. micrantha.

SPA, a Stigma-style-transmitting tract Physical microenvironment Assay for investigating mechano-signaling in pollen tubes.

Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it's naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.

The yield of common buckwheat (Fagopyrum esculentum Moench) depends on the genotype but not on the Pin-to-Thrum ratio.

Common buckwheat has a complicated flowering biology. It is characterized by a strong self-incompatibility resulting from heterostyly, i.e. the occurrence of two types of flowers: Pin and Thrum, differing in the length of pistils and stamens. Fertilization occurs only as a result of cross-pollination between these morphs. Suspicions exist that the disturbed ratio between plants producing Pin and Thrum flowers (with the latter type generating more seeds) causes low seed yield. The aim of the study was to analyze: (1) the ratio between plants with Pin and Thrum morphs, (2) flower and seed production, as well as abortion of flowers, (3) the composition of nectar collected at an early flowering stage and during full flowering. The study was performed under semi-controlled and field conditions on six Polish accessions. The results indicated that under semi-controlled conditions the Pin-to-Thrum ratio was indeed disproportionate; such a phenomenon is called anisoplethy. In the field, however, the Pin-to-Thrum ratio was well-balanced (isoplethy). The plants with both morphs aborted a similar percentage of flowers and produced a comparable number of empty seeds. The number of flowers, their abortion, and ripe seed production were independent of flower type, however, they were genotypically controlled. A strong correlation between the number of flowers produced by a plant, flower abortion and the number of empty seeds was found. The percentage of aborted flowers correlated positively with the weight of ripe seeds. Nectar composition was similar for all buckwheat genotypes, but we found some differences in the amount of individual sugars depending on the blooming stage. In the majority of accessions, the nectar produced at the early blooming stage was characterized by a greater mass and volume, and contained more individual sugars than at the full-flowering stage.

OsGA3ox genes regulate rice fertility and plant height by synthesizing diverse active GA.

Gibberellin (GA) is an important hormone, which is involved in regulating various growth and development. GA biosynthesis pathway and synthetase have been basically clarified. Gibberellin 3ß hydroxylase (GA3ox) is the key enzyme for the synthesis of various active GA. There are two GA3ox genes (OsGA3ox1 and OsGA3ox2) in rice, and their physiological functions have been preliminarily studied. However, it is not clear how they work together to synthesize active GA to regulate rice development. In this study, the knockout mutants ga3ox1 and ga3ox2 were obtained by CRISPR/Cas9 technology. The pollen fertility of ga3ox1 decreased significantly, while the plant height of ga3ox2 decreased significantly. It shows that OsGA3ox1 is necessary for normal pollen development, while OsGA3ox2 is necessary for stem and leaf elongation. Tissue expression analysis showed that OsGA3ox1 was mainly expressed in unopened flowers, while OsGA3ox2 was mainly expressed in unexpanded leaves. The GA in different tissues of wild type (WT), and two ga3ox mutants were detected. It was found that pollen fertility is most closely related to the content of GA7, and plant height is most closely related to the content of GA1. It was found that OsGA3ox1 catalyzes GA9 to GA7 in flowers, which is closely related to pollen fertility; OsGA3ox2 catalyzes the GA20 to GA1 in unexpanded leaves, thereby regulating plant height; OsGA3ox1 catalyzes the GA19 to GA20 in roots, regulating the generation of GA3. OsGA3ox1 and OsGA3ox2 respond to developmental and environmental signals, and cooperate to synthesize endogenous GA in different tissues to regulate rice development. This study provides a reference for clarifying its role in GA biosynthesis pathway and further understanding the function of OsGA3ox.

Profile and potential bioactivity of the miRNome and metabolome expressed in Malva sylvestris L. leaf and flower.

Malva sylvestris L. (common mallow) is a plant species widely used in phytotherapy and ethnobotanical practices since time immemorial. Characterizing the components of this herb might promote a better comprehension of its biological effects on the human body but also favour the identification of the molecular processes that occur in the plant tissues. Thus, in the present contribution, the scientific knowledge about the metabolomic profile of the common mallow was expanded. In particular, the phytocomplex of leaves and flowers from this botanical species and the extraction capacity of different concentrations of ethanol (i.e., 95%, 70%, 50%, and 0%; v/v in ddH2O) for it were investigated by spectrophotometric and chromatographic approaches. In detail, 95% ethanol extracts showed the worst capacity in isolating total phenols and flavonoids, while all the hydroalcoholic samples revealed a specific ability in purifying the anthocyanins. HPLC-DAD system detected and quantified 20 phenolic secondary metabolites, whose concentration in the several extracts depended on their own chemical nature and the percentage of ethanol used in the preparation. In addition, the stability of the purified phytochemicals after resuspension in pure ddH2O was also proved, considering a potential employment of them in biological/medical studies which include in vitro and in vivo experiments on mammalian models. Here, for the first time, the expressed miRNome in M. sylvestris was also defined by Next Generation Sequencing, revealing the presence of 33 microRNAs (miRNAs), 10 typical for leaves and 2 for flowers. Then, both plant and human putative mRNA targets for the detected miRNAs were predicted by bioinformatics analyses, with the aim to clarify the possible role of these small nucleic acids in the common mallow plant tissues and to try to understand if they could exert a potential cross-kingdom regulatory activity on the human health. Surprisingly, our investigations revealed that 19 miRNAs out of 33 were putatively able to modulate, in the plant cells, the expression of various chromosome scaffold proteins. In parallel, we found, in the human transcriptome, a total of 383 mRNAs involved in 5 fundamental mammalian cellular processes (i.e., apoptosis, senescence, cell-cycle, oxidative stress, and invasiveness) that theoretically could be bound and regulated by M. sylvestris miRNAs. The evidence collected in this work would suggest that the beneficial properties of the use of M. sylvestris, documented by the folk medicine, are probably linked to their content of miRNAs and not only to the action of phytochemicals (e.g., anthocyanins). This would open new perspectives about the possibility to develop gene therapies based on miRNAs isolated from medicinal plants, including M. sylvestris.

Integrated metabolic profiling and transcriptome analysis of Lonicera japonica flowers for chlorogenic acid, luteolin and endogenous hormone syntheses.

The active ingredients of many medicinal plants are the secondary metabolites associated with the growth period. Lonicera japonica Thunb. is an important traditional Chinese medicine, and the flower development stage is an important factor that influences the quality of medicinal ingredients. In this study, transcriptomics and metabolomics were performed to reveal the regulatory mechanism of secondary metabolites during flowering of L. japonica. The results showed that the content of chlorogenic acid (CGA) and luteolin gradually decreased from green bud stage (Sa) to white flower stage (Sc), especially from white flower bud stage (Sb) to Sc. Most of the genes encoding the crucial rate-limiting enzymes, including PAL, C4H, HCT, C3'H, F3'H and FNSII, were down-regulated in three comparisons. Correlation analysis identified some members of the MYB, AP2/ERF, bHLH and NAC transcription factor families that are closely related to CGA and luteolin biosynthesis. Furthermore, differentially expressed genes (DEGs) involved in hormone biosynthesis, signalling pathways and flowering process were analysed in three flower developmental stage.

Genome-wide identification and characterization of SRLK gene family reveal their roles in self-incompatibility of Erigeron breviscapus.

Self-incompatibility (SI) is a reproductive protection mechanism that plants acquired during evolution to prevent self-recession. As the female determinant of SI specificity, SRK has been shown to be the only recognized gene on the stigma and plays important roles in SI response. Asteraceae is the largest family of dicotyledonous plants, many of which exhibit self-incompatibility. However, systematic studies on SRK gene family in Asteraceae are still limited due to lack of high-quality genomic data. In this study, we performed the first systematic genome-wide identification of S-locus receptor like kinases (SRLKs) in the self-incompatible Asteraceae species, Erigeron breviscapus, which is also a widely used perennial medicinal plant endemic to China.52 SRLK genes were identified in the E. breviscapus genome. Structural analysis revealed that the EbSRLK proteins in E. breviscapus are conserved. SRLK proteins from E. breviscapus and other SI plants are clustered into 7 clades, and the majority of the EbSRLK proteins are distributed in Clade I. Chromosomal and duplication analyses indicate that 65% of the EbSRLK genes belong to tandem repeats and could be divided into six tandem gene clusters. Gene expression patterns obtained in E. breviscapus multiple-tissue RNA-Seq data revealed differential temporal and spatial features of EbSRLK genes. Among these, two EbSRLK genes having high expression levels in tongue flowers were cloned. Subcellular localization assay demonstrated that both of their fused proteins are localized on the plasma membrane. All these results indicated that EbSRLK genes possibly involved in SI response in E. breviscapus. This comprehensive genome-wide study of the SRLK gene family in E. breviscapus provides valuable information for understanding the mechanism of SSI in Asteraceae.

Pollen production per flower increases with floral display size across animal-pollinated flowering plants.

PREMISE: The number of open flowers on a plant (i.e., floral display size) can influence plant fitness by increasing pollinator attraction. However, diminishing marginal fitness returns with increasing floral display are expected as pollinators tend to visit more flowers per plant consecutively. An extended flower visitation sequence increases the fraction of ovules disabled by self-pollination (ovule discounting) and reduces the fraction of a plant's own pollen that is exported to sire seeds in other plants (pollen discounting). Hermaphroditic species with a genetic system that prevents self-fertilization (self-incompatibility) would avoid ovule discounting and its fitness cost, whereas species without such a genetically based barrier would not. Contrarily, pollen discounting would be an unavoidable consequence of a large floral display irrespective of selfing barriers. Nevertheless, the increasing fitness costs of ovule and pollen discounting could be offset by respectively increasing ovule and pollen production per flower. METHODS: We compiled data on floral display size and pollen and ovule production per flower for 1241 animal-pollinated, hermaphroditic angiosperm species, including data on the compatibility system for 779 species. We used phylogenetic general linear mixed models to assess the relations of pollen and ovule production to floral display size. RESULTS: Our findings provide evidence of increasing pollen production, but not of ovule production, with increasing display size irrespective of compatibility system and even after accounting for potentially confounding effects like flower size and growth form. CONCLUSIONS: Our comparative study supports the pollen-discount expectation of an adaptive link between per-flower pollen production and floral display across animal-pollinated angiosperms.

Comparative study on abortion characteristics of Nsa CMS and Pol CMS and analysis of long non-coding RNAs related to pollen abortion in Brassica napus.

Cytoplasmic male sterile system (CMS) is one of the important methods for the utilization of heterosisin Brassica napus. The involvement of long non-coding RNAs (lncRNAs) in anther and pollen development in B.napus has been recognized, but there is little data on the involvement of lncRNAs in pollen abortion in different types of rapeseed CMS. The present study compared the cytological, physiological and biochemical characteristics of Nsa CMS (1258A) and Pol CMS (P5A) during pollen abortion, and high-throughput sequencing of flower buds of different sizes before and after pollen abortion. The results showed that insufficient energy supply was an important physiological basis for 1258A and P5A pollen abortion, and 1258A had excessive ROS (reactive oxygen species) accumulation in the stage of pollen abortion. Functional analysis showed that Starch and sucrose metabolism and Sulfur metabolism were significantly enriched before and after pollen abortion in 1258A and P5A, and a large number of genes were down-regulated. In 1258A, 227 lncRNAs had cis-targeting regulation, and 240 cis-target genes of the lncRNAs were identified. In P5A, 116 lncRNAs had cis-targeting regulation, and 101 cis-target genes of the lncRNAs were identified. There were five lncRNAs cis-target genes in 1258A and P5A during pollen abortion, and LOC106445716 encodes ß-D-glucopyranosyl abscisate ß-glucosidase and could regulate pollen abortion. Taken together, this study, provides a new perspective for lncRNAs to participate in the regulation of Nsa CMS and Pol CMS pollen abortion.

Identifying Genes Associated with Female Flower Development of Phellodendron amurense Rupr. Using a Transcriptomics Approach.

Phellodendron amurense Rupr., a species of Rutaceae, is a nationally protected and valuable medicinal plant. It is generally considered to be dioecious. With the discovery of monoecious P. amurense, the phenomenon that its sex development is regulated by epigenetics has been revealed, but the way epigenetics affects the sex differentiation of P. amurense is still unclear. In this study, we investigated the effect of DNA methylation on the sexual development of P. amurense. The young inflorescences of male plants were treated with the demethylation agent 5-azaC, and the induced female flowers were obtained. The induced female flowers' morphological functions and transcriptome levels were close to those of normally developed plants. Genes associated with the development of female flowers were studied by comparing the differences in transcriptome levels between the male and female flowers. Referring to sex-related genes reported in other plants, 188 candidate genes related to the development of female flowers were obtained, including sex-regulating genes, genes related to the formation and development of sexual organs, genes related to biochemical pathways, and hormone-related genes. RPP0W, PAL3, MCM2, MCM6, SUP, PIN1, AINTEGUMENTA, AINTEGUMENTA-LIKE6, AGL11, SEUSS, SHI-RELATED SEQUENCE 5, and ESR2 were preliminarily considered the key genes for female flower development. This study has demonstrated that epigenetics was involved in the sex regulation of P. amurense, with DNA methylation as one of its regulatory modes. Moreover, some candidate genes related to the sexual differentiation of P. amurense were obtained with analysis. These results are of great significance for further exploring the mechanism of sex differentiation of P. amurense and studying of sex differentiation of plants.

Pollen-stigma incompatibility within and between species: Tread lightly, sedate the dogs, and don't wake the guards!

In a recent issue of Nature, Huang et al. identify and show how to overcome the barriers to successful pollen germination after interspecific crosses.1 Their findings answer a long-standing question about reproductive barriers in flowering plants and open the door to harnessing genetic diversity of distant relatives for crop improvement.

Cloning and Expression Analysis of Onion (Allium cepa L.) MADS-Box Genes and Regulation Mechanism of Cytoplasmic Male Sterility.

Flower organ development is one of the most important processes in plant life. However, onion CMS (cytoplasmic male sterility) shows an abnormal development of floral organs. The regulation of MADS-box transcription factors is important for flower development. To further understand the role of MADS-box transcription factors in the regulation of cytoplasmic male sterility onions. We cloned the full-length cDNA of five MADS-box transcription factors from the flowers of onion using RACE (rapid amplification of cDNA ends) technology. We used bioinformatics methods for sequence analysis and phylogenetic analysis. Real-time quantitative PCR was used to detect the expression patterns of these genes in different onion organs. The relative expression levels of five flower development genes were compared in CMS onions and wild onions. The results showed that the full-length cDNA sequences of the cloned MADS-box genes AcFUL, AcDEF, AcPI, AcAG, and AcSEP3 belonged to A, B, C, and E MADS-box genes, respectively. A phylogenetic tree construction analysis was performed on its sequence. Analysis of MADS-box gene expression in wild onion and CMS onion showed that the formation of CMS onion was caused by down-regulation of AcDEF, AcPI, and AcAG gene expression, up-regulation of AcSEP3 gene expression, and no correlation with AcFUL gene expression. This work laid the foundation for further study of the molecular mechanism of onion flower development and the molecular mechanism of CMS onion male sterility.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice.

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/ß Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1-1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

RD29A promoter constitutively drives a rice Hd3a expression to promote early-flowering in Saussurea involucrate Kar. et Kir. ex Maxim.

S. involucratae, an endemic and endangered plant, is a valuable and traditional Chinese medicinal herb. In order to control the flowering time of S. involucratae, we used the well-known stress inducible RD29A promoter to drive Hd3a (a FT ortholog from rice) expression in S. involucratae. Unexpectedly, the majority of regenerated buds in RD29A::Hd3a transgenic lines (S-RH) produced flowers in tissue culture stage under normal growth (25 ± 2 °C) condition. Their flowering time was not further influenced by salt treatment. Hd3a in S-RH was strongly expressed in MS media supplemented with or without 50 mM NaCl. RD29A::GUS transgenic experiments further revealed that RD29A constitutively promoted GUS expression in both S. involucrate and halophyte Thellungiella halophile, in contrast to glycophic plants Oryza sativa L. 'Zhonghua 11', in which its expression was up-regulated by cold, salinity, and drought stress. The results supported the hypothesis that RD29A promoter activity is inducible in stress-sensitive plants, but constitutive in stress-tolerant ones. Importantly, S-RH plants produced pollen grains and seeds under normal conditions. Additionally, we found that OsLEA3-1::Hd3a and HSP18.2::Hd3a could not promote S. involucrate to flower under either normal conditions or abiotic stresses. Taken together, we demonstrated the potential of RD29A::Hd3a might be served as a feasible approach in breeding S. involucrate under normal condition.