Correlation of sequential floral and male gametophyte development and preliminary results on anther culture in <Emphasis Type="Italic">Opuntia ficus-indica</Emphasis>

Before approaching anther culture as a tool to trigger an androgenic response in a new species, it is advisable to characterize and correlate flower and male gametophyte development to enable reproducible identification of the appropriate starting material. Buds and flowers of Opuntia ficus-indica cv. Gialla were classified in eight stages according to their total length at the earlier stages and the length of the corolla in flowers with emerging sepals. Due to the low condensation of chromatin in the microspore nucleus as well as in the vegetative nucleus of the bi- and tricellular pollen along with the high autofluorescence of the intricate exine, DAPI staining turned out not to be feasible in this species. Therefore an approach based on light-microscopy observation of semithin sections was used. These sections were stained with toluidine blue for general structure recognition and I₂KI to study starch deposition. Correlations were made between the sequential floral and male gametophyte development. Using this approach we determined the timing of pollen formation and observed that pollen development is impaired in plants producing seedless fruits. Furthermore, anther culture was carried out with anthers collected from flower buds at stages 2 and 3. Most of the anthers produced callus, however no regeneration was obtained.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice

Background and AimsCold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy.MethodsAfter exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities.Key ResultsWe observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress.ConclusionsThe pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.     

Up and down: stamen movements in Ruta graveolens (Rutaceae) enhance both outcrossing and delayed selfing

Background and Aims

Stamen movements directly determine pollen fates and mating patterns by altering positions of female and male organs. However, the implications of such movements in terms of pollination are not well understood. Recently, complex patterns of stamen movements have been identified in Loasaceae, Parnassiaceae, Rutaceae and Tropaeolaceae. In this study the stamen movements in Ruta graveolens (Rutaceae) and their impact on pollination are determined.

Methods

Pollination effects of stamen movements were studied in Ruta graveolens, in which one-by-one uplifting and falling back is followed by simultaneous movement of all stamens in some flowers. Using 30 flowers, one stamen was manipulated either to be immobilized or to be allowed to move freely towards the centre of the flower but be prevented from falling back. Pollen loads on stigmas and ovule fertilization in flowers with or without simultaneous stamen movement were determined.

Results

Pollen removal decreased dramatically (P < 0·001) when the stamen was stopped from uplifting because its anther was seldom contacted by pollinators. When a stamen stayed at the flower''s centre, pollen removal of the next freely moved anther decreased significantly (P < 0·005) because of fewer touches by pollinators and quick leaving of pollinators that were discouraged by the empty anther. Simultaneous stamen movement occurred only in flowers with low pollen load on the stigma and the remaining pollen in anthers dropped onto stigma surfaces after stamens moved to the flower''s centre.

Conclusions

In R. graveolens pollen removal is promoted through one-by-one movement of the stamen, which presents pollen in doses to pollinators by successive uplifting of the stamen and avoids interference of two consecutively dehisced anthers by falling back of the former stamen before the next one moves into the flower''s centre. Simultaneous stamen movement at the end of anthesis probably reflects an adaptation for late-acting self-pollination.     

Differential proteomic studies of the genic male-sterile line and fertile line anthers of upland cotton (Gossypium hirsutum L.)

Pollen development is disturbed in the microspore development stage of the double-recessive nuclear male-sterile line ms5ms6 (Gossypium hirsutum L.). This study aimed to identify differentially expressed anther proteins and their potential roles in pollen development and male sterility. We compared the proteomes of sterile and fertile anthers of the double recessive nuclear male-sterile line ms5ms6. Approximately 1,390 protein spots were detected by two-dimensional differential gel electrophoresis. Proteins with altered accumulation levels in sterile anthers compared with fertile anthers were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Down-regulated proteins in the sterile anthers included cytosolic ascorbate peroxidase 1 and glutaminyl-tRNA synthetase (glutamine-tRNA ligase). Several carbohydrate metabolism- and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. By contrast, ATP-dependent RNA helicase eIF4A-13, NADH dehydrogenase subunit 1, enolase, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, alcohol dehydrogenase 2d, 3-ketoacyl-CoA synthase, and trehalose 6-phosphate synthase were expressed at higher levels in sterile anthers than in fertile anthers. The regulation of upland cotton pollen development involves a complex network of differentially expressed genes. This study provides the foundation for future investigations of gene function in upland cotton pollen development and male sterility.     

Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.     

Biochemical and Ultrastructural Changes in Pollen ofZea maysL. Grown Under Enhanced UV-B Radiation

The influence of ultraviolet-B (UV-B) radiation on the developmentof the male gametophyte was studied inZea maysL. cv. LG12 grownin a growth chamber under PAR light supplemented with UV-B radiationand compared with a second set of plants grown under PAR light.Pollen samples collected from both groups of plants were culturedon germination medium and it was found that UV-B had no effecton pollen germination. Total pollen protein content was notaffected but UV-B absorbing pigments increased. Some ultrastructuralalterations were observed in pollen and pollen tubes, in particularlarge amounts of electron dense deposits were seen throughoutthe cytoplasm and in association with the pollen wall. In maturespikes of UV-B treated plants, anthers retained numerous pollengrains in their loculi while anthers of control plants werealmost empty. UV-B treatment delayed flowering by 2–3d. These results show that UV-B treatment of maize plants interfereswith flowering, pollen ultrastructure and anther maturationeven though pollen germination is unaffected. The significantincrease of UV-B absorbing pigments in pollen grains could representa defence mechanism that enables plants to complete their reproductivecycle.Copyright 1998 Annals of Botany Company Zea maysL., maize, UV-B radiation, pollen.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, <Emphasis Type="Italic">Pancratium maritimum</Emphasis>

Dynamic changes in the distribution of lipid and insoluble polysaccharide reserves of Pancratium maritimum L. (Amaryllidaceae) anthers were investigated throughout the successive stages of pollen development, using cytochemical methods, to determine whether the synthesis, transformation, and mobilization of reserve materials in developing anthers follow the regular pathway in angiosperms and support the physiological activities in developing pollen. Polysaccharides and lipid reserves exhibited a variable pattern of distribution from the sporogenous cell stage to the anthesis. Starch granules and lipid bodies were scarce in the cytoplasm of sporogenous cells, but their number increased significantly at the premeiotic stage. Conversely, starch and lipid reserves of meiocytes reduced at the early prophase of the first meiotic division, and then their amount showed fluctuations during the microsporogenesis. The cytoplasm of free and vacuolated microspores was poor regarding the polysaccharide and lipid reserves. However, at the late vacuolated microspore stage, small insoluble polysaccharides began to appear in the microspore cytoplasm, and their number increased remarkably in the cytoplasm of the bicellular pollen grain. During the maturation of pollen grains, polysaccharide reserves were replaced with lipids. The starch and lipid reserves of the staminal envelope also showed variations at different stages of the anther development. The dynamic changes in the polysaccharide and lipid reserves of P. maritimum anthers were consistent with the physiological activities such as differentiation, cell division and material synthesis that occur in the anther tissue at different stages of the male gametophyte development, and supported the normal pollen development.     

Quantitative shotgun proteomic analysis of cold-stressed mature rice anthers

Rice crops are vulnerable to low temperatures. During development, the reproductive stage is particularly sensitive to cold exposure, which causes abnormal pollen development and a high degree of male sterility. In this study, shotgun proteomic analysis was used to analyze rice anthers containing pollen grains from a cold-tolerant variety, Dianxi 4. Protein expression was compared between normal anthers and anthers exposed to cold temperatures at the young microspore stage. In total, 3835 non-redundant proteins were identified in the rice anther. Of these, 441 proteins were differentially expressed between normal and cold-treated anthers. Pollen allergens, ATP synthase, actin, profilin, and β-expansin proteins were highly abundant, reflecting anther development, pollen germination, and pollen tube elongation. Starch and sucrose metabolic proteins such as α-amylase precursor and 4-α-glucanotransferase exhibited reduced expression after cold exposure. Among the proteins that exhibited increased expression after cold exposure, C2 domain proteins, and GRPs were identified as candidate signaling factors for mediation of the cold tolerance response. Through high-throughput proteomic analysis we were able to reveal proteomic changes against cold stress and suggest two signaling factors as the candidate genes.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Male Sterility and Anther Wall Structure in Copper-deficient Plants

Anther development and pollen sterility were followed in plantsof wheat, oat, barley, sweetcorn, sunflower, petunia and subterraneumclover grown at a range of copper supplies. Copper-deficientplants had increased pollen sterility. Lignified wall thickenings were reduced or absent in the endotheciaof anthers from Cu-deficient plants. Reduced seed set may resultboth from reduced pollen fertility or failure of the stomiato rupture due to decreased lignification of anther walls. Triticum aestivum L., wheat, Hordeum vulgare L., barley, Avena sativa L., oat, Zea mays L., corn, sweetcorn, maize, Helianthus annuus L., sunflower, Petunia hybrida L., Trifolium subterraneum L., subterranean clover, male sterility, anther development, copper deficiency     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Temperature stress interferes with male reproductive system development in clementine (Citrus clementina Hort. ex. Tan.)

Male gametophyte development is a critical phase of the plant life cycle due to its high sensitivity to environmental stresses. The rise in the average global temperature, often accompanied by extreme fluctuations, has an important impact on biological processes. Among those, male gametophytes are particularly sensitive to temperature stress during flower bud development and anthesis. Male gametophyte development was extensively studied in several plant species, but little information is available about the effects of temperature stress on male gametophyte development in the genus Citrus. We evaluated the effects of cold and hot temperatures during microsporogenesis and microgametogenesis on one of the most economically valuable citrus species, the “Comune” clementine (Citrus clementina Hort. ex. Tan.). The effect of constant temperature on the androecium was evaluated by a time course histological analysis performed on the anthers and by monitoring in vitro pollen germination. The results revealed how suboptimal hot and cold temperatures induce drastic alterations on the morphology of the tapetal cells, microspores and mature pollen grains. Shifting from the optimal temperature affected the timing of starch depletion in the anther walls, such as epidermis, endothecium and middle layer, influencing the pollen germination rate and pollen tube growth. To the best of our knowledge this is the first study attempting to assess how temperature stress affects male reproductive development in citrus. A better understanding of the mechanisms underlining male sterility will provide novel insights to elucidate the physiology of this agronomical important quality trait.     

Identification of small RNAs in late developmental stage of rice anthers

Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA) are known as repressors of gene expression. There are many plant proteins involved in small RNA-mediated gene silencing, such as Dicer ribonucleases and RNA-dependent RNA polymerases. However, most of these proteins have been reported to be absent in the late developmental stage of the plant male gamete, pollen. In order to clarify the existence of the small RNAs during maturation of pollen, we cloned and sequenced small RNAs from rice anthers including tricellular pollen. From fifty six candidates of small RNAs, we identified two known miRNAs (miR166 and miR167), eight potential miRNAs, and ten putative heterochromatic siRNAs (hc-siRNAs). RNA gel blot analyses clearly showed that miR166 and miR167 were accumulated in the uninuclear pollen stage of anther development and remained until the tricellular pollen stage. Our cloning and RNA gel blot analyses of small RNAs led us to propose a possible function of small RNA-mediated gene regulation for the development of male gametes in rice.     

Small RNA profiling from meiotic and post-meiotic anthers reveals prospective miRNA-target modules for engineering male fertility in sorghum

Understanding male gametophyte development is essential to augment hybrid production in sorghum. Although small RNAs are known to critically influence anther/pollen development, their roles in sorghum reproduction have not been deciphered yet. Here, we report small RNA profiling and high-confidence annotation of microRNAs (miRNAs) from meiotic and post-meiotic anthers in sorghum. We identified 262 miRNAs (82 known and 180 novel), out of which 58 (35 known and 23 novel) exhibited differential expression between two stages. Out of 35 differentially expressed known miRNAs, 13 are known to regulate anther/pollen development in other plant species. We also demonstrated conserved spatiotemporal patterns of 21- and 24-nt phasiRNAs and their respective triggers, miR2118 and miR2275, in sorghum anthers as evidenced in other monocots. miRNA target identification yielded 5622 modules, of which 46 modules comprising 16 known and 8 novel miRNA families with 38 target genes are prospective candidates for engineering male fertility in grasses.     

Gibberellin Acts through Jasmonate to Control the Expression of MYB21, MYB24, and MYB57 to Promote Stamen Filament Growth in Arabidopsis

Precise coordination between stamen and pistil development is essential to make a fertile flower. Mutations impairing stamen filament elongation, pollen maturation, or anther dehiscence will cause male sterility. Deficiency in plant hormone gibberellin (GA) causes male sterility due to accumulation of DELLA proteins, and GA triggers DELLA degradation to promote stamen development. Deficiency in plant hormone jasmonate (JA) also causes male sterility. However, little is known about the relationship between GA and JA in controlling stamen development. Here, we show that MYB21, MYB24, and MYB57 are GA-dependent stamen-enriched genes. Loss-of-function of two DELLAs RGA and RGL2 restores the expression of these three MYB genes together with restoration of stamen filament growth in GA-deficient plants. Genetic analysis showed that the myb21-t1 myb24-t1 myb57-t1 triple mutant confers a short stamen phenotype leading to male sterility. Further genetic and molecular studies demonstrate that GA suppresses DELLAs to mobilize the expression of the key JA biosynthesis gene DAD1, and this is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant ga1-3 gai-t6 rga-t2 rgl1-1 is much lower than that in the WT. We conclude that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57. Therefore, we have established a hierarchical relationship between GA and JA in that modulation of JA pathway by GA is one of the prerequisites for GA to regulate the normal stamen development in Arabidopsis.     

Cover Caption: Rice Anther Development

At the early stage of rice flower development, the lemma, palea, stamen and pistil primordia are first formed, as shown by the cover picture taken under an electron microscope. The stamen primordia further develop into mature anthers, with viable pollen inside. In this issue, Guo and Liu (pp. 967–978) summarize recent advances in molecular genetic studies of rice anther development and male fertility/sterility control. Research in this field has significant implications in rice genetic improvement.