Heterologous Expression of Two Brassica campestris CCCH Zinc-Finger Proteins in Arabidopsis Induces Cytoplasmic Foci and Causes Pollen Abortion.

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism.

Polygalacturonase45 cleaves pectin and links cell proliferation and morphogenesis to leaf curvature in Arabidopsis thaliana.

Regulating plant architecture is a major goal in current breeding programs. Previous studies have increased our understanding of the genetic regulation of plant architecture, but it is also essential to understand how organ morphology is controlled at the cellular level. In the cell wall, pectin modification and degradation are required for organ morphogenesis, and these processes involve a series of pectin-modifying enzymes. Polygalacturonases (PGs) are a major group of pectin-hydrolyzing enzymes that cleave pectin backbones and release oligogalacturonides (OGs). PG genes function in cell expansion and separation, and contribute to organ expansion, separation and dehiscence in plants. However, whether and how they influence other cellular processes and organ morphogenesis are poorly understood. Here, we characterized the functions of Arabidopsis PG45 (PG45) in organ morphogenesis using genetic, developmental, cell biological and biochemical analyses. A heterologously expressed portion of PG45 cleaves pectic homogalacturonan in vitro, indicating that PG45 is a bona fide PG. PG45 functions in leaf and flower structure, branch formation and organ growth. Undulation in pg45 knockout and PG45 overexpression leaves is accompanied by impaired adaxial-abaxial polarity, and loss of PG45 shortens the duration of cell proliferation in the adaxial epidermis of developing leaves. Abnormal leaf curvature is coupled with altered pectin metabolism and autogenous OG profiles in pg45 knockout and PG45 overexpression leaves. Together, these results highlight a previously underappreciated function for PGs in determining tissue polarity and regulating cell proliferation, and imply the existence of OG-based signaling pathways that modulate plant development.

Arabinogalactan Proteins: Focus on the Role in Cellulose Synthesis and Deposition during Plant Cell Wall Biogenesis.

Arabinogalactan proteins (AGPs) belong to a family of glycoproteins that are widely present in plants. AGPs are mostly composed of a protein backbone decorated with complex carbohydrate side chains and are usually anchored to the plasma membrane or secreted extracellularly. A trickle of compelling biochemical and genetic evidence has demonstrated that AGPs make exciting candidates for a multitude of vital activities related to plant growth and development. However, because of the diversity of AGPs, functional redundancy of AGP family members, and blunt-force research tools, the precise functions of AGPs and their mechanisms of action remain elusive. In this review, we put together the current knowledge about the characteristics, classification, and identification of AGPs and make a summary of the biological functions of AGPs in multiple phases of plant reproduction and developmental processes. In addition, we especially discuss deeply the potential mechanisms for AGP action in different biological processes via their impacts on cellulose synthesis and deposition based on previous studies. Particularly, five hypothetical models that may explain the AGP involvement in cellulose synthesis and deposition during plant cell wall biogenesis are proposed. AGPs open a new avenue for understanding cellulose synthesis and deposition in plants.

FLA14 is required for pollen development and preventing premature pollen germination under high humidity in Arabidopsis.

BACKGROUND: As an important subfamily of arabinogalactan proteins (AGPs), fasciclin-like AGPs (FLAs) contribute to various aspects of growth, development and adaptation, yet their function remains largely elusive. Despite the diversity of FLAs, only two members, Arabidopsis FLA3 and rice MTR1, are reported to be involved in sexual reproduction. In this study, another Arabidopsis FLA-encoding gene, FLA14, was identified, and its role was investigated. RESULTS: Arabidopsis FLA14 was found to be a pollen grain-specific gene. Expression results from fusion with green fluorescent protein showed that FLA14 was localized along the cell membrane and in Hechtian strands. A loss-of-function mutant of FLA14 showed no discernible defects during male gametogenesis, but precocious pollen germination occurred inside the mature anthers under high moisture conditions. Overexpression of FLA14 caused 39.2% abnormal pollen grains with a shrunken and withered appearance, leading to largely reduced fertility with short mature siliques and lower seed set. Cytological and ultramicroscopic observation showed that ectopic expression of FLA14 caused disruption at the uninucleate stage, resulting in either collapsed pollen with absent intine or pollen of normal appearance but with a thickened intine. CONCLUSIONS: Taken together, our data suggest a role for FLA14 in pollen development and preventing premature pollen germination inside the anthers under high relative humidity in Arabidopsis.

Comprehensive Analysis of Arabinogalactan Protein-Encoding Genes Reveals the Involvement of Three BrFLA Genes in Pollen Germination in Brassica rapa.

Arabinogalactan proteins (AGPs) are a superfamily of hydroxyproline-rich glycoproteins that are massively glycosylated, widely implicated in plant growth and development. No comprehensive analysis of the AGP gene family has been performed in Chinese cabbage (Brassica rapa ssp. chinensis). Here, we identified a total of 293 putative AGP-encoding genes in B. rapa, including 25 classical AGPs, three lysine-rich AGPs, 30 AG-peptides, 36 fasciclin-like AGPs (FLAs), 59 phytocyanin-like AGPs, 33 xylogen-like AGPs, 102 other chimeric AGPs, two non-classical AGPs and three AGP/extensin hybrids. Their protein structures, phylogenetic relationships, chromosomal location and gene duplication status were comprehensively analyzed. Based on RNA sequencing data, we found that 73 AGP genes were differentially expressed in the floral buds of the sterile and fertile plants at least at one developmental stage in B. rapa, suggesting a potential role of AGPs in male reproductive development. We further characterized BrFLA2, BrFLA28 and BrFLA32, three FLA members especially expressed in anthers, pollen grains and pollen tubes. BrFLA2, BrFLA28 and BrFLA32 are indispensable for the proper timing of pollen germination under high relative humidity. Our study greatly extends the repertoire of AGPs in B. rapa and reveals a role for three members of the FLA subfamily in pollen germination.

Molecular characterization and expression analysis reveal the roles of Cys2/His2 zinc-finger transcription factors during flower development of Brassica rapa subsp. chinensis.

KEY MESSAGE: Conserved motif, gene structure, expression and interaction analysis of C2H2-ZFPs in Brassica rapa, and identified types of genes may play essential roles in flower development, and BrZFP38 was proved to function in flower development by affecting pollen formation. Flower development plays a central role in determining the reproduction of higher plants, and Cys2/His2 zinc-finger proteins (C2H2-ZFPs) widely participate in the transcriptional regulation of flower development. C2H2-ZFPs with various structures are the most widespread DNA-binding transcription factors in plants. In this study, conserved protein motif and gene structures were analyzed to investigate systematically the molecular features of Brassica rapa C2H2-ZFP genes. Expression of B. rapa C2H2-ZFPs in multiple tissues showed that more than half of the family members with different types ZFs were expressed in flowers. The specific expression profiles of these C2H2-ZFPs in different B. rapa floral bud stages were further evaluated to identify their potential roles in flower development. Interaction networks were constructed in B. rapa based on the orthology of flower-related C2H2-ZFP genes in Arabidopsis. The putative cis-regulatory elements in the promoter regions of these C2H2-ZFP genes were thoroughly analyzed to elucidate their transcriptional regulation. Results showed that the orthologs of known-function flower-related C2H2-ZFP genes were conserved and differentiated in B. rapa. A C2H2-ZFP was proved to function in B. rapa flower development. Our study provides a systematic investigation of the molecular characteristics and expression profiles of C2H2-ZFPs in B. rapa and promotes further work in function and transcriptional regulation of flower development.

AtC3H18L is a stop-codon read-through gene and encodes a novel non-tandem CCCH zinc-finger protein that can form cytoplasmic foci similar to mRNP granules.

The membraneless messenger ribonucleoprotein (mRNP) granules, including processing bodies (PBs) and stress granules (SGs), are important cytoplasmic structures in eukaryotes that can participate in gene expression through mRNA regulation. It has been verified that mRNP granules are mainly composed of proteins and translation-repressed mRNAs. Here, we reported a stop-codon read-through gene, At3g52980, in plants for the first time. At3g52980 encodes a novel non-tandem CCCH zinc-finger (non-TZF) protein named AtC3H18-Like (AtC3H18L), which contains two putative RNA-binding domains. By using transient expression system, we showed that heat treatment can induce the aggregation of diffuse distributed AtC3H18L to form cytoplasmic foci, which were similar to PBs and SGs in morphology. Further analysis did find that AtC3H18L can co-localize with markers of PB and SG. The aggregation of AtC3H18L was closely related to the cytoskeleton, and AtC3H18L-foci were highly dynamic and can move frequently along cytoskeleton. Moreover, analysis in transgenic plants showed that AtC3H18L was specifically expressed in pollen and can form cytoplasmic foci without heat treatment. It will be fascinating in future studies to discover whether and how AtC3H18L affects pollen development by participating in the assembly of mRNP granules as a protein component, especially under heat stress.

BcMF30a and BcMF30c, Two Novel Non-Tandem CCCH Zinc-Finger Proteins, Function in Pollen Development and Pollen Germination in Brassica campestris ssp. chinensis.

Chinese cabbage (Brassica campestris) is an economically important leaf vegetable crop worldwide. Mounting studies have shown that cysteine-cysteine-cysteine-histidine (CCCH) zinc-finger protein genes are involved in various plant growth and development processes. However, research on the involvement of these genes in male reproductive development is still in its infancy. Here, we identified 11 male fertility-related CCCH genes in Chinese cabbage. Among them, a pair of paralogs encoding novel non-tandem CCCH zinc-finger proteins, Brassica campestris Male Fertility 30a (BcMF30a) and BcMF30c, were further characterized. They were highly expressed in pollen during microgametogenesis and continued to express in germinated pollen. Further analyses demonstrated that both BcMF30a and BcMF30c may play a dual role as transcription factors and RNA-binding proteins in plant cells. Functional analysis showed that partial bcmf30a bcmf30c pollen grains were aborted due to the degradation of pollen inclusion at the microgametogenesis phase, and the germination rate of viable pollen was also greatly reduced, indicating that BcMF30a and BcMF30c are required for both pollen development and pollen germination. This research provided insights into the function of CCCH proteins in regulating male reproductive development and laid a theoretical basis for hybrid breeding of Chinese cabbage.

Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Complex Molecular Evolution and Expression of Expansin Gene Families in Three Basic Diploid Species of Brassica.

Expansins are a kind of structural proteins of the plant cell wall, and they enlarge cells by loosening the cell walls. Therefore, expansins are involved in many growth and development processes. The complete genomic sequences of Brassica rapa, Brassica oleracea and Brassica nigra provide effective platforms for researchers to study expansin genes, and can be compared with analogues in Arabidopsis thaliana. This study identified and characterized expansin families in B. rapa, B. oleracea, and B. nigra. Through the comparative analysis of phylogeny, gene structure, and physicochemical properties, the expansin families were divided into four subfamilies, and then their expansion patterns and evolution details were explored accordingly. Results showed that after the three species underwent independent evolution following their separation from A. thaliana, the expansin families in the three species had increased similarities but fewer divergences. By searching divergences of promoters and coding sequences, significant positive correlations were revealed among orthologs in A. thaliana and the three basic species. Subsequently, differential expressions indicated extensive functional divergences in the expansin families of the three species, especially in reproductive development. Hence, these results support the molecular evolution of basic Brassica species, potential functions of these genes, and genetic improvement of related crops.

The distinct functions of two classical arabinogalactan proteins BcMF8 and BcMF18 during pollen wall development in Brassica campestris.

Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline-rich glycoproteins ubiquitous in all plant tissues and cells. AtAGP6 and AtAGP11, the only two functionally known pollen-specific classical AGP encoding genes in Arabidopsis, are reported to have redundant functions in microspore development. BcMF18 and BcMF8 isolated from Brassica campestris are the orthologues of AtAGP6 and AtAGP11, respectively. In contrast to the functional redundancy of AtAGP6 and AtAGP11, single-gene disruption of BcMF8 led to deformed pollen grains with abnormal intine development and ectopic aperture formation in B. campestris. Here, we further explored the action of BcMF18 and its relationship with BcMF8. BcMF18 was specifically expressed in pollen during the late stages of microspore development. Antisense RNA transgenic lines with BcMF18 reduction resulted in aberrant pollen grains with abnormal cellulose distribution, lacking intine, cytoplasm and nuclei. Transgenic plants with repressive expression of both BcMF8 and BcMF18 showed a hybrid phenotype, expressing a mixture of the phenotypes of the single gene knockdown plant lines. In addition, we identified functional diversity between BcMF18/BcMF8 and AtAGP6/AtAGP11, mainly reflected by the specific contribution of BcMF18 and BcMF8 to pollen wall formation. These results suggest that, unlike the orthologous genes AtAGP6 and AtAGP11 in Arabidopsis, BcMF18 and BcMF8 are both integral to pollen biogenesis in B. campestris, acting through independent pathways during microspore development.

Systematic identification of long non-coding RNAs during pollen development and fertilization in Brassica rapa.

The importance of long non-coding RNAs (lncRNAs) in plant development has been established, but a systematic analysis of lncRNAs expressed during pollen development and fertilization has been elusive. We performed a time series of RNA-seq experiments at five developmental stages during pollen development and three different time points after pollination in Brassica rapa and identified 12 051 putative lncRNAs. A comprehensive view of dynamic lncRNA expression networks underpinning pollen development and fertilization was provided. B. rapa lncRNAs share many common characteristics of lncRNAs: relatively short length, low expression but specific in narrow time windows, and low evolutionary conservation. Gene modules and key lncRNAs regulating reproductive development such as exine formation were uncovered. Forty-seven cis-acting lncRNAs and 451 trans-acting lncRNAs were revealed to be highly coexpressed with their target protein-coding genes. Of particular importance are the discoveries of 14 lncRNAs that were highly coexpressed with 10 function-known pollen-associated coding genes. Fifteen lncRNAs were predicted as endogenous target mimics for 13 miRNAs, and two lncRNAs were proved to be functional target mimics for miR160 after experimental verification and shown to function in pollen development. Our study provides the systematic identification of lncRNAs during pollen development and fertilization in B. rapa and forms the foundation for future genetic, genomic, and evolutionary studies.

Comparative transcriptome analysis and ChIP-sequencing reveals stage-specific gene expression and regulation profiles associated with pollen wall formation in Brassica rapa.

BACKGROUND: Genic male sterility (GMS) line is an important approach to utilize heterosis in Brassica rapa, one of the most widely cultivated vegetable crops in Northeast Asia. However, the molecular genetic mechanisms of GMS remain to be largely unknown. RESULTS: Detailed phenotypic observation of 'Bcajh97-01A/B', a B. rapa genic male sterile AB line in this study revealed that the aberrant meiotic cytokinesis and premature tapetal programmed cell death occurring in the sterile line ultimately resulted in microspore degeneration and pollen wall defect. Further gene expression profile of the sterile and fertile floral buds of 'Bcajh97-01A/B' at five typical developmental stages during pollen development supported the result of phenotypic observation and identified stage-specific genes associated with the main events associated with pollen wall development, including tapetum development or functioning, callose metabolism, pollen exine formation and cell wall modification. Additionally, by using ChIP-sequencing, the genomic and gene-level distribution of trimethylated histone H3 lysine 4 (H3K4) and H3K27 were mapped on the fertile floral buds, and a great deal of pollen development-associated genes that were covalently modified by H3K4me3 and H3K27me3 were identified. CONCLUSIONS: Our study provids a deeper understanding into the gene expression and regulation network during pollen development and pollen wall formation in B. rapa, and enabled the identification of a set of candidate genes for further functional annotation.

Evolution of MIR159/319 genes in Brassica campestris and their function in pollen development.

KEY MESSAGE: MIR159/319 have conserved evolution and diversified function after WGT in Brassica campestris, both of them can lead pollen vitality and germination abnormality, Bra-MIR319c also can function in flower development. MiR159 and miR319 are extensively studied highly conserved microRNAs which play roles in vegetative development, reproduction, and hormone regulation. In this study, the effects of whole-genome triplication (WGT) on the evolution of the MIR159/319 family and the functional diversification of the genes were comprehensively investigated in Brassica campestris. We identified 11 MIR159/319 genes in B. campestris, which produced five mature sequences. After analyzing the precursor sequences and phylogenetic tree, we found that Bra-MIR159/319 have evolutionary conservatism. Furthermore, Bra-MIR159/319 show functional diversification after WGT, as indicated by their expression patterns and the cis-element in their promoter. GUS signal showed that Bra-MIR159a and Bra-MIR319c can be expressed in anther but in different development stages. In B. campestris, overexpressed MIR159a and MIR319c contribute to late anther development and promote pollen abortion. Moreover, Bra-MIR319c can partially assume the function of MIR319a in flower development.

Overexpression of a stamen-specific R2R3-MYB gene BcMF28 causes aberrant stamen development in transgenic Arabidopsis.

In flowering plants, stamen development is a complex multistage process, which is highly regulated by a series of transcription factors. In this study, BcMF28, which encodes a R2R3-MYB transcription factor, was isolated from Brassica campestris. BcMF28 is localized in the nucleus and cytoplasm, and acts as a transcriptional activator. Quantitative real-time PCR and promoter activity analysis revealed that BcMF28 was predominately expressed in inflorescences. The expression of BcMF28 was specifically detected in tapetum, developing microspores, anther endothecium, and filaments during late stamen development. The overexpression of BcMF28 in Arabidopsis resulted in aberrant stamen development, including filament shortening, anther indehiscence, and pollen abortion. Detailed analysis of anther development in transgenic plants revealed that the degeneration of septum and stomium did not occur, and endothecium lignification was affected. Furthermore, the expression levels of genes involved in the phenylpropanoid metabolism pathway were altered in BcMF28-overexpressing transgenic plants. Our results suggest that BcMF28 plays an important regulatory role during late stamen development.

Arabidopsis Cys2/His2 zinc-finger protein MAZ1 is essential for intine formation and exine pattern.

Cys2/His2 zinc-finger protein (C2H2-ZFP) is widely involved in the reproductive development of plants, but its role in pollen development is still elusive. Here, we identified a pollen-related C2H2-ZFP gene named as MALE FERTILITY-ASSOCIATED ZINC FINGER PROTEIN 1 (MAZ1), which was first isolated from Arabidopsis thaliana. MAZ1 showed a preferential expression pattern in early anther development. Its mutation resulted in aberrant primexine deposition at the tetrad stage, followed by a defective multiple-layer pattern of exine with irregular baculum and no tectum. Furthermore, microspore development was arrested, and no intine layer was formed. These developmental defects led to fertility reduction and pollen abortion. This study reveals the essential role of MAZ1 in pollen wall development.

BcPME37c is involved in pollen intine formation in Brassica campestris.

Pollen wall development is one of the key processes of pollen development. Several pectin methylesterase (PME) genes participate in pollen germination and pollen tube growth. However, the relationship between PME genes and pollen intine formation remains unclear. In this study, we investigated the expression and subcellular localization of the PME gene BcPME37c in Brassica campestris. Furthermore, morphology and cytology methods were used to examine the phenotype of the CRISPR/Cas9 system-induced BcPME37c mutant. We found that BcPME37c is predominately expressed in mature stamen and located at the cell wall. BcPME37c mutation causes the abnormal thickening of the pollen intine of B. campestris. Our study indicated that BcPME37c is required for pollen intine formation in B. campestris.

Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.

Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.

CircRNA Expression Pattern and ceRNA and miRNA-mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris.

Male-sterile plants provide an important breeding tool for the heterosis of hybrid crops, such as Brassicaceae. In the last decade, circular RNAs (circRNAs), as a novel class of covalently closed and single-stranded endogenous non-coding RNAs (ncRNAs), have received much attention because of their functions as "microRNA (miRNA) sponges" and "competing endogenous RNAs" (ceRNAs). However, the information about circRNAs in the regulation of male-sterility and anther development is limited. In this study, we established the Polima cytoplasm male sterility (CMS) line "Bcpol97-05A", and the fertile line, "Bcajh97-01B", in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis, and performed RNA expression profiling comparisons between the flower buds of the sterile line and fertile line by whole-transcriptome sequencing. A total of 31 differentially expressed (DE) circRNAs, 47 DE miRNAs, and 4779 DE mRNAs were identified. By using Cytoscape, the miRNA-mediated regulatory network and ceRNA network were constructed, and the circRNA A02:23507399|23531438 was hypothesized to be an important circRNA regulating anther development at the post-transcriptional level. The gene ontology (GO) analysis demonstrated that miRNAs and circRNAs could regulate the orderly secretion and deposition of cellulose, sporopollenin, pectin, and tryphine; the timely degradation of lipids; and the programmed cell death (PCD) of tapetum cells, which play key roles in anther development. Our study revealed a new circRNA-miRNA-mRNA network, which is involved in the anther development of B. campestris, which enriched the understanding of CMS in flowering plants, and laid a foundation for further study on the functions of circRNAs and miRNAs during anther development.

Genome-wide identification, phylogeny, evolution, and expression patterns of MtN3/saliva/SWEET genes and functional analysis of BcNS in Brassica rapa.

BACKGROUND: Members of the MtN3/saliva/SWEET gene family are present in various organisms and are highly conserved. Their precise biochemical functions remain unclear, especially in Chinese cabbage. Based on the whole genome sequence, this study aims to identify the MtN3/saliva/SWEETs family members in Chinese cabbage and to analyze their classification, gene structure, chromosome distribution, phylogenetic relationship, expression pattern, and biological functions. RESULTS: We identified 34 SWEET genes in Chinese cabbage and analyzed their localization on chromosomes and transmembrane regions of their corresponding proteins. Analysis of a phylogenetic tree indicated that there were at least 17 supposed ancestor genes before the separation in Brassica rapa and Arabidopsis. The expression patterns of these genes in different tissues and flower developmental stages of Chinese cabbage showed that they are mainly involved in reproductive development. The Ka/Ks ratio between paralogous SWEET gene pairs of B. rapa were far less than 1. In our previous study, At2g39060 homologous gene Bra000116 (BraSWEET9, also named BcNS, Brassica Nectary and Stamen) played an important role during flower development in Chinese cabbage. Instantaneous expression experiments in onion epidermal cells showed that the gene encoding this protein is localized to the plasma membrane. A basal nectary split is the phenotype of transgenic plants transformed with the antisense expression vector. CONCLUSION: This study is the first to perform a sequence analysis, structures analysis, physiological and biochemical characteristics analysis of the MtN3/saliva/SWEETs gene in Chinese cabbage and to verify the function of BcNS. A total of 34 SWEET genes were identified and they are distributed among ten chromosomes and one scaffold. The Ka/Ks ratio implies that the duplication genes suffered strong purifying selection for retention. These genes were differentially expressed in different floral organs. The phenotypes of the transgenic plants indicated that BcNs participates in the development of the floral nectary. This study provides a basis for further functional analysis of the MtN3/saliva/SWEETs gene family.