Pollen-Specific Protein PSP231 Activates Callose Synthesis to Govern Male Gametogenesis and Pollen Germination.

Spatiotemporally regulated callose deposition is an essential, genetically programmed phenomenon that promotes pollen development and functionality. Severe male infertility is associated with deficient callose biosynthesis, highlighting the significance of intact callose deposition in male gametogenesis. The molecular mechanism that regulates the crucial role of callose in production of functional male gametophytes remains completely unexplored. Here, we provide evidence that the gradual upregulation of a previously uncharacterized cotton (Gossypium hirsutum) pollen-specific SKS-like protein (PSP231), specifically at the post pollen-mitosis stage, activates callose biosynthesis to promote pollen maturation. Aberrant PSP231 expression levels caused by either silencing or overexpression resulted in late pollen developmental abnormalities and male infertility phenotypes in a dose-dependent manner, highlighting the importance of fine-tuned PSP231 expression. Mechanistic analyses revealed that PSP231 plays a central role in triggering and fine-tuning the callose synthesis and deposition required for pollen development. Specifically, PSP231 protein sequesters the cellular pool of RNA-binding protein GhRBPL1 to destabilize GhWRKY15 mRNAs, turning off GhWRKY15-mediated transcriptional repression of GhCalS4/GhCalS8 and thus activating callose biosynthesis in pollen. This study showed that PSP231 is a key molecular switch that activates the molecular circuit controlling callose deposition toward pollen maturation and functionality and thereby safeguards agricultural crops against male infertility.

Low temperature-induced aberrations in male and female reproductive organ development cause flower abortion in chickpea.

Chickpea (Cicer arietinum L.) is susceptible to low temperature (LT) at reproductive stage. LT causes flower abortion and delays pod set in chickpea until terminal drought becomes an issue, thereby decreasing yield potential. In chickpea, flower and anther/pollen development as well as LT-induced abnormalities on anther and pollen development are described inadequately. In the present manuscript, we report flower development stages, anther development stages, and aberrations in male gamete formation in chickpea under LT. Flower length was linearly correlated to flower and anther stages and can be used to predict these stages in chickpea. LT affected male gamete development in a flower/anther age-dependent manner where outcome ranged from no pollen formation to pollen sterility or no anther dehiscence to delayed dehiscence. In anthers, LT inhibited microsporogenesis, microgametogenesis, tapetum degeneration, breakage of septum and stomium, and induced pollen sterility. Whereas disruption of male function was the prime cause of abortion in flowers below vacuolated pollen stage, flower abortion was due to a combination of male and female reproductive functions in flowers with mature pollen. The study will help in elucidating mechanisms governing flower development, anther and pollen development, and tolerance/susceptibility to LT.

The Arabidopsis nucleoporin NUP1 is essential for megasporogenesis and early stages of pollen development.

KEY MESSAGE: Loss-of-function of nucleoporin NUP1 in Arabidopsis causes defect in both male and female gametogenesis. Its ovules are arrested during meiosis, and its pollen grains are aborted at mitosis I. Nuclear pore complex (NPC) plays crucial roles in nucleocytoplasmic trafficking of proteins and RNAs. The NPC contains approximately 30 different proteins termed nucleoporins (NUPs). So far, only a few of plant NUPs have been characterized. The Arabidopsis NUP1 was identified as an ortholog of the yeast NUP1 and animal NUP153. Loss-of-function of NUP1 in Arabidopsis caused fertility defect; however, the molecular mechanism of this defect remains unknown. Here, we found that both male and female gametogenesis of the nup1 mutants were defective. nup1 ovules were arrested from the meiosis stage onward; only approximately 6.7% and 3% ovules of the nup1-1 and nup1-4 mutants developed up to the FG7 stage, respectively. Pollen development of the nup1 mutants was arrested during the first mitotic division. In addition, enlarged pollen grains with increased DNA content were observed in the nup1 mutant. RNA-sequencing showed that expression levels of genes involved in pollen development or regulation of cell size were reduced dramatically in nup1 compared with wild type. These results suggest that NUP1 plays an important role in gametogenesis.

The stereotyped positioning of the generative cell associated with vacuole dynamics is not required for male gametogenesis in rice pollen.

During male gametogenesis in cereals, the generative cell undergoes a positioning process that parallels the dynamics of the central vacuole, which is believed to be associated with generative cell movement in the male gametophyte. However, the impact of the generative cell positioning and the central vacuole dynamics on male gametogenesis has remained poorly understood. Here, we report that OsGCD1 (GAMETE CELLS DEFECTIVE1) dysfunction influenced pollen development and disrupted pollen germination. Loss of function of OsGCD1 altered the central vacuole dynamics and the generative cell was mispositioned. Nevertheless, twin sperm cells were generated normally, indicating that gametogenesis does not rely on positional information as long as a generative cell is produced. The normal vacuole dynamics seems necessary only for pollen maturation and germination. Our findings also indicate that osgcd1 mutation resulted in rice male sterility in which pollen has full cell viability and generated normal gametes, but lacks the potential to germinate.

Protective effect of sodium selenite and zinc sulfate on intensive swimming-induced testicular gamatogenic and steroidogenic disorders in mature male rats.

To investigate the ameliorative potential of sodium selenite and zinc sulfate on intensive-swimming-induced testicular disorders, 48 Wistar male rats (age, 4 months; mass, 146.2 +/- 3.6 g) were randomly divided into 4 groups: the unexercised-control group (n = 12); the exercised group (n = 12); the control supplemented group (n = 12); and the exercised supplemented group (n = 12). For 10 weeks, the exercised rats underwent a protocol that consisted of 4 h.d-1 swimming, for 6 d.week-1; the control rats did not exercise. For 10 weeks, both the supplemented groups received an oral daily dose of a combination of sodium selenite and zinc sulfate (6 and 3 mg.kg body mass-1, respectively). After 10 weeks, a significant reduction (p < 0.05) was seen in rats in the exercised group, compared with rats in both control groups, in paired testicular masses; in epididymal sperm count; in testicular Delta5, 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD; in plasma levels of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin; in the numbers of preleptotine spermatocytes, midpachytene spermatocytes, and stage 7 spermatids of the stage VII seminiferous epithelium cycle; and in fertility performance. As well, a significant increase (p < 0.05) was seen in the exercised group, compared with both control groups, in plasma corticosterone levels and in testicular content of malondialdehyde and catalase activity. At the same time, there was a significant reduction (p < 0.05) in the exercised group, compared with both control groups, in plasma concentrations of zinc and selenium; in the testicular content of glutathione (GSH), the glutathione and glutathione disulphide (GSSG) ratio, ascorbic acid, and alpha-tocopherol; and in testicular activities of superoxide dismutase, glutathione-peroxidase, and glutathione-S-transferase in the testes. No significant changes were seen in the number of spermatogonia-A from the stage VII seminiferous epithelium cycle or the testicular content of GSSG among the groups. Sodium selenite and zinc sulfate supplementation significantly protected against exercise-induced testicular gamatogenic and spermatogenic disorders, prevented testicular oxidative stress, and increased antioxidant status. It can be concluded that intensive-swimming-induced oxidative stress causes dysfunctions in the male reproductive system, which can be protected by the coadministration of sodium selenite and zinc sulfate.

The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis.

Alpha-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes for four alpha-kleisin proteins encoded by SYN genes. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis. Our analysis of AtSYN3 shows that it localizes primarily in the nucleolus of both meiotic and mitotic cells. Furthermore, analysis of plants containing an AtSYN3 T-DNA knockout mutation demonstrated that it is essential for megagametogenesis and plays an important role in pollen. These results suggest that SYN3 may not function as part of a typical cohesin complex; rather it may have evolved a specialized role in controlling rDNA structure, transcription or rRNA processing.

Disruption of the Arabidopsis SMC4 gene, AtCAP-C, compromises gametogenesis and embryogenesis.

In higher eukaryotes, the condensin complex is a multisubunit apparatus that plays a pivotal role in the coordinated condensation of chromatin during mitosis. The catalytic subunits, CAP-E and CAP-C, members of the SMC family of ATPases, form a heterodimer, the activity of which is controlled by the non-SMC subunits CAP-D2, CAP-G and CAP-H. Here, we report the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfed heterozygotes revealed that the homozygous null genotype is embryo lethal, with arrest occurring at or before the globular stage of development. Patterning defects associated with altered planes of cytokinesis were found in both the embryo and the suspensor. Crosses of heterozygotes with wild type plants revealed both male and female gametophytic defects. Stretched chromatin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Additionally, some plants heterozygous for the T-DNA insertion exhibited loss of apical dominance and mild fasciation, indicating a semi-dominant effect of the mutation. These results reveal a critical role for AtCAP-C during cell division and, unlike our previous studies on the AtCAP-E genes, suggest that no redundant factors for AtCAP-C exist in the Arabidopsis genome.

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis.

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae).

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5 percent propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1 percent of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis.

Lysophosphatidyl acyltransferase (LPAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. We examined putative LPAT genes in Arabidopsis thaliana and characterized two related genes that encode the cytoplasmic LPAT. LPAT2 is the lone gene that encodes the ubiquitous and endoplasmic reticulum (ER)-located LPAT. It could functionally complement a bacterial mutant with defective LPAT. LPAT2 and 3 synthesized in recombinant bacteria and yeast possessed in vitro enzyme activity higher on 18:1-CoA than on 16:0-CoA. LPAT2 was expressed ubiquitously in diverse tissues as revealed by RT-PCR, profiling with massively parallel signature sequencing, and promoter-driven beta-glucuronidase gene expression. LPAT2 was colocalized with calreticulin in the ER by immunofluorescence microscopy and subcellular fractionation. LPAT3 was expressed predominately but more actively than LPAT2 in pollen. A null allele (lpat2) having a T-DNA inserted into LPAT2 was identified. The heterozygous mutant (LPAT2/lpat2) had minimal altered vegetative phenotype but produced shorter siliques that contained normal seeds and remnants of aborted ovules in a 1:1 ratio. Results from selfing and crossing it with the wild type revealed that lpat2 caused lethality in the female gametophyte but not the male gametophyte, which had the redundant LPAT3. LPAT2-cDNA driven by an LPAT2 promoter functionally complemented lpat2 in transformed heterozygous mutants to produce the lpat2/lpat2 genotype. LPAT3-cDNA driven by the LPAT2 promoter could rescue the lpat2 female gametophytes to allow fertilization to occur but not to full embryo maturation. Two other related genes, putative LPAT4 and 5, were expressed ubiquitously albeit at low levels in diverse organs. When they were expressed in bacteria or yeast, the microbial extract did not contain LPAT activity higher than the endogenous LPAT activity. Whether LPAT4 and 5 encode LPATs remains to be elucidated.

Selective transcriptional down-regulation of anther invertases precedes the failure of pollen development in water-stressed wheat.

Water deficit during male meiosis in wheat (Triticum aestivum L.) causes pollen sterility. With a view to identifying the internal trigger for this failure, it was found that water stress specifically impairs the activities of vacuolar and cell-wall invertases in anthers prior to the arrest of pollen development. The enzymes are affected only when water deficit occurs around meiosis. Three invertase cDNAs, two encoding the cell-wall (Ivr1, Ivr3) and one the vacuolar (Ivr5) isoform, were isolated from an anther cDNA library. RNA gel-blot analysis using floral organs of well-watered plants revealed that these genes were expressed preferentially, though not exclusively, in anthers. Semi-quantitative RT-PCR demonstrated that transitory water deficit during meiosis selectively down-regulated the transcription of two of the three genes, one encoding the vacuolar (Ivr5) and the other a cell-wall (Ivr1) isoform, without affecting the Ivr3 message. Their expression did not recover upon resumption of watering. Another homologue of Ivr1 was also down-regulated, but only during the post-stress period. The stress effects on invertase transcripts were consistent with those on the developmental profiles of the corresponding enzyme activities. In situ hybridization revealed that the stress-sensitive invertase genes, unlike an insensitive one, were expressed within the microspores. No evidence for an invertase inhibitor under stress was found. Together the results show that the decline in invertase activity is probably regulated primarily at the transcriptional level in a gene- and cell-specific manner.

[Inorganic ion dynamics in the microspore and pollen grain of tobacco during the development of the male gametophyte]. / Dinamika neorganicheskikh ionov v mikrospore i pyl'tsevom zerne tabaka v protsesse razvitiia muzhskogo gametofita.

We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the micro-spore and differentiation of the pollen grain in Nicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes. Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed. The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein synthesis in the pollen grain vegetative cell.

Gametocidal genes induce chromosome breakage in the interphase prior to the first mitotic cell division of the male gametophyte in wheat.

Male gametogenesis was cytologically analyzed in wheat lines homozygous or hemizygous for gametocidal (Gc) factors with different modes of action. The first and second meiotic divisions in all lines were cytologically normal. The postmeiotic mitoses were normal in the homozygous lines; however, chromosome fragments and bridges were observed in the mitoses of the hemizygous lines. The morphology of the chromosome fragments suggests that the Gc genes induce chromosome breaks in the G1 phase prior to DNA synthesis of the first postmeiotic mitosis. The age of an anther was correlated with the frequency of aberrant second mitosis. Younger anthers contained a higher number of pollen undergoing normal second mitosis. This observation suggests that the arresting of the cell cycle occurs as the result of chromosome breaks during the first mitosis. Because chromosome bridges were more frequent than fragments in the second mitosis, breakage-fusion-bridge cycles possibly occurred during gametogenesis, which led to further chromosomal rearrangements. The Gc factors located on chromosomes 2S of Aegilops speltoides and 4Ssh of Ae. sharonensis induce severe chromosome breakage in pollen lacking them. However, the Gc factor on telosome 2CcL of Ae. cylindrica only induced chromosome breaks at a low frequency. The observed partial fertility of Gc lines is presumably due to cell cycle arrest and the competition among gametes with and without chromosome breakage.

Plasmon analysis of Triticum (wheat) and Aegilops. 1. Production of alloplasmic common wheats and their fertilities.

Plasmons (= cytoplasms) of eight Triticum species (ten accessions) and 24 Aegilops species (36 accessions) have been introduced by repeated backcrosses to 12 genotypes of hexaploid, common wheat. At transfer problems such as crossing barrier, preferential transmission of a gametocidal or parthenogenesis-inducing chromosome, and mistagging of the material occurred, all of which hindered the plasmon transfer program. Of the 552 genotype-plasmon combinations produced, 532 (96.4%) had reached the B10 or a later backcross generation, 15 (2.7%) the B7 approximately B9 generation, and the remaining 5 (0.9%) the B4 approximately B6 generation by summer, 1996. Pollen and selfed seed fertilities were observed in plants of all the field-grown lines in the 1992-1993 winter crop season, and backcrossed and selfed seed fertilities of plants grown in a greenhouse under a long day condition (17-h light) were assessed in the five latest backcross generations. Selfed seed fertility was found to be a better parameter of male fertility than was pollen fertility. Female fertility, as estimated from the backcrossed seed fertility, was about three times more tolerant to genetic stress caused by the alien plasmon transfer than was male fertility evaluated from both the pollen and selfed seed fertilities. The plasmons studied could be classified into 14 fertility spectrum groups. Most, excluding 15 plasmons belonging to the B, D, D2, S, and Sb plasmon types, were considered the male sterile plasmon to common wheat.