Abnormal Pollen Development Research Articles

The miR159a-DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

The R2R3 MYB gene TaMYB305 positively regulates anther and pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

The loss of TaMYB305 function down-regulated the expression of jasmonic acid synthesis pathway genes, which may disturb the jasmonic acid synthesis, resulting in abnormal pollen development and reduced fertility. The MYB family, as one of the largest transcription factor families found in plants, regulates plant development, especially the development of anthers. Therefore, it is important to identify potential MYB transcription factors associated with pollen development and to study its role in pollen development. Here, the transcripts of an R2R3 MYB gene TaMYB305 from KTM3315A, a thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) wheat, was isolated. Quantitative real-time PCR (qRT-PCR) and promoter activity analysis revealed that TaMYB305 was primarily expressed in anthers. The TaMYB305 protein was localized in the nucleus, as determined by subcellular localization analysis. Our data demonstrated that silencing of TaMYB305 was related to abnormal development of stamen, including anther indehiscence and pollen abortion in KAM3315A plants. In addition, TaMYB305-silenced plants exhibited alterations in the transcriptional levels of genes involved in the synthesis of jasmonic acid (JA), indicating that TaMYB305 may regulate the expression of genes related to JA synthesis and play an important role during anther and pollen development of KTM3315A. These results provide novel insight into the function and molecular mechanism of R2R3-MYB genes in pollen development.

Abnormal Pollen Development in the Fragaria vesca Mutant fruitless 1

Knowledge of the genes underlying a given trait is highly useful for developing molecular markers for breeding and is the foundation for future genomic crop improvements. The cultivated strawberry, F. ×ananassa, is a valuable horticultural crop. Genome sequencing revealed that of the four diploid strawberry subgenomes contributing to the F. ×ananassa octoploid genome, the woodland strawberry, F. vesca, subgenome is dominant. Thus, F. vesca is an important system for determining gene function and should be used as a source of genetic diversity for F. ×ananassa breeding. Ethyl methanesulfonate mutagenesis of H4 F7-3, an inbred line of F. vesca, resulted in one M2 line that did not produce any strawberries over a 3-year period in the greenhouse. This line was named fruitless 1. The fruitless 1 phenotype results from a single gene recessive mutation. Microscopic characterization revealed that fruitless 1 failed to produce fruit because anthers fail to develop properly before meiosis, resulting in no pollen production. This report of fruitless 1 facilitates further studies of the line.

A single-nucleotide substitution of CjTKPR1 determines pollen production in the gymnosperm plant Cryptomeria japonica.

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution ("T" to "C" at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.

Apomictic Malus plants exhibit abnormal pollen development.

Apomixis is the asexual reproduction through seeds that leads to the production of genetically uniform progeny. It has become an important tool in plant breeding because it facilitates the retention of genotypes with desirable traits and allows seeds to be obtained directly from mother plants. Apomixis is rare in most economically important crops, but it occurs in some Malus species. Here, the apomictic characteristics of Malus were examined using four apomictic and two sexually reproducing Malus plants. Results from transcriptome analysis showed that plant hormone signal transduction was the main factor affecting apomictic reproductive development. Four of the apomictic Malus plants examined were triploid, and pollen was either absent or present in very low densities in the stamen. Variation in the presence of pollen was associated with variation in the apomictic percentage; specifically, pollen was absent in the stamens of tea crabapple plants with the highest apomictic percentage. Furthermore, pollen mother cells failed to progress normally into meiosis and pollen mitosis, a trait mostly observed in apomictic Malus plants. The expression levels of meiosis-related genes were upregulated in apomictic plants. Our findings indicate that our simple method of detecting pollen abortion could be used to identify apple plants that are capable of apomictic reproduction.

SlMYB72 affects pollen development by regulating autophagy in tomato.

The formation and development of pollen are among the most critical processes for reproduction and genetic diversity in the life cycle of flowering plants. The present study found that SlMYB72 was highly expressed in the pollen and tapetum of tomato flowers. Downregulation of SlMYB72 led to a decrease in the amounts of seeds due to abnormal pollen development compared with wild-type plants. Downregulation of SlMYB72 delayed tapetum degradation and inhibited autophagy in tomato anther. Overexpression of SlMYB72 led to abnormal pollen development and delayed tapetum degradation. Expression levels of some autophagy-related genes (ATGs) were decreased in SlMYB72 downregulated plants and increased in overexpression plants. SlMYB72 was directly bound to ACCAAC/ACCAAA motif of the SlATG7 promoter and activated its expression. Downregulation of SlATG7 inhibited the autophagy process and tapetum degradation, resulting in abnormal pollen development in tomatoes. These results indicated SlMYB72 affects the tapetum degradation and pollen development by transcriptional activation of SlATG7 and autophagy in tomato anther. The study expands the understanding of the regulation of autophagy by SlMYB72, uncovers the critical role that autophagy plays in pollen development, and provides potential candidate genes for the production of male-sterility in plants.

Transcription factor TaGAMYB from wheat (Triticum aestivum L.) regulates flowering time and fertility in transgenic Arabidopsis thaliana.

The loss of TaGAMYB function in Arabidopsis results in abnormal pollen development and leads to decreased fertility. This process may be regulated by microRNAs, which suppress the expression of fatty acid pathway genes. Development of the anthers and pollen is significantly influenced by the transcription factor GAMYB. However, our knowledge of GAMYB in wheat is limited. Here, under fertility and sterility conditions, we identified the distinct transcripts TaGAMYB-d and TaGAMYB-g in thermosensitive genic sterile wheat YanZhan 4110S and confirmed their functions. TaGAMYB-g overexpression decreased the pollen vigor and germination rates, thereby reducing fertility. TaGAMYB-d overexpression lines exhibited early flowering. Due to aberrant pollen germination, the mutant homologous TaGAMYB genes in Arabidopsis thaliana also resulted in lower fertility. Our findings indicate that TaGAMYB controls the fertility and flowering time in transgenic Arabidopsis thaliana.

Global transcriptome analysis reveals potential genes associated with genic male sterility of rapeseed (Brassica napus L.).

Rapeseed is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been widely used successfully for rapeseed hybrid production. The physiological and molecular mechanism of pollen development in GMS lines of rapeseed (Brassica napus L.) need to be determined for the creation of hybrids and cultivation of new varieties. However, limited studies have focused on systematically mining genes that regulate the pollen development of GMS lines in B. napus. In the present study, to determine the stage at which pollen development begins to show abnormality in the GMS lines, we performed semi-thin section analysis of the anthers with five pollen development stages. The results indicated that the abnormal pollen development in DGMS lines might start at the meiotic stage, and abnormal pollen development in RGMS lines probably occurred before the tetrad stage. To investigate the critical genes and pathways involved in pollen development in GMS lines, we constructed and sequenced 24 transcriptome libraries for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines (6251AB and 6284AB) and two dominant GMS (DGMS) lines (4001AB and 4006AB). A total of 23,554 redundant DEGs with over two-fold change between sterile and fertile lines were obtained. A total of 346 DEGs were specifically related to DGMS, while 1,553 DEGs were specifically related to RGMS. A total of 1,545 DEGs were shared between DGMS and RGMS. And 253 transcription factors were found to be differentially expressed between the sterile and fertile lines of GMS. In addition, 6,099 DEGs possibly related to anther, pollen, and microspore development processes were identified. Many of these genes have been reported to be involved in anther and microspore developmental processes. Several DEGs were speculated to be key genes involved in the regulation of fertility. Three differentially expressed genes were randomly selected and their expression levels were verified by quantitative PCR (qRT-PCR). The results of qRT-PCR largely agreed with the transcriptome sequencing results. Our findings provide a global view of genes that are potentially involved in GMS occurrence. The expression profiles and function analysis of these DEGs were provided to expand our understanding of the complex molecular mechanism in pollen and sterility development in B. napus.

Development of a highly efficient virus-free regeneration system of Salvia miltiorrhiza from Sichuan using apical meristem as explants

BcakgroundThe dry root and rhizome of Salvia miltiorrhiza are used to treat cardiovascular diseases, chronic pain, and thoracic obstruction over 2000 years in Asian countries. For high quality, Sichuan Zhongjiang is regarded as the genuine producing area of S. miltiorrhiza. Given its abnormal pollen development, S. miltiorrhiza from Sichuan (S.m.-SC) relies on root reproduction and zymad accumulation; part of diseased plants present typical viral disease symptoms and seed quality degeneration. This study aim to detected unknown viruses from mosaic-diseased plants and establish a highly efficient virus-free regeneration system to recover germplasm properties.ResultsTobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were detected from mosaic-diseased plants. Primary apical meristem with two phyllo podium in 0.15–0.5 mm peeled from diseased plants were achieved 73.33% virus-free rate. The results showed that the medium containing MS, 0.5 mg/L 6-BA, 0.1 mg/L NAA, 0.1 mg/L GA3, 30 g/L sucrose and 7.5 g/L agar can achieve embryonic-tissue (apical meristem, petiole and leaf callus) high efficient organogenesis. For callus induction, the optimal condition was detected on the medium containing MS, 2 mg/L TDZ, 0.1 mg/L NAA by using secondary petiole of virus-free plants under 24 h dark/d condition for 21 d. The optimal system for root induction was the nutrient solution with 1/2 MS supplemented with 1 mg/L NAA. After transplant, the detection of agronomic metric and salvianolic acid B content confirmed the great germplasm properties of S.m.-SC virus-free plants.ConclusionsA highly efficient virus-free regeneration system of S.m.-SC was established based on the detected viruses to recover superior seed quality. The proposed system laid support to control disease spread, recover good germplasm properties in S.m.-SC.

Overexpression of sesame polyketide synthase A leads to abnormal pollen development in Arabidopsis

BackgroundSesame is a great reservoir of bioactive constituents and unique antioxidant components. It is widely used for its nutritional and medicinal value. The expanding demand for sesame seeds is putting pressure on sesame breeders to develop high-yielding varieties. A hybrid breeding strategy based on male sterility is one of the most effective ways to increase the crop yield. To date, little is known about the genes and mechanism underlying sesame male fertility. Therefore, studies are being conducted to identify and functionally characterize key candidate genes involved in sesame pollen development. Polyketide synthases (PKSs) are critical enzymes involved in the biosynthesis of sporopollenin, the primary component of pollen exine. Their in planta functions are being investigated for applications in crop breeding.ResultsIn this study, we cloned the sesame POLYKETIDE SYNTHASE A (SiPKSA) and examined its function in male sterility. SiPKSA was specifically expressed in sesame flower buds, and its expression was significantly higher in sterile sesame anthers than in fertile anthers during the tetrad and microspore development stages. Furthermore, overexpression of SiPKSA in Arabidopsis caused male sterility in transgenic plants. Ultrastructural observation showed that the pollen grains of SiPKSA-overexpressing plants contained few cytoplasmic inclusions and exhibited an abnormal pollen wall structure, with a thicker exine layer compared to the wild type. In agreement with this, the expression of a set of sporopollenin biosynthesis-related genes and the contents of their fatty acids and phenolics were significantly altered in anthers of SiPKSA-overexpressing plants compared with wild type during anther development.ConclusionThese findings highlighted that overexpression of SiPKSA in Arabidopsis might cause male sterility through defective pollen wall formation. Moreover, they suggested that SiPKSA modulates vibrant pollen development via sporopollenin biosynthesis, and a defect in its regulation may induce male sterility. Therefore, genetic manipulation of SiPKSA might promote hybrid breeding in sesame and other crop species.

Overexpression of Two CCCH-type Zinc-Finger Protein Genes Leads to Pollen Abortion in Brassica campestris ssp. chinensis.

The pollen grains produced by flowering plants are vital for sexual reproduction. Previous studies have shown that two CCCH-type zinc-finger protein genes in Brassica campestris, BcMF30a and BcMF30c, are involved in pollen development. Due to their possible functional redundancy, gain-of-function analysis is helpful to reveal their respective biological functions. Here, we found that the phenotypes of BcMF30a and BcMF30c overexpression transgenic plants driven by their native promoters were similar, suggesting their functional redundancy. The results showed that the vegetative growth was not affected in both transgenic plants, but male fertility was reduced. Further analysis found that the abortion of transgenic pollen was caused by the degradation of pollen contents from the late uninucleate microspore stage. Subcellular localization analysis demonstrated that BcMF30a and BcMF30c could localize in cytoplasmic foci. Combined with the studies of other CCCH-type genes, we speculated that the overexpression of these genes can induce the continuous assembly of abnormal cytoplasmic foci, thus resulting in defective plant growth and development, which, in this study, led to pollen abortion. Both the overexpression and knockout of BcMF30a and BcMF30c lead to abnormal pollen development, indicating that the appropriate expression levels of these two genes are critical for the maintenance of normal pollen development.

A rice GDSL esterase/lipase protein (GELP) is required for anther and pollen development

Male reproductive development is vital for crop seed production. However, the mechanism underlying remains largely unknown. Here, we isolated a male sterile mutant osgelp34-1 in rice. Cytological analysis indicated that the mutant exhibited delayed degradation of anther wall layers and abnormal pollen development, which resulted in the production of shrunk and non-viable pollen grains. MutMap and CRISPR/Cas9 analysis confirmed that a point mutation in OsGELP34 is responsible for the male sterile phenotype. OsGELP34 is highly expressed in reproductive tissues and encodes a putative GDSL lipase. OsGELP34 protein is located to the endoplasmic reticulum (ER) and conserved in land plants. Collectively, our findings elaborated that OsGELP34 plays a vital role in rice male reproduction and has potential applications in rice hybrid breeding.

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis.

Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology. We used knock-down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana. Knock-down of RPS9M in the rps9m-3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m-3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m-3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS. Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

Identification and gene cloning of a male‐sterile mutant Oswbc11 in rice

AbstractCommercial application of nuclear genetic male sterility has been improved recently by a novel technique, Seed Production Technology (SPT), which incorporates transgenic maintainer lines capable of propagating non‐transgenic nuclear male‐sterile lines for use as female parents in hybrid production. Here, we identified a rice nuclear male‐sterile mutant, Oswbc11, with abnormal pollen development and lipid transport. We finely mapped the Oswbc11 gene into a 12.5‐kb region on chromosome 10 and found one candidate gene, which had a base substitution (C to T) resulting in a premature stop codon and was functionally confirmed by CRISPR/Cas9 technology. Gene OsWBC11 encodes a sub‐family member of adenosine triphosphate‐binding cassette transporter, which participates in the active transport of a wide range of molecules across membranes. Moreover, the agronomic traits of Oswbc11 mutants showed no significant differences compared to the wild‐type control except for the seed setting rate. These results indicated that Oswbc11 gene could be used in rice hybrid breeding as a recessive nuclear male‐sterile gene combined with CRISPR/Cas9 and the SPT technology and applied in different rice varieties.

Transcriptomic signature reveals mechanism of flower bud distortion in witches\u2019-broom disease of soybean (Glycine max)

BackgroundSoybean (Glycine max L. Merril) crop is major source of edible oil and protein for human and animals besides its various industrial uses including biofuels. Phytoplasma induced floral bud distortion syndrome (FBD), also known as witches’ broom syndrome (WBS) has been one of the major biotic stresses adversely affecting its productivity. Transcriptomic approach can be used for knowledge discovery of this disease manifestation by morpho-physiological key pathways.ResultsWe report transcriptomic study using Illumina HiSeq NGS data of FBD in soybean, revealing 17,454 differentially expressed genes, 5561 transcription factors, 139 pathways and 176,029 genic region putative markers single sequence repeats, single nucleotide polymorphism and Insertion Deletion. Roles of PmbA, Zn-dependent protease, SAP family and auxin responsive system are described revealing mechanism of flower bud distortion having abnormalities in pollen, stigma development. Validation of 10 randomly selected genes was done by qPCR. Our findings describe the basic mechanism of FBD disease, right from sensing of phytoplasma infection by host plant triggering molecular signalling leading to mobilization of carbohydrate and protein, phyllody, abnormal pollen development, improved colonization of insect in host plants to spread the disease. Study reveals how phytoplasma hijacks metabolic machinery of soybean manifesting FBD.ConclusionsThis is the first report of transcriptomic signature of FBD or WBS disease of soybean revealing morphological and metabolic changes which attracts insect for spread of disease. All the genic region putative markers may be used as genomic resource for variety improvement and new agro-chemical development for disease control to enhance soybean productivity.

Meiotic Defects and Premature Tapetal Degeneration Are Involved in the Low Fertility of Oncidesa Gower Ramsey, an Important Cut-flower Orchid

The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

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.

Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth.

Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.

Discovery, utilization and perspective of Honglian cytoplasmic male sterile rice

Hybrid rice has been researched and utilized in China for 50 years. Three mainly cytoplasmic male sterile rice are wild abortive (WA), boro-II (BT), and Honglian (HL). Honglian (HL) type hybrid rice has made important contributions to the development of hybrid rice by combining scientific research, application research and industrial utilization. We identified Honglian cytoplasmic male sterile (HL-CMS) gene, orfH79 , which located downstream of atp6 in mitochondrial genome. CMS RNA, atp6-orfH79 , were translated into a cytotoxic protein, ORFH79. Data showed ORFH79 interacts with P61, which is a subunit of complex III of mitochondrial electron transfer chain. This interaction may impair energy production and leads to the accumulation of reactive oxygen species (ROS) in mitochondria, consequently resulting in abnormal pollen development. Furthermore, we cloned two restorer of fertility ( Rf ) genes, Rf5 and Rf6 , both of which are PPR genes. Neither RF5 nor RF6 binds to CMS RNA, atp6-orfH79 , directly. RF5 interacts with glycine rich protein 162 (GRP162) and RFC3 (restoration of fertility complex subunit 3) thus forming a restoration of fertility complex (RFC) to process CMS RNA. GRP162 can form homodimer and bind to CMS RNA with two binding sites directly. RCF3 is transported into the mitochondria as a membrane protein and functions as a scaffold protein for the assembly of the RFC complex. The RFC complex was detected with a size ranging from 400 to 500 kD. Moreover, Rf6 encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3 to 5. Results show RF6 is also targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS RNA, atp6-orfH79 , to restore the fertility of CMS line. To create more available germplasm in hybrid rice, we developed a molecular marker of orfH79 to screen wild rice and bred some novel CMS lines. We also developed molecular markers of Rf5 and Rf6 to breed novel stronger restore lines which contain two Rf genes. Consequently, we bred several combinations such as Honglian you 6, Yueyou 938, Luoyou 8 and Luoyou 9348 with high yield, good quality, multiple resistance and wide adaptation. HL hybrid rice has been widely planted in Yangzte river areas and South China with the yield of 10 to 12 tons per hectare. HL hybrid rice combinations also have been planted in Southeast Asian countries, such as Vietnam, Indonesia, Malaysia, Bangladesh and Phillippine. Most combinations exhibited 140% or more of yield compared with the local rice cultivars in Southeast Asian countries. HL hybrid rice even more is becoming the leading hybrid rice and priority choice in most of Asian countries and other Africa countries.

Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis.

Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2β revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock-out mutants of atipk2α and atipk2β were indistinguishable from the wild type, whereas the atipk2α atipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2α atipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.