Yuan-Yuan Tan

Disruption of OsSULTR3;3 reduces phytate and phosphorus concentrations and alters the metabolite profile in rice grains.

Two low phytic acid (lpa) mutants have been developed previously with the aim to improve the nutritional value of rice (Oryza sativa) grains. In the present study, the impacts of lpa mutations on grain composition and underlying molecular mechanisms were investigated. Comparative compositional analyses and metabolite profiling demonstrated that concentrations of both phytic acid (PA) and total phosphorus (P) were significantly reduced in lpa brown rice, accompanied by changes in other metabolites and increased concentrations of nutritionally relevant compounds. The lpa mutations modified the expression of a number of genes involved in PA metabolism, as well as in sulfate and phosphate homeostasis and metabolism. Map-based cloning and complementation identified the underlying lpa gene to be OsSULTR3;3. The promoter of OsSULTR3;3 is highly active in the vascular bundles of leaves, stems and seeds, and its protein is localized in the endoplasmic reticulum. No activity of OsSULTR3;3 was revealed for the transport of phosphate, sulfate, inositol or inositol 1,4,5 triphosphate by heterologous expression in either yeast or Xenopus oocytes. The findings reveal that OsSULTR3;3 plays an important role in grain metabolism, pointing to a new route to generate value-added grains in rice and other cereal crops.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis

Rice is one of the world’s most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.Serotonin, a well-known neurotransmitter in animals, is also produced by plants. In rice, biosynthesis of serotonin through the cytochrome P450 CYP71A1 gene increases the susceptibility of plants by promoting growth of destructive insects.

Stability of the Metabolite Signature Resulting from the OsSULTR3;3 Mutation in Low Phytic Acid Rice (Oryza sativa L.) Seeds upon Cross-breeding

The low phytic acid ( lpa) rice ( Oryza sativa L.) mutant Os-lpa-MH86-1, resulting from the mutation of the putative sulfate transporter gene OsSULTR3;3, was crossed with a commercial rice cultivar. The obtained progenies of generations F4 to F7 were subjected to a nontargeted metabolite profiling approach allowing the analyses of a broad spectrum of lipophilic and hydrophilic low-molecular-weight constituents. The metabolite profiles of the homozygous lpa progenies were characterized not only by a decreased concentration of phytic acid but also by increased contents of constituents from various classes, such as sugars, sugar alcohols, amino acids, phytosterols, and biogenic amines. Statistical assessments of the data via multivariate and univariate approaches demonstrated that this mutation-induced metabolite signature was nearly unaffected by the cross-breeding step and consistently expressed over several generations. The data demonstrate that even for complex metabolic changes resulting from a mutation, cross-breeding can be employed as a tool to generate progeny rice seeds stably exhibiting the mutation induced traits.

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa1 in rice

The OsLpa1 gene (LOC_Os02g57400) was identified to be involved in phytic acid (PA) metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa1 in rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter::GUS transgenic plants (GUS: β-glucuronidase); GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts (OsLpa1.1, OsLpa1.2, OsLpa1.3) are produced due to alternative splicing; quantitative reversetranscriptase polymerase chain reaction (RT-PCR) analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa1.2 in all tissues. OsLpa1.2 is predominant in germinating seeds (about 5 times that of OsLpa1.1), but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa1.2 in both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF (day after flowering) seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein (GFP) in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.中文概要目 的揭示水稻低植酸基因OsLpa1 的分子生物学特征, 特别是深化对其可变剪切和表达的时空和组织 特征, 以及蛋白亚细胞定位的认识。创新点确定了OsLpa1 存在的三种剪切方式, 明确了三 种转录本在不同组织和发育时期丰度的变化; 揭 示了OsLpa1 表达的组织和时空差异, 确定其在 根、种子糊粉层细胞和花丝中高度表达; 明确了 三种转录本编码的蛋白均定位于亚叶绿体。方 法通过培育OsLpa1 启动子与β-葡萄糖醛酸糖苷酶 (GUS)杂合基因的转基因植株, 通过不同组织 的GUS 组织化学染色确定OsLpa1 表达的组织特 异性; 通过设计特异性引物确定OsLpa1 存在的 转录方式, 采用实时荧光定量聚合酶链式反应 (PCR)分析三种转录本在不同组织和发育时期 的丰度; 采用OsLpa1 三种转录本与绿色荧光蛋 白(GFP)基因构建杂合基因并在水稻原生质体 中的瞬时表达, 在共聚焦显微镜下观察蛋白的亚 细胞定位。结 论OsLpa1 在根、茎、叶和花丝有强烈的表达。它存 在三种可变剪切方式, 产生的三种转录本存在明 显的时空和组织差异, 但其编码的蛋白均定位于 叶绿体。