Anther Wall Cells Research Articles

The development of yellow lupin anthers depends on the relationship between jasmonic acid and indole-3-acetic acid.

The main purpose of this study was to demonstrate that the course of anther development, including post-meiotic maturation, dehiscence and senescence, is ensured by the interdependencies between jasmonic acid (JA) and indole-3-acetic acid (IAA) in yellow lupin (Lupinus luteus L.). The concentration of JA peaked during anther dehiscence when IAA level was low, whereas the inverse relationship was specific to anther senescence. Cellular and tissue localization of JA and IAA, in conjunction with broad expression profile for genes involved in biosynthesis, signalling, response, and homeostasis under different conditions, allowed to complete and define the role of studied phytohormones during late anther development, as well as predict events triggered by them. The development/degeneration of septum and anther wall cells, dehydration of epidermis, and rupture of stomium may involve JA signalling, while the formation of secondary thickening in endothecial cell walls is rather JA independent. The IAA is involved in programmed cell death (PCD)-associated processes during anther senescence but does not exclude its participation in the anther dehiscence processes, mainly related to cell disintegration and degeneration. A detailed understanding of these multistage processes, especially at the level of phytohormonal interplay, can contribute to the effective control of male fertility, potentially revolutionizing the breeding of L. luteus.

Mobile ARGONAUTE 1d binds 22-nt miRNAs to generate phasiRNAs important for low-temperature male fertility in rice.

Phased small interfering RNAs (phasiRNAs) are abundantly expressed in anthers and linked to environment-related male fertility in grasses, yet how they function under different environmental conditions remains unclear. Here, we identified a rice (Oryza sativa) low temperature-induced Argonaute (AGO) protein, OsAGO1d, that is responsible for generating phasiRNAs and preserving male fertility at low temperature. Loss of OsAGO1d function causes low-temperature male sterility associated with delayed programmed cell death of tapetal cells during anther development. OsAGO1d binds miR2118 and miR2275 family members and triggers phasiRNA biogenesis; it also binds 21-nt phasiRNAs with a 5' terminal U. In total, phasiRNAs from 972 loci are OsAGO1d-dependent. OsAGO1d protein moves from anther wall cells into meiocytes, where it loads miR2275 to produce 24-nt phasiRNAs. Together, our results show that OsAGO1d acts as a mobile signal to fine-tune phasiRNA production and this function is important for male fertility at low temperature.

Temperature-sensitive male sterility in rice determined by the roles of AGO1d in reproductive phasiRNA biogenesis and function.

Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial-temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive. Here, through histological and high-throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis. AGO1d preferentially binds to miR2118-triggered 21-nucleotide (nt) phasiRNAs with a 5'-terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21- and 24-nt phasiRNAs and temperature-sensitive male sterility. At lower temperatures, anthers of the ago1d mutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism. These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

Functional analysis of flower development related gene SvGLOBOSA from Senecio vulgaris.

The unique capitulum of Asteraceae has important ornamental and research value. Few studies have described the complex molecular mechanism of flower development. In this study, SvGLOBOSA(SvGLO), the MADS-box gene of Senecio vulgaris, was identified by screening the transcriptome data, and its function was examined. The gene structure was analyzed and its function was predicted through bioinformatics. The relative expression levels in different tissues of wild-type S. vulgaris were analyzed by qRT-PCR. SvGLO was overexpressed in Solanum nigrum and morphological observations were made. Histological staining was used in analyzing the histological changes in the ovary of transgenic S. nigrum. The results showed that the open reading frame of SvGLO was 591 bp long, encoding 196 amino acids. It has typical MADS-box and K-box domains and contains a PI motif at the C-terminal. SvGLO belongs to the PI/GLO subfamily of class B MADS-box genes. qRT-PCR results showed that SvGLO was highly expressed in inflorescence tissues but not in vegetative organs. In SvGLO-overexpressed S. nigrum, the sepals showed some characteristics of petals, carpels transformed into stamen-like organs, and fruit development was abnormal. Histological staining revealed that the morphology of ovary wall cells of transgenic S. nigrum was similar to that of anther wall cells of the stamen of wild-type S. vulgaris. Therefore, SvGLO may be involved in the regulation of petal and stamen development in S. vulgaris.

MiR2118-dependent U-rich phasiRNA production in rice anther wall development

Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.

Autophagy mitigates high-temperature injury in pollen development of Arabidopsis thaliana

Autophagy is one of the cellular processes that break down cellular components during senescence, starvation, and stress. The susceptibility of plant pollen development to high-temperature (HT) stress is well known, but the involvement of autophagy in HT injury is yet to be clarified. Here, we found that following transfer to 30 °C, all autophagy-deficient (atg) mutants (atg2-1, 5-1, 7-2, and 10-1) of Arabidopsis thaliana tested displayed visibly impaired pollen development and anther dehiscence. HT-induced male sterility significantly increased in the atg mutants, but the degree of HT-induced obstacles did not change between the wild type (WT) and mutants from the seedling stage to the bolting stage. Cytological analyses showed that 30 °C promoted autophagy and autolysosome formation in both anther wall cells and microspores in developing anthers of WT, but the atg5-1 mutant did not show completion of tapetum degeneration and microspore maturation. HT upregulated hydrogen peroxide and dehydroascorbate reductase 1 production in both WT and atg5-1 anthers, but the basal levels were already higher in the mutant. HT repressed expression of UNDEAD and its regulator MYB80, which are required for tapetal programmed cell death (PCD) for proper pollen development. Taken together, our results suggest that autophagy functions in tapetum degeneration and pollen development during HT-caused tapetal PCD abortion.

Transcriptomic and Proteomic Analysis of Shaan2A Cytoplasmic Male Sterility and Its Maintainer Line in Brassica napus.

Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

CIK Receptor Kinases Determine Cell Fate Specification during Early Anther Development in Arabidopsis.

Appropriate cell division and differentiation ensure normal anther development in angiosperms. BARELY ANY MERISTEM 1/2 (BAM1/2) and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), two groups of leucine-rich repeat receptor-like protein kinases, are required for early anther cell specification. However, little is known about the molecular mechanisms underlying these two RLK-mediated signaling pathways. Here, we show that CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs), a group of novel coreceptor protein kinase-controlling stem cell homeostasis, play essential roles in BAM1/2- and RPK2-regulated early anther development in Arabidopsis thaliana The archesporial cells of cik1/2/3 triple and cik1/2/3/4 quadruple mutant anthers perform anticlinal division instead of periclinal division. Defective cell division and specification of the primary and inner secondary parietal cells occur in these mutant anthers. The disordered divisions and specifications of anther wall cells finally result in excess microsporocytes and a lack of one to three parietal cell layers in mutant anthers, resembling rpk2 or bam1/2 mutant anthers. Genetic and biochemical analyses indicate that CIKs function as coreceptors of BAM1/2 and RPK2 to regulate archesporial cell division and determine the specification of anther parietal cells.

Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Nutrients and Heavy Metals in Flowers and Corms of the Saffron Crocus (Crocus sativus L.)

We used ICP mass spectrometry and gas chromatography to examine the nutritional properties of the flower organs of saffron crocus grown in two cultivation fields. The pollenkitt consisted of a variety of mainly unsaturated fatty acids of dietary value, and fats of the same type were also on the anther wall. Microelements important for the human body were at concentrations allowable in both pollen and anther wall cells. Lead and mercury were below allowable levels, unlike arsenic which in pollen of the Field A was 1.70 μg/g, and cadmium which in the Field B was 0.29 μg/g in pollen and 0.43 μg/g in the anther wall cells. The contamination of the stamens was associated with the high concentration of heavy metals found in the soil. Phosphorus, magnesium and calcium were high and sodium low in both pollen and anther wall cells, regardless of the cultivation fields. Styles and corms were rich in macroelements and not contamined by heavy metals. If on one hand pollen and anthers of saffron deserve attention as dietary supplements, on the other it must be taken into due account their tendency to absorb toxic metals from the soil.

Dynamics of Polysaccharides and Neutral Lipids during Anther Development in Castor (Ricinus communis)

Anthers contain starch and neutral lipids, which have key roles in microspore ontogeny and gametophyte development. In this study, we observed the dynamic changes in starch and neutral lipids in the anther developmental processes of castor (Ricinus communis) by cytochemical methods. Starch grains and neutral lipids presented a regular dynamic distribution during anther development. In young anthers, some neutral lipids accumulated in sporogenous cells, whereas neutral lipids disappeared with microspore growth. At the late microspore stage, starch grains began to accumulate in microspores, and the starch content of bicellular pollen significantly increased after microspore mitosis. At anthesis, starch grains and neutral lipids accumulated in the mature pollen grains. Visible changes occurred in anther wall cells. The epidermis, middle layer, and tapetum were degenerated, and only a single layer of endothecium remained at anthesis. The dynamic variation of starch grains and neutral lipids in tapetal cells was consistent with the changes in microspores and pollen during anther development. All these findings demonstrated that tapetal cells directly interacted with the developing gametophytes. The tapetal cells play an important role in supplying nutritional substances for microspore absorption. Moreover, the endothecium protects the pollen and contributes to anther dehiscence. The results of this study provide a foundation for the further research on sexual reproduction in angiosperms.

Calcium distribution during pollen development of Uncaria hirsuta Havil. (Rubiaceae), with special reference to the function of the protruding oncus

Calcium distribution in the developing anthers of Uncaria hirsuta was studied with the potassium pyroantimonite method. The amount and distribution of calcium precipitates change along with the developing anthers and pollen grains in U. hirsuta. Calcium distribution is closely correlated with the anther wall differentiation, exine and intine formations. In mature pollen grains, abundant calcium is in intine, protruding onci and nucleus. A generative cell has more calcium than a vegetative cell does. The calcium storage is likely to contribute to pollen germination, and mitosis of the generative cell. Ubisch bodies formed at the same period with the pollen exine development, and some of them are in close contact with the exine. Abundant calcium is present in the Ubisch bodies, protruding onci and tapetum. These results might support the idea that Ubisch bodies and protruding onci participate in calcium transportation during pollen development. The protruding oncus is likely to have function of calcium or other ions transportation, although further experiment and additional data are required to confirm this. The present study also provides confirmatory evidence to the previous knowledge that calcium participates in regulation of cell differentiation, degradation in each layer of anther wall cells, and pollen development.

Cytochemical study of developing anthers of Amomum villosum Lour.

We analyzed anther development in Amomum villosum Lour. (Zingiberaceae) using the periodic acid-Schiff's technique and Sudan black staining to test for the presence of starch and lipids, respectively. Our analyses showed that microspore mother cells of A. villosum lack typical callose walls, and numerous lipid granules appear in the cells early in development. Some starch granules are present in anther wall cells, but not in tapetal cells. After meiosis, numerous lipid granules remain unchanged in the microspores. During microspore development, some small starch granules first appear in the central cell region, and then the starch granules increase in size. After microspore division, the bicellular pollen grains become filled with starch and lipids, and remain in this state until the pollen grains reach maturity. At anthesis, the anther wall of A. villosum consists of several layers of endothecium cells with an evidently thickened radial wall, and some layers of parenchyma cells containing numerous starch granules.

少花龙葵花药发育中多糖和脂滴的组织化学研究

对少花龙葵花药发育中淀粉粒和脂滴的分布和转化过程进行组织化学研究。结果显示：在造孢细胞时期幼小花药中没有营养物质积累；在小孢子母细胞时期，花药表皮和药室内壁细胞中出现淀粉粒，同时在绒毡层细胞中出现了较多的脂滴；在四分体时期，中层细胞中出现淀粉粒，而绒毡层细胞中的脂滴减少；到小孢子早期，花药表皮细胞中除淀粉粒外还出现了脂滴；但到小孢子晚期，药壁细胞中的淀粉和脂滴都明显减少；在二胞花粉早期，花粉开始积累淀粉粒，药壁细胞中的淀粉和脂滴几乎消失；在即将开花的成熟花粉中积累了大量淀粉粒和少量脂滴作为其存储物。 The distribution of polysaccharides and lipid drops in developing anther of Solanum photeinocarpum was studied. At the sporogenous cell stage, neither starches nor lipids were in anther wall cells. At the stage of microspore mother cells, some starches appeared in epidemic cells and endothecium cells and many lipids appeared in tapetal cells. At the tetrad stage, some polysaccharides appeared in middle layer cells. After microspores were released from tetrads, both starches and lipids appeared in epidemic cells, but both materials decreased at the late microspore stage. After the microspore division, early bicellular pollen began to accumulate polysaccharides, and the starches and lipids disappeared in anther wall cells. With the pollen development, many starches and some lipids were accumulated in the vegetative cell of the nearly mature pollen.

Variations of total protein content and protein synthesis during microsporogenesis in Larix europaea L.

The variations of the total protein content and protein synthesis were analyzed in successive stages of microsporogenesis in <em>Larix europaea</em> L. Pure fractions of microsporocytes containing the cells in successive phases of meiosis and fractions of the anther wall cells were used to perform the assays. It was revealed, that the protein content and the intensity of protein synthesis undergo considerable changes in the course of meiosis. It was proved also, by means of electrophoretic separation of protein fractions, that no new proteins are synthesised during differentiation of microsporocytes of larch, and that the microsporogenesis process is related rather to disappearance of some protein fractions.

Improvement of Selected Induction Culture Media on Callus Induction in Anther Culture of Anthurium and a Histological Study on its Callus Formation

Improvement of selected induction culture media on callus induction in anther culture of anthurium and a histologicalstudy on its callus formation were studied at the tissue culture laboratory of the Indonesian Ornamental CropsResearch Institute from February to October 2008. The objectives of the study were to optimize selected media forcallus formation, reveal cell origin of callus derived from anther culture and shoot formation process. Selectedmedia improved in the study were 1) MMS-TBN containing 0,5 mg/l TDZ, 1,0 mg/l BAP and 0,01 mg/l NAA (Winartomedium, WM) and 2) MMS III supplemented with 1,5 mg/l TDZ, 0,75 mg/l BAP and 0,02 mg/l NAA (Winarto andRachmawati medium, WRM). Improvement treatments were carried out by omission and application of 2,4-D in 0.5mg/l and reduction of medium strength of full, half, quarter, one eighth, one sixteenth, and zero strength. Afactorial experiment was arranged using a randomized complete block design with four replications. Results ofthis study indicated that the highest callus induction was clearly established in WRM. The medium stimulatedpotential growth of anther (PGA) up to 81% with 49% of percentage of anther regeneration (PAR) and 2.7 number ofcallus formed per replication (NCF). Significant improvement in callus formation was also recorded by reduction ofmedium strength of WRM to one eighth compared to others. The reduction induced PGA up to 58% with 29% of PARand 1.8 NCF. From histological studies it was well recognized that regenerated callus on half anthers cultured wasoriginated from middle layer cells of anther wall. The morphogenic response of anther wall cells caused primarilyon no androgenesis effect in microspore cells.

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.

MIL2 (MICROSPORELESS2) regulates early cell differentiation in the rice anther

The formation of diverse, appropriately patterned cell types is critical in the development of all complex multicellular organisms. In flowering plants, anther patterning is a complex process essential for successful sexual reproduction. However, few genes regulating this process have been characterized to date. We report here that the gene MICROSPORELESS2 (MIL2) regulates early anther cell differentiation in rice (Oryza sativa). The anthers of mil2 mutants were characterized using molecular markers and cytological examination. The MIL2 gene was cloned and its expression pattern was analyzed through RNA insitu hybridization. The localization of the MIL2 protein was observed by immunostaining. MIL2 encodes the rice homolog of the Arabidopsis TAPETUM DETERMINANT1 (TPD1) protein. However, mil2 anthers display phenotypes different from those of tpd1 mutants, with only two layers of anther wall cells formed. MIL2 has an expression pattern distinct from that of TPD1. Its transcripts and proteins predominate in inner parietal cells, but show little accumulation in reproductive cells. Our results demonstrate that MIL2 is responsible for the differentiation of primary parietal cells into secondary parietal cells in rice anthers, and suggest that rice and Arabidopsis anthers might share similar regulators in anther patterning, but divergent mechanisms are employed in these processes.

Pseudo-embryogenic structures in anther and isolated microspore cultures in vitro: a cautionary guide

This review describes sources of structures of non-microspore origin observed in anther and microspore cultures. Various characteristics of these structures may cause a wrong diagnosis of these structures as embryos or cell/tissue clusters of microspore origin. Here we suggest such structures to be named as pseudo-embryogenic structures. The introduction of pseudo-embryogenic structures and their origins could be helpful to distinguish them from true microspore-derived structures. Prompted by certain environmental cues, somatic cells existing as a contamination in immature pollen (microspores) cultures can lead to the formation of ‘pseudo-embryos’ commonly known as embryoids. The pseudo-embryogenic structures may be classified in the following groups: (i) pseudo-star-like structures; pseudo-multicellular structures; (ii) pseudo-embryos with pseudo-suspensors; (iii) contaminating bacteria appearing as callus colonies; (iv) calli and embryos of somatic origin; (v) giant tetrad-like structures; (vi) anther wall cells. The exact origin of these structures is discussed in this paper, and some recommendations are proposed in order to avoid misinterpretation.

Meiotic chromosome behaviour and karyomorphology of Aloe vera (L.) Burm. f.

Aloe vera (L.) Burm. f. (the family Aloaceae) indigenous to Africa is an admired medicinal plant used as a folk medicine. Meiotic division was investigated in A. vera with a view to decipher the behavior of chromosomes during prophase I and the subsequent stages of meiosis following acetocarmine squash technique. The various meiotic stages from leptotene to pollen development were studied. Seven bivalents were clearly visible at diplotene, and the chiasma frequency averaged to 1.86 per bivalent and 13.1 per pollen mother cell (PMC). Prophase I stages are difficult to identify due to clumping of chromosome threads. Second nuclear division, however, allows clear distinction of chromosome configuration. Besides clumping, various other types of chromosomal aberrations such as stickiness and fragments at metaphase I, precocious movement of chromosomes, bridges, laggards and unequal separation of chromosomes at anaphase I and unequal separation of chromosomes at telophase II were noted. Karyomorphological study on the other hand, was carried out in anther wall (mitotic) cells of A. vera. Mitotic as well as meiotic studies in the species showed 2n=14 chromosomes including two pairs of sat-chromosomes. The karyotype of the species was adjudged to be typically asymmetric.