Separation Of Microspores Research Articles

Degradation of de-esterified pctin/homogalacturonan by the polygalacturonase GhNSP is necessary for pollen exine formation and male fertility in cotton.

SummaryThe pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map‐based cloning and confirmed by genetic analysis (co‐segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de‐esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de‐esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de‐esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de‐esterified homogalacturonan in pollen exine formation.

Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction

Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction

BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus

7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus.

Isolation and Characterization of an Anther-Specific Polygalacturonase Gene, BcMF16, in Brassica campestris ssp. chinensis

Many genes in the genic male sterile A/B line (Bajh97-01A/B) of Chinese cabbage pak choi (Brassica campestris L. subsp. chinensis Makino) are expressed differentially, and some play critical roles in the formation of pollen walls. In this study, one of these genes, Brassica campestris Male Fertility 16 (BcMF16), has been isolated and characterized. The BcMF16 gene shares approximately 85% nucleotide sequence homology with two exopolygalacturonase (EC3.2.1.67) genes of Arabidopsis thaliana. Cluster analysis of polygalacturonase peptides indicate that BcMF16 belongs to the pollen polygalacturonase clade. Quantitative real-time PCR analysis has revealed that BcMF16 is specifically expressed in reproductive tissues of the fertile line of genic male sterile A/B line of Chinese cabbage pak choi, and that expression levels dramatically increased during later stages of pollen development. In situ hybridization has demonstrated that BcMF16 is specifically and transiently expressed in both tapetum and pollen following microspore separation at the tetrad stage.

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C4 and C3 photosynthesis

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.

Cytological study of a male-sterile Cryptomeria japonica that does not release microspores from tetrads

To determine the mechanism of male-sterility Cryptomeria japonica tree Shindai3, the process of microspore development was observed under light and fluorescence microscopy. Microspore development in the Shindai3 was normal until the tetrad stage, but separation of the microspores from the tetrads was not observed even after callose had been degraded. In contrast to the microspore stage in a male-fertile tree, amorphous substances were observed around tetrads in the Shindai3, and the amount of the amorphous substance increased from the middle of November to the middle of December. The substances are hypothesized to prevent the separation of individual microspores from tetrads by inducing adhesion between microspore cells within the tetrad or enclosing the tetrads. The form of the tetrads in the Shindai3 was maintained until just before the pollen dispersal season, but they ultimately degenerated. The results of this study indicate that the mechanism causing male sterility in Shindai3 differs from that previously reported for other male-sterile trees of C. japonica.

Transcript profiling of a dominant male sterile mutant ( Ms-cd1) in cabbage during flower bud development

A spontaneous male-sterile mutation affecting anther development has been identified in cabbage line 79-399-3. The male sterility was associated with abnormal callose degeneration and failure of microspore separation during anther development. Three pairs near-isogenic lines (NILs) with or without the dominant male sterility allele ( MS-cd1) were profiled by cDNA-AFLP to detect differentially expressed genes during flower bud development. Out of 6500 transcript derived fragments (TDFs) inspected, 27 were differentially expressed between fertile and male sterile plants in all three sets of NILs. All 27 TDFs were sequenced and BLAST searching revealed that 14 out of 27 fragments were highly homologous to genes with known or predicted function in Arabidopsis. Moreover, three of these TDFs were homologous to genes involved in cell wall formation and degradation, including plant invertase/pectin methylesterase inhibitor (PMEI) and polysaccharide lyase. RT-PCR experiments revealed that PMEI was expressed strongly in the anther and filament. Analysis of a set of NILs with different male sterility types showed that PMEI was also not expressed in a Nigra CMS NIL, a recessive male sterile NIL and an Ogura CMS NIL compared to their corresponding fertile NILs. Based on both cytological and transcriptional data we suggest that the dominant MS-cd1 mutant gene may disrupts proper separation of pollens from tetrads, which leads to the suppressed expression of a number of genes including BoPMEI1, a gene likely involved in the degradation of pectin.

Plant separation: 50 ways to leave your mother

One of the remarkable features of plants is their ability to shed organs, such as leaves, seeds, flowers, and fruit. Genetic analysis of fruit dehiscence and floral organ shedding in Arabidopsis is revealing the pathways that underlie these distinct separation events. The transcriptional network that patterns the fruit links factors that regulate organ polarity and growth with those that control differentiation of the three cell types that are required for dehiscence. Transcriptional regulators that pattern the proximal-distal axis in developing leaves are required for floral organ shedding, and chromatin-modifying complexes might globally regulate genes that affect flower senescence and abscission. Ground-breaking studies have also recently identified a hydrolytic enzyme that is required for microspore separation during pollen development, and the first transcription factor controlling seed abscission.

Pollen-wall Formation in Arum alpinum

Arum alpinum has a quite uncommon pollen wall. A sporopolleninous ektexine is missing. The outermost pollen wall layer is formed by the endexine which is covered by polysaccharidic ornamentation elements. An ontogenetical investigation was accomplished to clarify pollen-wall development, with special reference to callose and pollen-wall development. Plants of Arum alpinum grown in their natural habitat were collected once a week within the vegetative period and processed for semi- and ultra-thin sectioning. At any stage of pollen-wall formation callose is missing. Microspores are released from the tetrad by invagination of the amoeboid tapetum. The polysaccharidic wall ornamentations are formed by the tapetum. There appears to be no truth in the dogma that callose is essential for microspore separation and release from the tetrad. The lack of callose does not influence fertility but could be the reason for the uncommon pollen wall, where a sporopolleninous ektexine is missing.

Pollen wall ultrastructure and ontogeny in Heliotropium europaeum L. (Boraginaceae)

The pollen grains of Heliotropium europaeum are heterocolpate, with alternation of 3 colpori and 3 pseudocolpi. The exine is characterized by a scabrate and thick tectum, massive columellae with a granular appearance and a thick nexine. The thickening of the intine at the apertural level makes the interpretation of this zone difficult. The ontogenetic study helped to understand the ultrastructure of the exine and the apertures. The different steps are as follows. The primexine matrix is formed during the beginning of the tetrad stage; it consists of an outer thick and electron dense zone and an inner one, less dense to electrons. The tectum and the infratectum begin to form in the outer zone of the matrix, towards the middle of the tetrad stage. The infratectum consists of a network of columellae variable in thickness and oriented in different directions. The foot layer is lacking. The endexine is formed on a lamella system during the callose loss and microspore separation. The endexine becomes compact very early on its inner part. The apertures are initiated during the tetrad stage; a granulo-fibrillar oncus develops. At the free microspore stage, the oncus gets fibrillar and is bordered by endexine lamellae on its outer side and by endexine granulations on its inner one and laterally. The intine is set at the end of this stage. At the vacuolated microspore stage, the intine shows three layers: two thin, clear and homogeneous layers, one outside and the other inside, and a thick middle layer that forms the zwischenkörper, crossed by trabecula, in the apertural areas.

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation.

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.

TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis.

A key step in pollen formation is the segregation of the products of male meiosis into a tetrad of microspores, each of which develops into a pollen grain. Separation of microspores does not occur in tetraspore (tes) mutants of Arabidopsis thaliana, owing to the failure of male meiotic cytokinesis. tes mutants thus generate large 'tetraspores' containing all the products of a single meiosis. Here, we report the positional cloning of the TES locus and details of the role played by the TES product in male cytokinesis. The predicted TES protein includes an N-terminal domain homologous to kinesin motors and a C-terminus with little similarity to other proteins except for a small number of plant kinesins. These include the Arabidopsis HINKEL protein and NACK1 and two from tobacco (Nishihama et al., 2002), which are involved in microtubule organization during mitotic cytokinesis. Immunocytochemistry shows that the characteristic radial arrays of microtubules associated with male meiotic cytokinesis fail to form in tes mutants. The TES protein therefore is likely to function as a microtubule-associated motor, playing a part either in the formation of the radial arrays that establish spore domains following meiosis, or in maintaining their stability.

Study of the gametophyte of fuchsia cv [formula omitted

The pollen grains of Heliotropium europaeum are heterocolpate, with alternation of 3 colpori and 3 pseudocolpi. The exine is characterized by a scabrate and thick tectum, massive columellae with a granular appearance and a thick nexine. The thickening of the intine at the apertural level makes the interpretation of this zone difficult. The ontogenetic study helped to understand the ultrastructure of the exine and the apertures. The different steps are as follows. The primexine matrix is formed during the beginning of the tetrad stage; it consists of an outer thick and electron dense zone and an inner one, less dense to electrons. The tectum and the infratectum begin to form in the outer zone of the matrix, towards the middle of the tetrad stage. The infratectum consists of a network of columellae variable in thickness and oriented in different directions. The foot layer is lacking. The endexine is formed on a lamella system during the callose loss and microspore separation. The endexine becomes compact very early on its inner part. The apertures are initiated during the tetrad stage; a granulo-fibrillar oncus develops. At the free microspore stage, the oncus gets fibrillar and is bordered by endexine lamellae on its outer side and by endexine granulations on its inner one and laterally. The intine is set at the end of this stage. At the vacuolated microspore stage, the intine shows three layers: two thin, clear and homogeneous layers, one outside and the other inside, and a thick middle layer that forms the zwischenkörper, crossed by trabecula, in the apertural areas.

A Cytochemical Study of Anther Development in <i>Iphigenia pallida</i> Bak

Microsporogenesis and anther development in Iphigenia pallida Bak. were investigated using the cytochemical staining methods for the demonstration of insoluble polysaccharides, RNA, proteins and DNA. The insoluble polysaccharides and proteins are at low level in sporogenous tissue and PMCs. Both of them gradually increase in dyads, tetrads and microspores. Large number of starch grains appearing in the-vegetative cell of the young 2-celled pollen grain disintegrate and form densely PAS positive cytoplasm in the shedding pollen grain. A protein and PAS positive layer deposited internally around the PMCs at the beginning of meiosis, disappears prior to the separation of microspores from the tetrads. The cytoplasm of PMCs, dyads, tetrads, microspores and pollen grains is rich in RNA. The nucleus of the generative cell is more Feulgen positive than that of the vegetative cell. The tapetal cells have rich RNA and considerable concentration of proteins as well as polysaccharides. The fibrous endothecial thickenings are lignified.

THE INFLUENCE OF GLUTATHIONE AND GLUTATHIONE ANTAGONISTS ON MEIOSIS IN EXCISED ANTHERS OF LILIUM HENRYI1

Young anthers, in early prophase and in the premeiotic mitotic stage, were excised and cultivated under aseptic conditions on media containing glutathione and/or several sulfhydryl antagonists. 1 Meiotic induction in excised anthers, not yet in prophase, is possible and is not dependent on addition of glutathione. 2 The formation of the wall structures of young microspores is dependent on the availability of sulfhydryl groups. Formation is inhibited reversibly by p-chloromercuribenzoic acid and irreversibly by cystine and iodoacetic acid. 3 It is suggested that inhibition of cross wall formation and separation of microspores in cultured anthers may be due to a lack of energy or substrate reserves.