The Effects of Low-Energy Nitrogen Ion Implantation on Pollen Exine Substructure and Pollen Germination of Cedrus deodara

The aim of this study is to investigate the biological effects of ion beams on pollen. Pollen grains of Cedrus deodara were implanted with 30 keV nitrogen ion beams at doses ranging from 1 × 10^15 ions/cm^2 to 15 × 10^15 ions/cm^2. The effects of N^＋ implantation on the pollen exine substructure were examined using an atomic force microscope （AFM）, and the structure and morphology of pollen and pollen tubes were observed using a laser scanning confocal microscope （LSCM）. AFM observations distinctly revealed the erosion of the pollen exine caused by N^＋ implantation in the micrometer to nanometer range. Typical results showed that the erosion degree was linearly proportional to the ion dose. Pollen germination experiments in vitro indicated that N^＋ implantation within a certain dose range increased the rate of pollen germination. The main abnormal phenomena in pollen tubes were also analyzed. Our results suggest that low energy ion implantation with suitable energy and dosage can be used to break the pollen wall to induce a transfer of exogenous DNA into the pollen without any damage to the cytoplasm and nuclei of the pollen. The present study suggests that a combination of the method of ion-beam-induced gene transfer and the pollen-tube pathway method （PTPW） would be a new plant transformation method.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development

Because of its significance for plant male fertility and,hence,direct impact on crop yield,pollen exine development has inspired decades of scientific inquiry.However,the molecularmechanismunderlying exine formation and thickness remains elusive.In this study,we identified that a previously unrecognized repressor,ZmMS1/ZmLBD30,controls proper pollen exine development in maize.Using an ms1 mutant with aberrantly thickened exine,we cloned a male-sterility gene,ZmMs1,which encodes a tapetum-specific lateral organ boundary domain transcription factor,ZmLBD30.Weshowed thatZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7,and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine.This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum,and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis.Collectively,these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine

Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum‐expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice（Oryza sativa L.）. However,the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3‐2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3‐2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in‐chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7‐hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in‐chain hydroxylation of lauric acid required for the development of male organ in higher plants.

Atomic force microscopic observation on substructure of pollen exine in Cedrus deodara and Metasequoia glyptostroboides

The substructure of pollen exine in Cedrus deodara (Roxb.) Loud, and Metasequoia glyptostroboides Hu et Cheng has been examined with an atomic force microscope (AFM). The results indicate that the exine substructure units containing sporopollenin in two species are similar in shape, which are granular, but slightly different in size. In Cedrus the substructure unit of pollen exine appears to be 56-99 nm long and 42-74 nm wide, while in Metasequoia it appears to be 81-118 nm long and 43-98 nm wide. It has been observed that the subunits of pollen exine in Cedrus arranged tightly to form short-rod-like or spheroidal pollen exine units, several or more than ten of which formed an island-like structure. There are various spaces among these island-like structures which are interconnected to occupy the entire pollen exine. In Metasequoia, the subunits of pollen exine also arranged tightly with a distribution tendency of cluster of 3-10, however, no obvious boundary exists among these clusters. From our

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases （PKSs） are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase （OsPKS2） in male reproduction of rice （Oryza sativa）. Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl- CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT （wild type）, ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/laps, in which most detected phenolics were substantially decreased, ospks2 accumu- lated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/laps, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.

Rice Glucose 6-Phosphate/Phosphate Translocator 1 is required for tapetum function and pollen development

In plants, non-green plastids in heterotrophic tissues are sites for starch and fatty acids biosynthesis,which are essential for plant development and reproduction. Distinct from chloroplasts, the metabolites for these processes in non-green plastids have to be imported through specific transporters. Glucose 6-Phosphate/Phosphate Translocator 1 is required for the uptake of cytosolic Glucose 6-Phosphate into non-green plastids. In Arabidopsis, GPT1 has been demonstrated to play essential roles in male, female gametophyte and embryo development. However, the roles of GPTs in other species are yet largely unknown. Here, we reported that rice OsGPT1 is indispensable for normal tapetal degeneration and pollen exine formation during anther and pollen development. OsGPT1 is localized in the plastid and distributed in the anther wall layers and late-stage pollen grains. Different from the gametic defects caused by mutation in At GPT1, disruption of OsGPT1 does not affect male and female gamete transmission as well as embryo development. On the contrary, osgpt1 mutant exhibits delayed tapetum degeneration,decreased Ubisch bodies formation and thinner pollen exine, leading to pollen abortion at the mature stage. Furthermore, the expression of several genes involved in tapetal programmed cell death(PCD)and sporopollenin formation is decreased in osgpt1. Our study suggests that OsGPT1 coordinates the development of anther sporophytic tissues and the male gametophyte by integrating carbohydrate and fatty acid metabolism in the plastid.

Understanding the Phylomorphological Implications of Pollinia from <i>Dendrobium</i>(Orchidaceae)

We aim to evaluate the evolutionary trends of pollen ultra-structures and microstructures in the light of molecular phylogeny, and to test whether pollen micromorphology is a predictor of interspecific phylogenetic relationship or a consequence of ecological influences. Using scanning electron microscopy pollinia micromorphologies of 18 species of the genus Dendrobium were comprehensively examined. A phylogenetic tree from ribosomal-ITS2 sequences was constructed reflecting molecular phylogeny. In result, two major clusters were identified representing the sections Aporum, Formoase Dendrobium and Holochrysa. The section Dendrobium could be further classified, for the first time, into two clades sharing the same root of origin. Variations in the shape of pollinia were distinct and consistent constituting six novel groups. Other qualitative/quantitative keys to pollinia showed congruence with molecular phylogeny and supported the correlated evolution of these traits. Concurrently pollen sculpturing also showed large variation in exine morphology (rugular/psilate to psilate-scabrate/rugulate-scabrate) providing explicit clues for phylogenetic trend of exine evolution. For the characteristic sculpturing on the pollinia surface, a simple version of pre-pattern model is also hypothesized for deeper insight into pattern formation in other biological systems. Results indicated that distinct groups of Dendrobium species have parallel and independent evolution accompanied with genetic changes occurred during species diversification. Thus pollen micromorphology is a good candidate for inferring the genetic relationship at inter-specific level having phylogenetic implications even using abundant palynological records.

Pollen morphology of the genus Cornukaempferia (Zingiberaceae) in Thailand

Cornukaempferia is a recently described genus of Zingiberaceae which only occurs in Thailand as a rare genus with limited geographical distribution. Only three species have been described so far, including a recently described new species, C. larsenii. These three species are morphologically very similar and additional data on other biological aspects are useful for the elucidation of their relationship. Pollen morphology of all three species of genus Cornukaempferia has been studied by light microscopy and scanning electron microscopy. The pollen grains are monad, spherical, inaperturate. The exine sculpture is echinate with psilate between the spines for C. aurantiflora and C. longipetiolata, or echinate with regulate between the spines for C. larsenii. This observation helps support the taxonomic status of C. larsenii.

Glycerol-3-Phosphate Acyltransferase 6 （GPAT6） Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility

Glycerol-3-phosphate acyltransferase （GPAT） mediates the initial synthetic step for the formation of glycer- olipids, which act as the major components of biological membranes and the principal stored forms of energy. GPAT6 is a member of the Arabidopsis GPAT family, which is crucial for cutin biosynthesis in sepals and petals. In this work, a func- tional analysis of GPAT6 in anther development and plant fertility was performed. GPAT6 was highly expressed in the tapetum and microspores during anther development. The knockout mutant, gpat6, caused a massive reduction in seed production. This report shows that the ablation of GPAT6 caused defective tapetum development with reduced endoplas- mic reticulum （ER） profiles in the tapetum, which largely led to the abortion of pollen grains and defective pollen wall formation. In addition, pollen germination and pollen tube elongation were affected in the mutant plants. Furthermore, the double mutant analysis showed that GPAT6 and GPAT1 make joint effects on the release of microspores from tetrads and stamen filament elongation. This work shows that GPAT6 plays multiple roles in stamen development and fertility in Arabidopsis.

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice （Oryza sativa L.） male sterile mutant. Based on map- based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate （ATP）-binding cassette （ABC） transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcgl5. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 （meiosis II stage） to stage 10 （late microspore stage）. Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcgl5 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther Iocules. Consistently, histological analysis showed that osabcgl5 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.

The Post-meiotic Deficicent Anther1 (PDA1) gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 （pda1） from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,the pda1 mutant displayed obvious defects in postmeiotic tapetal development,abnormal degeneration occurred in the tapetal cells at stage 9 of anther development.Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant,causing no obvious pollen exine formation.RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB,OsC4 and Wax-deficient anther1 （WDA1） was greatly reduced in the pda1 mutant anther.Using map-based cloning approach,the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F2 population.The physical distance between HLF610 and HLF627 was about 194 kb.This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.

DEAP1 encodes a fasciclin-like arabinogalactan protein required for male fertility in rice

Arabinogalactan proteins(AGPs)are widely distributed in plant cells.Fasciclin-like AGPs(FLAs)belong to a subclass of AGPs that play important roles in plant growth and development.However,little is known about the biological functions of rice FLA.Herein,we report the identification of a male-sterile mutant of DEFECTIVE EXINE AND APERTURE PATTERNING1(DEAP1)in rice.The deap1 mutant anthers produced aberrant pollen grains with defective exine formation and a flattened aperture annulus and exhibited slightly delayed tapetum degradation.DEAP1 encodes a plasma membrane-associated member of groupⅢplant FLAs and is specifically and temporally expressed in reproductive cells and the tapetum layer during male development.Gene expression studies revealed reduced transcript accumulation of genes related to exine formation,aperture patterning,and tapetum development in deap1 mutants.Moreover,DEAP1 may interact with two rice D6 PROTEIN KINASE-LIKE3 s(OsD6PKL3s),homologs of a known Arabidopsis aperture protein,to affect rice pollen aperture development.Our findings suggested that DEAP1 is involved in male reproductive development and may affect exine formation and aperture patterning,thereby providing new insights into the molecular functions of plant FLAs in male fertility.

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.

PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris

PECTATE LYASE‐LIKE10（PLL10） was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage（Brassica campestris ssp. chinensis）. Here, antisense‐RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines（bcpll10‐4, ‐5, and ‐6）. In fertilization experiments, fewer seeds were harvested when the antisense‐RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10.Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed thenormal proportional distribution of the two layers in the non‐germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture.