Peptide Transporter OsNPF8.1 Contributes to Sustainable Growth under Salt and Drought Stresses,and Grain Yield under Nitrogen Deficiency in Rice

Peptide transport is important for plant tissues where rapid proteolysis occurs, especially during germination and senescence, to enhance redistribution of organic nitrogen (N). However, the biological role of peptide transporters is poorly investigated in rice. We characterized the function of the peptide transporter OsNPF8.1 of rice nitrate transporter 1/peptide transporter family (NPF). Ectopic expression of OsNPF8.1 in yeast revealed that OsNPF8.1 encoded a high-affinity di-/tri-peptide transporter, and the osnpf8.1 mutants had a lower uptake rate of the fluorescent-labelled dipeptide c in leaves of rice seedlings. Histochemical assays showed that OsNPF8.1 was highly expressed in mesophyll cells and vascular parenchyma cells, but not detected in root hairs and epidermises. Expression of OsNPF8.1 was induced by N deficiency, drought, NaCl and abscisic acid, and kept at a high level in senescing leaves. Under N deficiency conditions, compared with the wild type Zhonghua 11, the osnpf8.1 mutants grew slower at the seedling stage, and had lower grain yield and lower N content in the grains. In contrast, OsNPF8.1-over-expressing rice (OsNPF8.1-OE) grew faster at the seedling stage and had a higher grain yield. The osnpf8.1 seedlings were less tolerant to salt and drought stresses. These results suggested that stress-induced organic N transportation mediated by OsNPF8.1 might contribute to balance plant growth and tolerate to salt/drought stress and N-deficiency.     

Glucose Uptake and Oxidative Stress in Caco-2 Cells: Health Benefits from Posidonia oceanica (L.) Delile

Posidonia oceanica (L.) Delile is an endemic Mediterranean marine plant of extreme ecological importance. Previous in vitro and in vivo studies have demonstrated the potential antidiabetic properties of P. oceanica leaf extract. Intestinal glucose transporters play a key role in glucose homeostasis and represent novel targets for the management of diabetes. In this study, the ability of a hydroalcoholic P. oceanica leaf extract (POE) to modulate intestinal glucose transporters was investigated using Caco-2 cells as a model of an intestinal barrier. The incubation of cells with POE significantly decreased glucose uptake by decreasing the GLUT2 glucose transporter levels. Moreover, POE had a positive effect on the barrier integrity by increasing the Zonulin-1 levels. A protective effect exerted by POE against oxidative stress induced by chronic exposure to high glucose concentrations or tert-butyl hydroperoxide was also demonstrated. This study highlights for the first time the effect of POE on glucose transport, intestinal barrier integrity, and its protective antioxidant effect in Caco-2 cells. These findings suggest that the P. oceanica phytocomplex may have a positive impact by preventing the intestinal cell dysfunction involved in the development of inflammation-related disease associated with oxidative stress.     

Positive Regulation of Phytochrome B on Chlorophyll Biosynthesis and Chloroplast Development in Rice

Phytochromes in rice are encoded by a gene family composed of three members,PHYA,PHYB,and PHYC.Through characterizing the phytochrome mutants and wild type (WT) in terms of photomorphogenesis,roles of individual phytochromes have been preliminarily explored in regulating rice de-etiolation,flowering time and fertility.However,little information has been reported about whether or how phytochromes affect chlorophyll biosynthesis and chloroplast development in rice.In this study,we compared the chlorophyll contents of wild type and the phyA,phyB and phyAphyB mutants grown under either white light (WL) or red light (R).The results suggest that phyB perceives R to positively regulate chlorophyll biosynthesis,while the role of phyA can be detected only in the phyB-deficient mutant.Analyses of the expression levels of genes involved in chlorophyll biosynthesis revealed that phytochromes affected the chlorophyll biosynthesis by regulating protochlorophyll oxidoreductase A (PORA) expression.The role of phyB in chloroplast development was also analyzed,and the results suggest that phyB perceives R to regulate chloroplast development by affecting the numbers of chloroplasts and grana,as well as the chloroplast membrane system.     

Mechanisms for coping with submergence and waterlogging in rice

Background

Oligopeptide transporters (OPTs) play important roles in the mobilization of organic nitrogenous compounds and usually associate with tissues that show signs of rapid protein hydrolysis, such as germinating seeds and senescing leaves. This study is to investigate rice OPT genes.

Results

A total of sixteen OsOPT genes (Os for Oryza sative L.) were identified in the rice genome, which were then classified into six sections that belong to two subfamilies (the PT and YSL subfamily). The major mechanisms for evolutionary expansion of the sixteen genes during the rice genome evolution include segmental and tandem duplication. Calculation of the duplication event dates indicated that the sixteen genes originated from nine original OsOPT genes, and the duplication events could be classified into three evolutionary stages. The first evolutionary stage occurred approximately 50 million years ago (Mya) and involved the evolution of four new genes. The second evolutionary stage was approximately 20 Mya and was marked by the appearance of two new genes, and the third evolutionary stage was approximately 9 Mya when two new genes evolved. Mining of the expression database and RT-PCR analysis indicated that the expression of most duplicated OsOPT genes showed high tissue specificities. Diverse expression patterns for the sixteen genes were evaluated using both semi-quantitative RT-PCR and the MPSS data. Expression levels of some OsOPT genes were regulated by abiotic and biotic stresses suggesting the potential involvement of these gene products in rice stress adaptation. Five OsOPT gene mutants showed abnormal development and growth, the primary analysis of five OsOPT gene mutants suggested that they may be necessary for rice development.

Conclusions

These results suggested that rice-specific OsOPT genes might be potentially useful in improving rice.     

Screening of Rice Genes Interacting with p5b of Rice BlackStreaked Dwarf Virus

Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus,family Reoviridae.Its genome has ten double-stranded RNA (dsRNA) segments (S1-S10),in which the fifth genome segment (S5) contains two open reading frames (ORFs) with a partially overlapping region.The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function.To reveal the function of p5b,its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay.The bait protein p5b was detected in yeast by western blot,and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast.Then the bait protein p5b was used for screening the cDNA expression libraries of rice.Gene fragments of some pivotal enzymes involved in photosynthesis,respiration and other important metabolic processes,were identified to interact with p5b in yeast,suggesting that these interactions may play roles in symptom development in infected plants.     

寒地冬小麦TaEXPA7同源基因的遗传转化及其功能分析

膨胀素是一类具有非酶活性的细胞壁松弛蛋白,广泛参与植物生长的各个阶段,其表达受非生物胁迫如低温等调控。为了探究寒地冬小麦(Triticum aestivum)膨胀素基因 TaEXPA7-A/B/D的功能,本研究将 TaEXPA7-A/B/D三个基因分别转入拟南芥(Arabidopsis thaliana)中,利用抗性筛选和PCR鉴定,分别获得了过表达 TaEXPA7-A/B/D三个基因的转基因拟南芥植株,分别观察并测定了 TaEXPA7-A/B/D三个基因对转基因拟南芥生长和低温胁迫下生理指标的影响。结果表明,转基因拟南芥的根长、侧根数目、株高、开花后2周内的花序和角果数目均显著高于野生型;4℃低温处理后,转基因植株中抗氧化酶SOD、POD和CAT的活性及可溶性糖的含量相比于野生型均维持在较高的水平,且MDA的积累量较低。以上结果表明, TaEXPA7-A/B/D三个基因的过表达显著促进了转基因拟南芥的生长,并使植株在低温条件下维持更好的抗氧化和渗透调节能力。     

Constitutive expression of <Emphasis Type="Italic">REL1</Emphasis> confers the rice response to drought stress and abscisic acid

Leaf rolling is one of the most significant symptoms of drought stress in plant. Previously, we identified a dominant negative mutant, termed rolled and erect 1 (hereafter referred to rel1-D), regulating leaf rolling and erectness in rice. However, the role of REL1 in drought response is still poorly understood. Here, our results indicated that rel1-D displayed higher tolerance to drought relative to wild type, and the activity of superoxide dismutase (SOD) and drought responsive genes were significantly up-regulated in rel1-D. Moreover, our results revealed that rel1-D was hypersensitive to ABA and the expression of ABA associated genes was significantly increased in rel1-D, suggesting that REL1 likely coordinates ABA to regulate drought response. Using the RNA-seq approach, we identified a large group of differentially expressed genes that regulate stimuli and stresses response. Consistently, we also found that constitutive expression of REL1 alters the expression of biotic and abiotic stress responsive genes by the isobaric tags for relative and absolute quantification (iTRAQ) analysis. Integrative analysis demonstrated that 8 genes/proteins identified by both RNA-seq and iTRAQ would be the potential targets in term of the REL1-mediated leaf morphology. Together, we proposed that leaf rolling and drought tolerance of rel1-D under normal condition might be caused by the endogenously perturbed homeostasis derived from continuous stressful dynamics.     

Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by γ-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel) markers C1-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene D10 within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the D10 allele in gsor23 revealed that the base cytosine (C) at the 404^th position in the coding region was deleted, which would cause frameshift mutation after the 134^th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8), a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs). After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene D10 was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.     

QTL Analysis for Grain Iron and Zinc Concentrations in Two O. nivara Derived Backcross Populations

Identification of quantitative trait loci (QTLs) for grain mineral elements can assist in faster and more precise development of micronutrient dense rice varieties through marker-assisted breeding. In the present study, QTLs were mapped for Fe and Zn concentrations in two BC₂F₃ mapping populations derived from the crosses of O. sativa cv Swarna with two different accessions of O. nivara. In all, 10 and 8 QTLs were identified for grain Fe and Zn concentrations in population 1, and 7 and 5 QTLs were identified in population 2, respectively. Eighty percent of the QTLs detected in both populations were derived from O. nivara. Five QTLs for Fe and three QTLs for Zn explained more than 15% phenotypic variance either in interval or composite interval mapping. The locations of O. nivara derived QTLs such as qFe2.1, qFe3.1, qFe8.2 and qZn12.1 were consistently identified in both the populations. Epistatic interaction was observed only between RM106 and RM6 on chromosome 2 and between RM22 and RM7 on chromosome 3 for Fe concentration in population 1. Sixteen candidate genes for metal homeostasis were found to co-locate with 10 QTLs for Fe and Zn concentrations in both the populations. Most of the Fe and Zn QTLs were found to co-locate with QTLs for grain yield and grain quality traits. Some of the major effect QTLs identified can be used to improve rice grain Fe and Zn concentrations.     

水稻中的磷转运蛋白基因在异源表达系统中的功能分析

为了更好地研究植物在磷素吸收过程中的分子机制以及生物化学过程的变化,将水稻中分离得到的一个磷转运蛋白基因（OsPT6）用于互补实验。互补实验结果表明,OsPT6能够与缺失磷转运功能的酵母突变体实现互补,并在低磷条件下促进酵母突变体对磷的吸收。进一步分析表明OsPT6属于水稻Pht1家族运输蛋白基因,所编码的蛋白对磷酸盐（Pi）的吸收Km值为96 μmol/L,属于高亲和的磷转运蛋白。不同的酵母转化子对不同pH环境的响应实验显示,OsPT6是一个与质子相偶联的磷运输蛋白,其吸收磷素的最佳pH为6.0。对OsPT6在人的胚胎肾细胞（HEK293）中的表达分析表明,该基因能够编码蛋白并定位于细胞膜,证明OsPT6的功能与酵母磷转运子PHO84相似,是一个定位于细胞膜上的具有吸收转运磷素作用的运输蛋白。     

小麦TaHKT家族基因的生物信息学分析

高亲和性钾离子转运蛋白（high-affinity K⁺ transporter，HKT）是植物体内一种非常重要的离子转运蛋白，具有运输Na⁺/K⁺的能力，在植物响应盐胁迫过程中发挥重要作用。为系统了解小麦TaHKT家族基因，挖掘有效的小麦耐盐基因，本研究采用生物信息学的方法，对小麦TaHKT家族基因进行了全基因组分析，鉴定了家族成员，构建了系统进化树,并对其跨膜结构域、染色体定位、基因结构、Motif、上游顺式作用元件、共线性以及表达谱等进行了分析。结果鉴定出23个TaHKT基因，其编码蛋白质长度为443~590 aa，等电点范围8.2~10.4，跨膜结构域为6~8个。23个基因分布于小麦的2、4、6、7号染色体上，根据亲缘关系可将其分为3个亚族，同一亚族的成员具有较为相似的Motif组成和基因结构。23个基因中，发现了4对串联重复基因，10对大片段复制基因，具有良好的共线性。顺式作用元件分析发现，大部分TaHKT成员含有盐胁迫响应元件MYB、G-box、ABRE和DRE。表达谱分析发现，TaHKT基因在小麦的16种组织中均有表达，在根、茎中的表达量较高，其中TraesCS4D02G361300（ID,下同）在根中的表达量最高。在盐胁迫处理后，不同成员对盐胁迫响应程度不同，TraesCS7B02G318400在盐胁迫处理后表达量逐渐降低；TraesCS2B02G451400和TraesCS2D02G428200在盐胁迫处理后的表达量也明显降低；TraesCS2B02G451800在根部几乎不表达，但在受到盐胁迫处理后表达量逐渐提高，推测它们在小麦抵御盐胁迫过程中发挥不同作用。     

The ATP-binding cassette (ABC) transporter OsABCG3 is essential for pollen development in rice

Background

The pollen wall, which protects male gametophyte against various stresses and facilitates pollination, is essential for successful reproduction in flowering plants. The pollen wall consists of gametophyte-derived intine and sporophyte-derived exine. From outside to inside of exine are tectum, bacula, nexine I and nexine II layers. How these structural layers are formed has been under extensive studies, but the molecular mechanisms remain obscure.

Results

Here we identified two osabcg3 allelic mutants and demonstrated that OsABCG3 was required for pollen development in rice. OsABCG3 encodes a half-size ABCG transporter localized on the plasma membrane. It was mainly expressed in anther when exine started to form. Loss-function of OsABCG3 caused abnormal degradation of the tapetum. The mutant pollen lacked the nexine II and intine layers, and shriveled without cytoplasm. The expression of some genes required for pollen wall formation was examined in osabcg3 mutants. The mutation did not alter the expression of the regulatory genes and lipid metabolism genes, but altered the expression of lipid transport genes.

Conclusions

Base on the genetic and cytological analyses, OsABCG3 was proposed to transport the tapetum-produced materials essential for pollen wall formation. This study provided a new perspective to the genetic regulation of pollen wall development.

     

Phytochromes are Involved in Elongation of Seminal Roots and Accumulation of Dry Substances in Rice Seedlings

Phytochromes have been reported to play important roles in seedling de-etiolation and flowering in rice.To identify the roles of phytochromes in regulating root growth and accumulation of dry substances,the lengths of seminal roots and the dry weights of seedlings were measured in the wild type as well as the phytochrome A(phyA) and phytochrome B(phyB) mutants grown under different conditions.When the whole seedlings were exposed to white light,the elongation of the seminal roots was significantly photoinhibited in the wild type,whereas this inhibitory effect was clearly reduced in the phyA and phyB mutants.When the roots of the seedlings were blocked from white light,the phyA and phyB mutants exhibited significantly longer seminal roots than the wild type.These results suggest that both the root-localized and shoot-localized PHYA and PHYB are involved in the photoinhibition of seminal root elongation in rice seedlings.By measuring the dry weights of roots and shoots,it is revealed that PHYB positively regulates the accumulation of dry substances in shoots,however,PHYA exerts the contrary effects on the accumulation of dry substances in roots and shoots of rice seedlings.