Hai-Jun Zhao

Mutations of the multi-drug resistance-associated protein ABC transporter gene 5 result in reduction of phytic acid in rice seeds

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is important to the nutritional quality of cereal and legume seeds. PA and its salts with micronutrient cations, such as iron and zinc, cannot be digested by humans and non-ruminant animals, and hence may affect food/feed nutritional value and cause P pollution of groundwater from animal waste. We previously developed a set of low phytic acid (LPA) rice mutant lines with the aim of increasing the nutritional quality of rice. Two of these lines, Os-lpa-XS110-2 (homozygous non-lethal) Os-lpa-XS110-3 (homozygous lethal), contain two mutant alleles of a LPA gene (hereafter XS-lpa2-1 and XS-lpa2-2, respectively). In this study, we mapped the XS-lpa2-1 gene to a region on chromosome 3 between microsatellite markers RM14360 and RM1332, where the rice orthologue (OsMRP5) of the maize lpa1 gene is located. Sequence analysis of the OsMRP5 gene revealed a single base pair change (C/G–T/A transition) in the sixth exon of XS-lpa2-1 and a 5-bp deletion in the first exon of XS-lpa2-2. OsMRP5 is expressed in both vegetative tissues and developing seeds, and the two mutations do not change the level of RNA transcription. A T-DNA insertion line, 4A-02500, in which OsMRP5 was disrupted, also showed the same high inorganic phosphorus phenotype as Os-lpa-XS110-3 and appeared to be homozygous lethal. PA is significantly reduced in Os-lpa-XS110-2 (~20%) and in 4A-02500 (~90%) seeds compared with their wild type lines, and no PA was detected in Os-lpa-XS110-3 using HPLC analysis. This evidence indicates that the OsMRP5 gene plays an important role in PA metabolism in rice seeds.

Gene identification and allele-specific marker development for two allelic low phytic acid mutations in rice (Oryza sativa L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is an important anti-nutritional component in cereal and legume grains. PA forms of phosphorus (P) and its salts with micronutrient cations, such as iron and zinc, are indigestible in humans and non-ruminant animals, and hence could affect food/feed nutritional value and cause P pollution of ground water from animal waste. We previously developed a set of low phytic acid (LPA) rice mutants with the aim to increase their nutritional quality. Among them, one line, i.e., Os-lpa-XQZ-1 (hereafter lpa 1-2), was identified to have a mutation allelic to the KBNT lpa 1-1 mutation (hereafter lpa 1-1), which was already delimited to a 47-kb region on chromosome 2. In this study, we searched the candidate gene for these two allelic LPA mutations using T-DNA insertion mutants, mutation detection by CEL I facilitated mismatch cleavage, and gene sequencing. The TIGR locus LOC_Os02g57400 was revealed as the candidate gene hosting these two mutations. Sequence analysis showed that the lpa 1-1 is a single base pair substitution mutation, while lpa 1-2 involves a 1,475-bp fragment deletion. A CAPS marker (LPA1_CAPS) was developed for distinguishing the lpa 1-1 allele from lpa 1-2 and WT alleles, and InDel marker (LPA1_InDel) was developed for differentiating the lpa 1-2 allele from lpa 1-1 and WT ones. Analysis of two populations derived from the two mutants with wild-type varieties confirmed the complete co-segregation of these two markers and LPA phenotype. The LOC_Os02g57400 is predicted to encode, through alternative splicing, four possible proteins that are homologous to the 2-phosphoglycerate kinase reported in hyperthermophilic and thermophilic bacteria. The identification of the LPA gene and development of allele-specific markers are of importance not only for breeding LPA varieties, but also for advancing genetics and genomics of phytic acid biosynthesis in rice and other plant species.

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.

Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application

The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.摘要目的分析CYP81A6基因在苯达松及甲磺隆处理下的诱导表达模式, 解释该基因与两种除草剂代谢相关的可能原因。创新点从两种除草剂降解途径中产生的小分子物质的结构相似性出发, 通过基因诱导表达的特点分析, 解释CYP81A6和两种除草剂降解相关的原因。方法通过实时定量聚合酶链反应(PCR)来分析基因表达的特点; 利用CYP81A6启动子与GUS报告基因构建的载体来分析组织特异性表达; 通过亚细胞定位来确定CYP81A6发挥功能的场所。结论CYP81A6基因受苯达松及甲磺隆诱导, 在不同的时间点开始上调, 说明了甲磺隆的降解中间产物可以诱导这个基因的表达; CYP81A6是组成型表达, 在根、 茎、 叶中均有表达; 亚细胞定位结果证明CYP81A6是一个内质网上的蛋白。

Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants

中文概要目 的揭示转基因水稻全基因组遗传变异的特征与频率。创新点通过单核苷酸分辨率揭示了农杆菌介导法转化水 稻植株全基因组水平遗传变异的类型和频率以 及外源DNA 的整合模式。方 法应用Illumina Hiseq2000 高通量测序技术测定了 5 个T0 代转基因水稻株系的基因组序列。结合生 物信息学分析和聚合酶链反应(PCR)扩增, 以 及Sanger 测序, 我们检测和验证单核苷酸多态 性(SNP)和Indel 变化类型和数量, 转移DNA (T-DNA) 及其载体骨架序列和转座子整合位点 及特征, 大片段的结构变异等遗传变异。结 论结果表明, 农杆菌介导的水稻遗传转化, 除T-DNA 整合到水稻基因组外, 还存在载体骨架整合现 象; 每个转基因水稻基因组的变异(SNP 和小片 段的缺失插入)数目为338–1774, 与报道的组 培过程中产生的变异类似; 转基因水稻基因组仅 存在Tos17 转座子数量的变化, 未检测到其他转 座子数目和位置的变化。

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。