Calreticulin expression and localization in relation to exchangeable Ca2+ during pollen development in Petunia

Background Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata)

Background Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. Results In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. Conclusions The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers

In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. By integrating RNA and small RNA (sRNA) profiling plus single-molecule and sRNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum. Our data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.

Evaluation of mitotic activity in tapetal cells of grapevine (Vitis L.)

The knowledge with reference to the grapevine tapetum has been centered on its anatomy/morphology and hardly anything at all is known about its mitotic activity throughout the microsporogenesis. The aim of this study was to ascertain the mitotic activity in tapetal cells of some grapevines (Vitis L.) broadening knowledge about this tissue and simultaneously corroborating the viability of its use as an alternative tissue for further cytogenetic studies. Young buds of 12 grapevine varieties at different meiotic stages were squashed and tapetal cells a prometaphase/metaphase scored in each meiotic stage. Mitotic activity was observed since the beginning of microsporogenesis, where it was more intense, decreasing toward tetrad. Polyploid tapetal cells arose through endomitosis while the microsporogenesis advanced. Two types of polyploid cells were evidenced, those with two or more individualized diploid chromosome groups and those with only one polyploid group. The percentage of diploid cells and of polyploid cells with two or more individualized diploid groups was higher during the first stage of microsporogenesis, though decreasing and giving way to cells with one large polyploid group as microsporogenesis moved toward tetrad. The nucleolus number was scored at interphase at different stages. Two and four nucleoli prevailed in tapetal cells at all stages except at tetrad where one large nucleolus was seen. The results showed that despite of the squashing technique applied, grapevine tapetum has a substantial amount of cells with mitotic activity with a satisfactory chromosome spreading therefore establishing an interesting alternative and promising tissue for later cytomolecular studies.

Conserved and non-conserved triggers of 24-nt reproductive phasiRNAs in eudicots

Plant small RNAs (sRNAs) play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: pre-meiotic 21-nt phasiRNAs triggered by miR2118, and meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine are produced by the canonical trigger, miR2275, as well as by other non-conserved triggers, miR482/2118 and aco_cand81. These triggering miRNAs are localized in microspore mother cells (MMC) and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a new trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry. We validated the presence of miR2275-derived 24-nt phasiRNAs pathway in rose. Finally, we evaluated all the eudicots that have been validated for the presence of 24-nt phasiRNAs as models to study biogenesis and function of 24-nt phasiRNAs and conclude that columbine would be an excellent model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs.

Genome-wide identification of GH3 genes in Brassica oleracea and identification of a promoter region for anther-specific expression of a GH3 gene

Background: The Gretchen Hagen 3 (GH3) genes encode acyl acid amido synthetases, many of which have been shown to modulate the amount of active plant hormones or their precursors. GH3 genes, especially Group Ⅲ subgroup 6 GH3 genes, and their expression patterns in economically important B. oleracea var. oleracea have not been systematically identified. Results: As a first step to understand regulation and molecular functions of Group Ⅲ subgroup 6 GH3 genes, 34 GH3 genes including four subgroup 6 genes were identified in B. oleracea var. oleracea. Synteny found around subgroup 6 GH3 genes in B. oleracea var. oleracea and Arabidopsis thaliana indicated that these genes are evolutionarily related. Although expression of four subgroup 6 GH3 genes in B. oleracea var. oleracea is not induced by auxin, gibberellic acid, or jasmonic acid, the genes show different organ-dependent expression patterns. Among subgroup 6 GH3 genes in B. oleracea var. oleracea, only BoGH3.13-1 is expressed in anthers when microspores, polarized microspores, and bicellular pollens are present, similar to two out of four syntenic A. thaliana subgroup 6 GH3 genes. Detailed analyses of promoter activities further showed that BoGH3.13-1 is expressed in tapetal cells and pollens in anther, and also expressed in leaf primordia and floral abscission zones. Conclusions: Sixty-two base pairs (bp) region (-340 ~ -279 bp upstream from start codon) and about 450 bp region (-1489 to -1017 bp) in BoGH3.13-1 promoter are important for expressions in anther and expressions in leaf primordia and floral abscission zones, respectively. The identified anther-specific promoter region can be used to develop male sterile transgenic Brassica plants.

Genome-wide identification of GH3 genes in Brassica oleracea and identification of a promoter region for anther-specific expression of a GH3 gene

Background: The Gretchen Hagen 3 ( GH3 ) genes encode acyl acid amido synthetases, many of which have been shown to modulate the amount of active plant hormones or their precursors. GH3 genes, especially Group Ⅲ subgroup 6 GH3 genes, and their expression patterns in economically important B. oleracea var. oleracea have not been systematically identified. Results: As a first step to understand regulation and molecular functions of Group Ⅲ subgroup 6 GH3 genes, 34 GH3 genes including four subgroup 6 genes were identified in B. oleracea var. oleracea . Synteny found around subgroup 6 GH3 genes in B. oleracea var. oleracea and Arabidopsis thaliana indicated that these genes are evolutionarily related. Although expression of four subgroup 6 GH3 genes in B. oleracea var. oleracea is not induced by auxin, gibberellic acid, or jasmonic acid, the genes show different organ-dependent expression patterns. Among subgroup 6 GH3 genes in B. oleracea var. oleracea , only BoGH3.13-1 is expressed in anthers when microspores, polarized microspores, and bicellular pollens are present, similar to two out of four syntenic A. thaliana subgroup 6 GH3 genes. Detailed analyses of promoter activities further showed that BoGH3.13-1 is expressed in tapetal cells and pollens in anther, and also expressed in leaf primordia and floral abscission zones. Conclusions: Sixty-two base pairs (bp) region (-340 ~ -279 bp upstream from start codon) and about 450 bp region (-1489 to -1017 bp) in BoGH3.13-1 promoter are important for expressions in anther and expressions in leaf primordia and floral abscission zones, respectively. The identified anther-specific promoter region can be used to develop male sterile transgenic Brassica plants.

Genome-Wide Identification of GH3 Genes in Brassica Oleracea and Identification of a Promoter Region for Anther-Specific Expression of a GH3 Gene

Background:The Gretchen Hagen 3 (GH3) genes encode acyl acid amido synthetases, many of which have been shown to modulate the amount of active plant hormones or their precursors. GH3 genes, especially Group Ⅲ subgroup 6 GH3 genes, and their expression patterns in economically important kale-type Brassica oleracea have not been systematically identified. Results:As a first step to understand regulation and molecular functions of Group Ⅲ subgroup 6 GH3 genes, thirty-four GH3 genes including four subgroup 6 genes were identified In B. oleracea var. oleracea, using TO1000. Synteny found around subgroup 6 GH3 genes in TO1000 and Arabidopsis indicated that these genes are evolutionarily related. Although expression of four subgroup 6 GH3 genes in TO1000 is not induced by auxin, gibberellic acid, and jasmonic acid, the genes show different organ-dependent expression patterns. Only one TO1000 subgroup 6 GH3 gene, Bo2g011210, is expressed in anthers when microspores, polarized microspores, and bicellular pollens are present, similar to two out of four syntenic Arabidopsis subgroup 6 GH3 genes. Detailed analyses of promoter activities of Bo2g011210 further showed that Bo2g011210 is expressed in tapetal cells and pollens in anther, and also expressed in leaf primordia and floral abscission zones. Conclusions:Sixty-two base pair (bp) region (-340 ~ -279 bp upstream from start codon) and about 450 bp region (-1489 to -1017 bp) in Bo2g011210 promoter were found to be important for expressions in anther and expressions in leaf primordia and floral abscission zones, respectively. The identified anther-specific promoter region will be useful to develop male sterile transgenic Brassica plants.

The mitochondrial aldehyde dehydrogenase OsALDH2b negatively regulates tapetum degeneration in rice

Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Although several genes involved in tapetum development have been identified, the molecular basis of tapetum degeneration regulation remains poorly understood. In this study, we identified and characterized the nucleus-encoded, conserved mitochondrial aldehyde dehydrogenase OsALDH2b as a key regulator of tapetum degeneration in rice (Oryza sativa). OsALDH2b was highly expressed in anthers from meiosis to the early microspore stage. Mutation of OsALDH2b resulted in excess malonaldehyde accumulation and earlier programmed cell death in the tapetum, leading to premature tapetum degeneration and abnormal microspore development. These results demonstrate that OsALDH2b negatively regulates tapetal programmed cell death and is required for male reproductive development, providing insights into the regulation of tapetum development in plants.