Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

PINOID regulates floral organ development by modulating auxin transport and interacts with MADS16 in rice.

In rice (Oryza sativa L.), floral organ development is an important trait. Although a role for PINOID in regulating floral organ development was reported recently, the underlying molecular mechanism remains unclear. Here, we isolated and characterized an abnormal floral organ mutant and mapped the causative gene through an improved MutMap method. Molecular study revealed that the observed phenotype is caused by a point mutation in OsPINOID (OsPID) gene; therefore, we named the mutation as ospid-4. Our data demonstrate that OsPID interacts with OsPIN1a and OsPIN1b to regulate polar auxin transport as shown previously. Additionally, OsPID also interacts with OsMADS16 to regulate transcription during floral organ development in rice. Together, we propose a model that OsPID regulates floral organ development by modulating auxin polar transport and interaction with OsMADS16 and/or LAX1 in rice. These results provide a novel insight into the role of OsPID in regulating floral organ development of rice, especially in stigma development, which would be useful for genetic improvement of high-yield breeding of rice.

Gnp4/LAX2, a RAWUL protein, interferes with the OsIAA3-OsARF25 interaction to regulate grain length via the auxin signaling pathway in rice.

Grain length is one of the determinants of yield in rice and auxin plays an important role in regulating it by mediating cell growth. Although several genes in the auxin pathway are involved in regulating grain length, the underlying molecular mechanisms remain unclear. In this study we identify a RING-finger and wd40-associated ubiquitin-like (RAWUL) domain-containing protein, Gnp4/LAX2, with a hitherto unknown role in regulation of grain length by its influence on cell expansion. Gnp4/LAX2 is broadly expressed in the plant and subcellular localization analysis shows that it encodes a nuclear protein. Overexpression of Gnp4/LAX2 can significantly increase grain length and thousand-kernel weight. Moreover, Gnp4/LAX2 physically interacts with OsIAA3 and consequently interferes with the OsIAA3-OsARF25 interaction in vitro and in vivo. OsIAA3 RNAi plants consistently exhibit longer grains, while the mutant osarf25 has small grains. In addition, OsARF25 binds to the promoter of OsERF142/SMOS1, a regulator of organ size, and positively regulates its expression. Taken together, the results reveal that Gnp4/LAX2 functions as a regulator of grain length through participation in the OsIAA3-OsARF25-OsERF142 pathway and that it has potential value for molecular breeding in rice.

OsLG3 contributing to rice grain length and yield was mined by Ho-LAMap.

BACKGROUND: Most agronomic traits in rice are complex and polygenic. The identification of quantitative trait loci (QTL) for grain length is an important objective of rice genetic research and breeding programs. RESULTS: Herein, we identified 99 QTL for grain length by GWAS based on approximately 10 million single nucleotide polymorphisms from 504 cultivated rice accessions (Oryza sativa L.), 13 of which were validated by four linkage populations and 92 were new loci for grain length. We scanned the Ho (observed heterozygosity per locus) index of coupled-parents of crosses mapping the same QTL, based on linkage and association mapping, and identified two new genes for grain length. We named this approach as Ho-LAMap. A simulation study of six known genes showed that Ho-LAMap could mine genes rapidly across a wide range of experimental variables using deep-sequencing data. We used Ho-LAMap to clone a new gene, OsLG3, as a positive regulator of grain length, which could improve rice yield without influencing grain quality. Sequencing of the promoter region in 283 rice accessions from a wide geographic range identified four haplotypes that seem to be associated with grain length. Further analysis showed that OsLG3 alleles in the indica and japonica evolved independently from distinct ancestors and low nucleotide diversity of OsLG3 in indica indicated artificial selection. Phylogenetic analysis showed that OsLG3 might have much potential value for improvement of grain length in japonica breeding. CONCLUSIONS: The results demonstrated that Ho-LAMap is a potential approach for gene discovery and OsLG3 is a promising gene to be utilized in genomic assisted breeding for rice cultivar improvement.

SLOW WALKER2, a NOC1/MAK21 homologue, is essential for coordinated cell cycle progression during female gametophyte development in Arabidopsis.

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.

The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator in plant cells.

Turgor pressure plays pivotal roles in the growth and movement of walled cells that make up plants and fungi. However, the molecular mechanisms regulating turgor pressure and the coordination between turgor pressure and cell wall remodelling for cell growth remain poorly understood. Here, we report the characterization of Arabidopsis TurgOr regulation Defect 1 (TOD1), which is preferentially expressed in pollen tubes and silique guard cells. We demonstrate that TOD1 is a Golgi-localized alkaline ceramidase. tod1 mutant pollen tubes have higher turgor than wild type and show growth retardation both in pistils and in agarose medium. In addition, tod1 guard cells are insensitive to abscisic acid (ABA)-induced stomatal closure, whereas sphingosine-1-phosphate, a putative downstream component of ABA signalling and product of alkaline ceramidases, promotes closure in both wild type and tod1. Our data suggest that TOD1 acts in turgor pressure regulation in both guard cells and pollen tubes.

[A study on introduction of chitinase gene and beta-1,3-glucanase gene into restorer line of dian-type hybrid rice (Oryza sativa L.) and enhanced resistance to blast (Magnaporthe grisea)].

Plasmid pBLGC containing chitinase gene from Phaseolus limensis and beta-1,3-glucanase gene from Nicotiana tabacum was bombarded into the restorer line "Nan29" of Dian-type hybrid rice (Oryza sativa L. ssp. japonica) from Yunnan province of South-west China. 93 regenerants were obtained from the calli that were resistant to G418 (100 to 150 mg/L) on NB medium. Using beta-1,3 glucanase gene as the probe, 17 of the regenerants were identified to be transgenic lines by dot blotting and the foreign genes construction were integrated into the genomes of T1 lines by Southern blotting hybridization. Two foreign genes were inherited stably to T4 generation according to PCR results of the lines. The resistance to rice blast of six transgenic lines were evaluated by inoculating four violent biological races of Magnaporthe grisea from Yunnan province and inducing the disease in the field. The results indicated that the resistance to rice blast of transgenic lines were enhanced to varying degrees compared with the receptor line and the transgenic lines could be used in rice blast resistant breeding.

[The germinating characters of the transgenic rice seeds in the stress condition of antibiotic G418 and their application in crop breeding].

The seeds of transgenic rice line D2-1-2 and the receptor cultivar Zhonghua No.9 were germinated on the stress condition of the antibiotic G418. The number of taking root seed, the length of root and the length of shoot of two used materials were checked in different concentrations of the antibiotic G418, but the ratio of germinating seed was not affected. At the 100 mg/L level of G418, the transgenic line D2-1-2 could take longer root (mean 1.45 cm) but Zhonghua No.9 very short (mean 0.27 cm). 88.46% of the total long-root (<0.5 cm) seeds selected from the mixing population of D2-1-2 and Zhonghua No.9 at the 100 mg/L level of antibiotic G418 were real transgenic ones.

Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III.

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by beta-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.

EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice.

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.