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TaHPPR Gene in Wheat: Cloning and Expression Analysis
Hao Xiaocong, Wang Weiwei, Zhang Fengting, Sun Rui, Fang Zhaofeng, Liu Shan, Cao Zhishen, Zhu Wengen, Zhao Changping, Wang Dezhou, Tang Yimiao
TaHPPR Gene in Wheat: Cloning and Expression Analysis
To identify the role of hydroxyphenylpyruvate reductase (HPPR) in wheat stress, this study used homologous cloning to obtain a wheat HPPR gene which named TaHPPR. At the same time, the expressions of HPPR gene in tissues and under stress were analyzed. ‘Taiyuan 806’, ‘Xiaobaimai’, ‘Jingdong 8’ and ‘Jing 411’ were used as materials. We performed RT-qPCR analysis to determine TaHPPR gene expression levels in tissue and under stress. Sequence analysis showed that the TaHPPR gene contained a complete open reading frame of 975 bp, and encoded 324 amino acids. TaHPPR protein contained a NAD-binding domain structure. In addition, phylogenetic analysis indicated that TaHPPR gene was in the same branch of Triticum dicoccoides and their genetic relationships were extremely familiar. Expression profiling revealed that TaHPPR expressed in roots, spikelets (excluding stamens), and leaf sheaths, of which the expression level in roots was the highest. The expression of TaHPPR gene decreased under cold, drought, high salt and ABA stress treatment, In the high-salt resistance variety ‘Jingdong 8’, TaHPPR gene expression increased, while it was suppressed in the sensitive variety ‘Xiaobaimai’ and the moderate sensitive variety ‘Jing411’. The results of subcellular localization show that TaHPPR protein is mainly expressed in mitochondria, and the overexpression of TaHPPR gene may increase the salt tolerance in wheat. This study clarifies the expression characteristics of TaHPPR under adversity stress response and salt stress treatment in different varieties, and provides new gene resources and new ideas for studying the molecular mechanism of wheat resistance breeding.
wheat / hydroxyphenylpyruvate reductase / abiotic stress / salt resistance / subcellular localization {{custom_keyword}} /
表1 留树保鲜对油甘氨基酸的影响 mg/g |
氨基酸 | 11月 | 1月 | 4月 |
---|---|---|---|
半胱氨酸/(mg/g) | 12.54±2.93a | 17.32±1.88b | 24.14±1.04c |
谷氨酸/(mg/g) | 1.66±0.01a | 1.91±0.23b | 2.05±0.28c |
赖氨酸/(mg/g) | 7.87±0.08a | 8.14±0.16a | 12.40±0.95b |
脯氨酸/(mg/g) | 0.21±0.01a | 0.37±0.02b | 0.51±0.01c |
羟脯氨酸/(μg/g) | 66.64±6.90a | 89.31±4.87b | 104.05±18.24c |
注:同行数据后小写英文字母不同表示差异显著,P<0.05,下同。 |
表2 留树保鲜对油甘糖类的影响 mg/g |
糖类 | 11月 | 1月 | 4月 |
---|---|---|---|
总糖 | 119.16±4.40a | 98.35±6.47b | 78.34±3.97c |
蔗糖 | 42.34±1.83a | 37.61±2.94b | 33.59±4.52c |
还原糖 | 58.67±4.81a | 44.85±3.76b | 31.12±0.88c |
表3 留树保鲜对油甘营养成分的影响 |
营养成分 | 11月 | 1月 | 4月 |
---|---|---|---|
水溶性果胶 | 1.87±0.18a | 1.86±0.18a | 1.81±0.05a |
还原型抗坏血酸 | 6.05±0.18a | 7.80±0.11b | 7.43±0.23b |
单宁 | 5.08±0.07a | 3.92±0.26b | 3.33±0.48b |
纤维素 | 95.28±8.45a | 80.41±2.99b | 78.02±3.38b |
表4 留树保鲜对油甘功能性成分的影响 |
功能性成分 | 11月 | 1月 | 4月 |
---|---|---|---|
总抗氧化能力/(μmol/mg) | 5.92±0.37a | 2.75±0.22b | 1.85±0.05c |
原花青素/(mg/g) | 7.80±0.11a | 4.21±0.09b | 3.91±0.15b |
类黄酮/(mg/g) | 34.21±1.84a | 17.46±0.97b | 9.33±0.48c |
总酚/(mg/g) | 58.02±3.38a | 25.57±0.75b | 12.91±0.07c |
表5 留树保鲜对油甘酶活性的影响 |
酶活性 | 11月 | 1月 | 4月 |
---|---|---|---|
碱性磷酸酶/[μmol/(min·g)] | 50.64±1.58a | 61.51±1.09b | 72.73±3.06c |
羧酸酯酶/(U/g) | 77.62±6.39a | 89.43±4.21b | 91.06±9.38b |
超氧化物歧化酶/(U/g) | 166.40±21.27a | 180.56±11.09b | 186.72±9.23c |
过氧化物酶/(U/g) | 1020.44±91.48a | 1333.57±72.64b | 1539.67±86.22c |
多酚氧化酶/(U/g) | 27.68±4.12a | 20.79±3.01b | 19.08±2.63b |
酸性磷酸酶/[μmol/(min·g)] | 4.72±0.01a | 1.78±0.03b | 1.16±0.04b |
过氧化氢酶/[nmol/(min·g)] | 291.15±26.22a | 202.28±18.49b | 162.35±18.23c |
乙酰胆碱酯/[nmol/(min·g)] | 20.58±4.36a | 11.37±3.27b | 7.35±2.14c |
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The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.
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In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.
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Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.
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Tyrosine serves as a precursor to several types of plant natural products of medicinal or nutritional interests. Hydroxyphenylpyruvate reductase (HPPR), which catalyzes the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), has been shown to be the key enzyme in the biosynthesis of rosmarinic acid (RA) from tyrosine and, so far, HPPR activity has been reported only from the RA-accumulating plants. Here, we show that HPPR homologs are widely distributed in land plants. In Arabidopsis thaliana, which does not accumulate RA at detectable level, two homologs (HPPR2 and HPPR3) are functional in reducing pHPP. Phylogenetic analysis placed HPPR2 and HPPR3 in two separate groups within the HPPR clade, and HPPR2 and HPPR3 are distinct from HPR1, a peroxisomal hydroxypyruvate reductase (HPR). In vitro characterization of the recombinant proteins revealed that HPPR2 has both HPR and HPPR activities, whereas HPPR3 has a strong preference for pHPP, and both enzymes are localized in the cytosol. Arabidopsis mutants defective in either HPPR2 or HPPR3 contained lower amounts of pHPL and were impaired in conversion of tyrosine to pHPL. Furthermore, a loss-of-function mutation in tyrosine aminotransferase (TAT) also reduced the pHPL accumulation in plants. Our data demonstrate that in Arabidopsis HPPR2 and HPPR3, together with TAT1, constitute to a probably conserved biosynthetic pathway from tyrosine to pHPL, from which some specialized metabolites, such as RA, can be generated in specific groups of plants. Our finding may have broad implications for the origins of tyrosine-derived specialized metabolites in general.
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The tapetum plays an important role in anther development by providing necessary enzymes and nutrients for pollen development. However, it is difficult to identify tapetum-specific genes on a large-scale because of the difficulty of separating tapetum cells from other anther tissues. Here, we reported the identification of tapetum-specific genes by comparing the gene expression patterns of four male sterile (MS) lines of Brassica oleracea. The abortive phenotypes of the four MS lines revealed different defects in tapetum and pollen development but normal anther wall development when observed by transmission electron microscopy. These tapetum displayed continuous defective characteristics throughout the anther developmental stages. The transcriptome from flower buds, covering all anther developmental stages, was analyzed and bioinformatics analyses exploring tapetum development-related genes were performed. We identified 1,005 genes differentially expressed in at least one of the MS lines and 104 were non-pollen expressed genes (NPGs). Most of the identified NPGs were tapetum-specific genes considering that anther walls were normally developed in all four MS lines. Among the 104 NPGs, 22 genes were previously reported as being involved in tapetum development. We further separated the expressed NPGs into different developmental stages based on the MS defects. The data obtained in this study are not only informative for research on tapetum development in B. oleracea, but are also useful for genetic pathway research in other related species.
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Wheat is the most widely adapted crop to abiotic stresses and considered an excellent system to study stress tolerance in spite of its genetic complexity. Recent studies indicated that several hundred genes are either up- or down-regulated in response to stress treatment. To elucidate the function of some of these genes, an interactome of proteins associated with abiotic stress response and development in wheat was generated using the yeast two-hybrid GAL4 system and specific protein interaction assays. The interactome is comprised of 73 proteins, generating 97 interactions pairs. Twenty-one interactions were confirmed by bimolecular fluorescent complementation in Nicotiana benthamiana. A confidence-scoring system was elaborated to evaluate the significance of the interactions. The main feature of this interactome is that almost all bait proteins along with their interactors were interconnected, creating a spider web-like structure. The interactome revealed also the presence of a
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碱性亮氨酸拉链(basic leucine zipper, bZIP)是植物中广泛存在的一类转录因子,参与多种胁迫响应与生长发育过程。本研究从小麦(Triticum aestivum)中克隆到一个热胁迫诱导的bZIP家族转录因子基因TabZIP28 (GenBank登录号: KT753298.1),ORF长度为1 713 bp,编码570个氨基酸。生物信息学分析结果表明,TabZIP28与拟南芥bZIP家族转录因子中B亚组的3个基因AtbZIP17、AtbZIP28 和AtbZIP49归为一类。氨基酸序列比对结果表明,该蛋白具有bZIP和跨膜结构域(transmembrane domain, TMD)两个保守结构域以及规范的位点1蛋白酶(site 1 protease, S1P)剪切位点。对该基因起始密码子ATG上游1 699 bp的序列进行顺式作用元件分析,发现该基因的启动子区域包含众多激素和逆境胁迫响应元件。通过qRT-PCR对该基因在逆境胁迫下的表达模式进行分析,结果表明,TabZIP28在热胁迫处理1 h即上调表达且达到最大值;用20% PEG 6000模拟干旱环境处理小麦幼苗后,TabZIP28在处理6 h达到最大值,并在12 h时急剧下降;对5 mmol/L H2O2处理响应比较缓慢,在处理12 h才上调表达;该基因不受到二硫苏糖醇(dithiothreitol, DTT)处理的诱导表达。在拟南芥(Arabidopsis thaliana)中过量表达TabZIP28基因,转基因株系在高温胁迫后的成活率和种子发芽率较野生型明显提高,说明该基因可能对植物的耐热性有贡献,可以作为耐热性育种的候选基因。
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