Transcriptome Dynamics of Brassica juncea Leaves in Response to Omnivorous Beet Armyworm (Spodoptera exigua, Hübner).

Cruciferous plants manufacture glucosinolates (GSLs) as special and important defense compounds against insects. However, how insect feeding induces glucosinolates in Brassica to mediate insect resistance, and how plants regulate the strength of anti-insect defense response during insect feeding, remains unclear. Here, mustard (Brassica juncea), a widely cultivated Brassica plant, and beet armyworm (Spodoptera exigua), an economically important polyphagous pest of many crops, were used to analyze the changes in GSLs and transcriptome of Brassica during insect feeding, thereby revealing the plant-insect interaction in Brassica plants. The results showed that the content of GSLs began to significantly increase after 48 h of herbivory by S. exigua, with sinigrin as the main component. Transcriptome analysis showed that a total of 8940 DEGs were identified in mustard challenged with beet armyworm larvae. The functional enrichment results revealed that the pathways related to the biosynthesis of glucosinolate and jasmonic acid were significantly enriched by upregulated DEGs, suggesting that mustard might provide a defense against herbivory by inducing JA biosynthesis and then promoting GSL accumulation. Surprisingly, genes regulating JA catabolism and inactivation were also activated, and both JA signaling repressors (JAZs and JAMs) and activators (MYCs and NACs) were upregulated during herbivory. Taken together, our results indicate that the accumulation of GSLs regulated by JA signaling, and the regulation of active and inactive JA compound conversion, as well as the activation of JA signaling repressors and activators, collectively control the anti-insect defense response and avoid over-stunted growth in mustard during insect feeding.

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.

The complete chloroplast genome sequence of Plectranthus hadiensis (Lamiaceae) and phylogenetic analysis.

Plants of the genus Plectranthus are used for the treatment of digestive problems, skin diseases, and allergies, with a wide variety of uses. Here, the complete chloroplast genome sequence of Plectranthus hadiensis (Benth. ex E.Mey) Codd. 1788 was assembled and characterized for the first time. The full length of the chloroplast genome is 152,484 bp, consisting of a small single-copy region of 17,686 bp, a large single-copy region of 83,380 bp, and a pair of inverted repeats of 51,418 bp. The overall GC content is 37.73%. The chloroplast genome contains 131 unique genes, including 87 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes. Phylogenetic tree construction based on the complete chloroplast genome sequences of 25 species (23 Nepetoideae, two Ajugoideae) of the Lamiaceae family showed that P. hadiensis exhibited the closest relationship with Isodon.

Highly Overexpressed AtC3H18 Impairs Microgametogenesis via Promoting the Continuous Assembly of mRNP Granules.

Plant CCCH zinc-finger proteins form a large family of regulatory proteins function in many aspects of plant growth, development and environmental responses. Despite increasing reports indicate that many CCCH zinc-finger proteins exhibit similar subcellular localization of being localized in cytoplasmic foci, the underlying molecular mechanism and the connection between this specific localization pattern and protein functions remain largely elusive. Here, we identified another cytoplasmic foci-localized CCCH zinc-finger protein, AtC3H18, in Arabidopsis thaliana. AtC3H18 is predominantly expressed in developing pollen during microgametogenesis. Although atc3h18 mutants did not show any abnormal phenotype, possibly due to redundant gene(s), aberrant AtC3H18 expression levels caused by overexpression resulted in the assembly of AtC3H18-positive granules in a dose-dependent manner, which in turn led to male sterility phenotype, highlighting the importance of fine-tuned AtC3H18 expression. Further analyzes demonstrated that AtC3H18-positive granules are messenger ribonucleoprotein (mRNP) granules, since they can exhibit liquid-like physical properties, and are associated with another two mRNP granules known as processing bodies (PBs) and stress granules (SGs), reservoirs of translationally inhibited mRNAs. Moreover, the assembly of AtC3H18-positive granules depends on mRNA availability. Combined with our previous findings on the AtC3H18 homologous genes in Brassica campestris, we concluded that appropriate expression level of AtC3H18 during microgametogenesis is essential for normal pollen development, and we also speculated that AtC3H18 may act as a key component of mRNP granules to modulate pollen mRNAs by regulating the assembly/disassembly of mRNP granules, thereby affecting pollen development.

Integrative Analysis of Transcriptomic and Proteomic Changes Related to Cytoplasmic Male Sterility in Spring Stem Mustard (Brassica juncea var. tumida Tsen et Lee).

The development of flower and pollen is a complex biological process that involves multiple metabolic pathways in plants. In revealing novel insights into flower and pollen development underlying male sterility (MS), we conducted an integrated profiling of gene and protein activities in developing buds in cytoplasmic male sterile (CMS) mutants of mustard (Brassica juncea). Using RNA-Seq and label-free quantitative proteomics, 11,832 transcripts and 1780 protein species were identified with significant differential abundance between the male sterile line 09-05A and its maintainer line 09-05B at the tetrad stage and bi-nucleate stage of B. juncea. A large number of differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) involved in carbohydrate and energy metabolism, including starch and sucrose metabolism, tricarboxylic acid (TCA) cycle, glycolysis, and oxidoreductase activity pathways, were significantly downregulated in 09-05A buds. The low expression of these DEGs or functional loss of DAPs, which can lead to an insufficient supply of critical substrates and ATP, could be associated with flower development, pollen development, and changes in fertility in B. juncea. Therefore, this study provided transcriptomic and proteomic information of pollen abortion for B. juncea and a basis for further research on the molecular regulatory mechanism of MS in plants.

Effects of BrMYC2/3/4 on Plant Development, Glucosinolate Metabolism, and Sclerotinia sclerotiorum Resistance in Transgenic Arabidopsis thaliana.

MYC2/3/4, known as a basic helix-loop-helix (bHLH) transcription factor, directly activate the genes involved in diverse plant development and secondary metabolites biosynthesis. In this study, we identified and cloned five MYC paralogs (BrMYC2/3-1/3-2/4-1/4-2) from Chinese cabbage (Brassica rapa ssp. pekinensis). In-silico analyses for the physicochemical properties suggested that BrMYC2/3-1/3-2/4-2/4-3 are unstable hydrophobic and acidic proteins, while BrMYC4-1 is an unstable hydrophobic and basic protein. BrMYC2/3/4 belong to the bHLH superfamily and are closely related to AthMYC2/3/4 orthologs that mediate the regulation of various secondary metabolites. It was demonstrated that BrMYC2/3/4-GFP fusion protein localized in the nucleus and expression levels of five BrMYC2/3/4 homologous genes all elevated relative to control (Ctrl). When expressed in Arabidopsis under the control of 35S promoter, each of the BrMYC2/3-1/3-2/4-1/4-2 transgenes differentially influenced root and shoot elongation, vegetative phase change, flowering time, plant height and tiller number after flowering, and seed production. Despite the variation of phenotypes between the transgenic lines, all the lines except for BrMYC4-2 exhibited shorter seed length, less seed weight, higher accumulation of glucosinolates (GSs), and resistance to Sclerotinia sclerotiorum than Ctrl. Notably, BrMYC2 overexpression (OE) line significantly reduced the lengths of root and hypocotyl, seed length, and weight, along with faster bolting time and strikingly higher accumulation of total GSs. Accumulation of GSs at the highest levels in the BrMYC2 OE line conferred the highest resistance to S. sclerotiorum. Unlike BrMYC3 OE and BrMYC4 OE , BrMYC2 OE stimulated the growth of plant height after fluorescence. The results of this study point to the BrMYC2 overexpression that may provide a beneficial effect on plant growth and development via plant resistance to the fungal pathogen.

Complete chloroplast genome and phylogenetic analysis of Glebionis coronaria (L.) Cass. ex Spach (Asteraceae).

Glebionis coronaria (Asteraceae) is widely distributed in China, and it regulates the stomach, strengthens the spleen, reduces blood pressure, and reinforces the brain. In this study, the complete chloroplast genome sequence of G. coronaria was reported. The total chloroplast genome cycle was 149,750 bp, and it formed a large single-copy (LSC, 82,290 bp), a small single-copy (SSC, 18,414 bp), and two inverted repeats (IR, 24,523 bp) regions. The GC content of this genome was 36.35%. The whole-genome contained 128 genes, including 84 protein-coding genes, 36 tRNA genes, and eight rRNA genes. Phylogenetic analysis indicated that G. coronaria appeared within a clade comprised of Chrysanthemum species.

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.

Melatonin elevated Sclerotinia sclerotiorum resistance via modulation of ATP and glucosinolate biosynthesis in Brassica rapa ssp. pekinensis.

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SIGNIFICANCE: Melatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

Overexpression of Two CCCH-type Zinc-Finger Protein Genes Leads to Pollen Abortion in Brassica campestris ssp. chinensis.

The pollen grains produced by flowering plants are vital for sexual reproduction. Previous studies have shown that two CCCH-type zinc-finger protein genes in Brassica campestris, BcMF30a and BcMF30c, are involved in pollen development. Due to their possible functional redundancy, gain-of-function analysis is helpful to reveal their respective biological functions. Here, we found that the phenotypes of BcMF30a and BcMF30c overexpression transgenic plants driven by their native promoters were similar, suggesting their functional redundancy. The results showed that the vegetative growth was not affected in both transgenic plants, but male fertility was reduced. Further analysis found that the abortion of transgenic pollen was caused by the degradation of pollen contents from the late uninucleate microspore stage. Subcellular localization analysis demonstrated that BcMF30a and BcMF30c could localize in cytoplasmic foci. Combined with the studies of other CCCH-type genes, we speculated that the overexpression of these genes can induce the continuous assembly of abnormal cytoplasmic foci, thus resulting in defective plant growth and development, which, in this study, led to pollen abortion. Both the overexpression and knockout of BcMF30a and BcMF30c lead to abnormal pollen development, indicating that the appropriate expression levels of these two genes are critical for the maintenance of normal pollen development.

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.

Identification of microRNAs and their targets in inflorescences of an Ogura-type cytoplasmic male-sterile line and its maintainer fertile line of turnip (Brassica rapa ssp. rapifera) via high-throughput sequencing and degradome analysis.

Cytoplasmic male sterility (CMS) is a widely used trait in angiosperms caused by perturbations in nucleus-mitochondrion interactions that suppress the production of functional pollen. MicroRNAs (miRNAs) are small non-coding RNAs that act as regulatory molecules of transcriptional or post-transcriptional gene silencing in plants. The discovery of miRNAs and their possible implications in CMS induction provides clues for the intricacies and complexity of this phenomenon. Previously, we characterized an Ogura-CMS line of turnip (Brassica rapa ssp. rapifera) that displays distinct impaired anther development with defective microspore production and premature tapetum degeneration. In the present study, high-throughput sequencing was employed for a genome-wide investigation of miRNAs. Six small RNA libraries of inflorescences collected from the Ogura-CMS line and its maintainer fertile (MF) line of turnip were constructed. A total of 120 pre-miRNAs corresponding to 89 mature miRNAs were identified, including 87 conversed miRNAs and 33 novel miRNAs. Among these miRNAs, the expression of 10 differentially expressed mature miRNAs originating from 12 pre-miRNAs was shown to have changed by more than two-fold between inflorescences of the Ogura-CMS line and inflorescences of the MF line, including 8 down- and 2 up-regulated miRNAs. The expression profiles of the differentially expressed miRNAs were confirmed by stem-loop quantitative real-time PCR. In addition, to identify the targets of the identified miRNAs, a degradome analysis was performed. A total of 22 targets of 25 miRNAs and 17 targets of 28 miRNAs were identified as being involved in the reproductive development for Ogura-CMS and MF lines of turnip, respectively. Negative correlations of expression patterns between partial miRNAs and their targets were detected. Some of these identified targets, such as squamosa promoter-binding-like transcription factor family proteins, auxin response factors and pentatricopeptide repeat-containing proteins, were previously reported to be involved in reproductive development in plants. Taken together, our results can help improve the understanding of miRNA-mediated regulatory pathways that might be involved in CMS occurrence in turnip.

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.

Genome-Wide Identification and Characterization of Pectin Methylesterase Inhibitor Genes in Brassica oleracea.

The activities of pectin methylesterases (PMEs) are regulated by pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. However, the role of PMEI genes in Brassica oleracea, an economically important vegetable crop, is poorly understood. In this study, 95 B. oleracea PMEI (BoPMEI) genes were identified. A total of 77 syntenic ortholog pairs and 10 tandemly duplicated clusters were detected, suggesting that the expansion of BoPMEI genes was mainly attributed to whole-genome triplication (WGT) and tandem duplication (TD). During diploidization after WGT, BoPMEI genes were preferentially retained in accordance with the gene balance hypothesis. Most homologous gene pairs experienced purifying selection with ω (Ka/Ks) ratios lower than 1 in evolution. Five stamen-specific BoPMEI genes were identified by expression pattern analysis. By combining the analyses of expression and evolution, we speculated that nonfunctionalization, subfunctionalization, neofunctionalization, and functional conservation can occur in the long evolutionary process. This work provides insights into the characterization of PMEI genes in B. oleracea and contributes to the further functional studies of BoPMEI genes.

Genome-Wide Identification and Analysis of Polygalacturonase Genes in Solanum lycopersicum.

Polygalacturonase (PG), a large hydrolase family in plants, is involved in pectin disassembly of the cell wall in plants. The present study aims to characterize PG genes and investigate their expression patterns in Solanum lycopersicum. We identified 54 PG genes in the tomato genome and compared their amino acid sequences with their Arabidopsis counterpart. Subsequently, we renamed these PG genes according to their Arabidopsis homologs. Phylogenetic and evolutionary analysis revealed that these tomato PG genes could be classified into seven clades, and within each clade the exon/intron structures were conserved. Expression profiles analysis through quantitive real-time polymerase chain reaction (qRT-PCR) revealed that most SlPGs had specific or high expression patterns in at least one organ, and particularly five PG genes (SlPG14, SlPG15, SlPG49, SlPG70, and SlPG71) associated with fruit development. Promoter analysis showed that more than three cis-elements associated with plant hormone response, environmental stress response or specific organ/tissue development exhibited in each SlPG promoter regions. In conclusion, our results may provide new insights for the further study of PG gene function during plant development.

A Profusion of Molecular Scissors for Pectins: Classification, Expression, and Functions of Plant Polygalacturonases.

In plants, the construction, differentiation, maturation, and degradation of the cell wall are essential for development. Pectins, which are major constituents of primary cell walls in eudicots, function in multiple developmental processes through their synthesis, modification, and degradation. Several pectin modifying enzymes regulate pectin degradation via different modes of action. Polygalacturonases (PGs), which function in the last step of pectin degradation, are a crucial class of pectin-modifying enzymes. Based on differences in their hydrolyzing activities, PGs can be divided into three main types: exo-PGs, endo-PGs, and rhamno-PGs. Their functions were initially investigated based on the expression patterns of PG genes and measurements of total PG activity in organs. In most plant species, PGs are encoded by a large, multigene family. However, due to the lack of genome sequencing data in early studies, the number of identified PG genes was initially limited. Little was initially known about the evolution and expression patterns of PG family members in different species. Furthermore, the functions of PGs in cell dynamics and developmental processes, as well as the regulatory pathways that govern these functions, are far from fully understood. In this review, we focus on how recent studies have begun to fill in these blanks. On the basis of identified PG family members in multiple species, we review their structural characteristics, classification, and molecular evolution in terms of plant phylogenetics. We also highlight the diverse expression patterns and biological functions of PGs during various developmental processes, as well as their mechanisms of action in cell dynamic processes. How PG functions are potentially regulated by hormones, transcription factors, environmental factors, pH and Ca2+ is discussed, indicating directions for future research into PG function and regulation.

Genome-Wide Identification, Molecular Evolution, and Expression Profiling Analysis of Pectin Methylesterase Inhibitor Genes in Brassica campestris ssp. chinensis.

Pectin methylesterase inhibitor genes (PMEIs) are a large multigene family and play crucial roles in cell wall modifications in plant growth and development. Here, a comprehensive analysis of the PMEI gene family in Brassicacampestris, an important leaf vegetable, was performed. We identified 100 BrassicacampestrisPMEI genes (BcPMEIs), among which 96 BcPMEIs were unevenly distributed on 10 chromosomes and nine tandem arrays containing 20 BcPMEIs were found. We also detected 80 pairs of syntenic PMEI orthologs. These findings indicated that whole-genome triplication (WGT) and tandem duplication (TD) were the main mechanisms accounting for the current number of BcPMEIs. In evolution, BcPMEIs were retained preferentially and biasedly, consistent with the gene balance hypothesis and two-step theory, respectively. The molecular evolution analysis of BcPMEIs manifested that they evolved through purifying selection and the divergence time is in accordance with the WGT data of B. campestris. To obtain the functional information of BcPMEIs, the expression patterns in five tissues and the cis-elements distributed in promoter regions were investigated. This work can provide a better understanding of the molecular evolution and biological function of PMEIs in B. campestris.

The putative pectin methylesterase gene, BcMF23a, is required for microspore development and pollen tube growth in Brassica campestris.

KEY MESSAGE: BcMF23a contributes to pollen wall development via influencing intine construction, which, in turn, influences pollen tube growth. Pollen wall, the morphological out face of pollen, surrounds male gametophyte and plays an important role in plant reproduction. Pectin methylesterases (PMEs) are involved in pollen wall construction by de-esterifying pectin of the intine. In this study, the function of a putative pectin methylesterase gene, Brassica campestris Male Fertility 23a (BcMF23a), was investigated. Knockdown of BcMF23a by artificial microRNA (amiRNA) technology resulted in abnormal pollen intine formation outside of the germinal furrows at the binucleate stage. At the trinucleate stage, 20.69% of pollen possessed the degradation of nuclei, cytoplasm and the intine, resulting in shrunken pollen, whereas the remaining 75.86% were wall-disrupted with degrading cytoplasm and broken exine inside the germinal furrows. In addition, pollen abortion in transgenic plants caused germination percentage reduction by 19% in vitro and pollen tube growth disruption in natural stigma in vivo. Taken together, BcMF23a is involved in pollen development and pollen tube growth, possibly via participating in intine construction. This study may contribute towards understanding the function of pollen-specific PMEs and the molecular regulatory network of pollen wall development.

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.

A comparative analysis of the evolution, expression, and cis-regulatory element of polygalacturonase genes in grasses and dicots.

Cell walls are a distinguishing characteristic of plants essential to their survival. The pectin content of primary cell walls in grasses and dicots is distinctly different. Polygalacturonases (PGs) can degrade pectins and participate in multiple developmental processes of plants. This study comprehensively compared the evolution, expression, and cis-regulatory element of PGs in grasses and dicots. A total of 577 PGs identified from five grasses and five dicots fell into seven clades. Evolutionary analysis demonstrated the distinct differences between grasses and dicots in patterns of gene duplication and loss, and evolutionary rates. Grasses generally contained much fewer clade C and F members than dicots. We found that this disparity was the result of less duplication and more gene losses in grasses. More duplications occurred in clades D and E, and expression analysis showed that most of clade E members were expressed ubiquitously at a high overall level and clade D members were closely related to male reproduction in both grasses and dicots, suggesting their biological functions were highly conserved across species. In addition to the general role in reproductive development, PGs of clades C and F specifically played roles in root development in dicots, shedding light on organ differentiation between the two groups of plants. A regulatory element analysis of clade C and F members implied that possible functions of PGs in specific biological responses contributed to their expansion and preservation. This work can improve the knowledge of PGs in plants generally and in grasses specifically and is beneficial to functional studies.