Isolation and functional analysis of MxNAS3 involved in enhanced iron stress tolerance and abnormal flower in transgenic Arabidopsis

Metal trace elements, such as Fe, Zn, and Mn, are necessary micronutrients required by all plants. In this study, the MxNAS3 gene was cloned from Malus xiaojinensis and MxNAS3 was localized in the cytoplasmic membrane. The expression level of MxNAS3 in root and new leaf was higher than in mature leaf and phloem, which was greatly influenced by high and low Fe stresses, IAA and ABA treatments in M. xiaojinensis. Over-expression of MxNAS3 in transgenic Arabidopsis thaliana contributed to enhanced Fe stress tolerance, as well as higher levels of root length, fresh weight, concentrations of chlorophyll, nicotianamine, Fe, Zn, and Mn, especially under high and low Fe stresses. More importantly, it was the first time for us to find that higher expression of MxNAS3 in transgenic A. thaliana contributed to misshappen flowers. Moreover, the MxNAS5-OE A. thaliana had increased expression levels of flowering-related genes (AtYSL1, AtYSL3, AtAFDL, AtAP1, ATMYB21, and AtSAP).     

Genomic mosaicism underlies the adaptation of marine Synechococcus ecotypes to distinct oceanic iron niches

Phytoplankton are limited by iron (Fe) in ~40% of the world's oceans including high-nutrient low-chlorophyll (HNLC) regions. While low-Fe adaptation has been well-studied in large eukaryotic diatoms, less is known for small, prokaryotic marine picocyanobacteria. This study reveals key physiological and genomic differences underlying Fe adaptation in marine picocyanobacteria. HNLC ecotype CRD1 strains have greater physiological tolerance to low Fe congruent with their expanded repertoire of Fe transporter, storage and regulatory genes compared to other ecotypes. From metagenomic analysis, genes encoding ferritin, flavodoxin, Fe transporters and siderophore uptake genes were more abundant in low-Fe waters, mirroring paradigms of low-Fe adaptation in diatoms. Distinct Fe-related gene repertories of HNLC ecotypes CRD1 and CRD2 also highlight how coexisting ecotypes have evolved independent approaches to life in low-Fe habitats. Synechococcus and Prochlorococcus HNLC ecotypes likewise exhibit independent, genome-wide reductions of predicted Fe-requiring genes. HNLC ecotype CRD1 interestingly was most similar to coastal ecotype I in Fe physiology and Fe-related gene content, suggesting populations from these different biomes experience similar Fe-selective conditions. This work supports an improved perspective that phytoplankton are shaped by more nuanced Fe niches in the oceans than previously implied from mostly binary comparisons of low- versus high-Fe habitats and populations.     

中间锦鸡儿WRKY75基因对拟南芥耐受盐和ABA能力的影响

该研究从旱生灌木中间锦鸡儿中克隆得到1个CiWRKY75基因。序列分析显示,CiWRKY75开放阅读框长570bp,编码189个氨基酸,含有1个WRKYGQK基序和1个C2H2型锌指结构,属于第二类WRKY转录因子。亚细胞定位显示,CiWRKY75定位于细胞核。实时荧光定量PCR检测表明,CiWRKY75基因的表达受盐胁迫和ABA诱导。在拟南芥中过量表达CiWRKY75后,与野生型拟南芥相比,转基因株系种子的萌发率在盐胁迫下降低,并且对盐胁迫的耐受能力明显减弱;ABA处理下,2个转基因株系的种子萌发率(10.3%、9.6%)较野生型(25.9%)明显降低。研究表明,CiWRKY75是中间锦鸡儿对盐和ABA响应的重要调控因子。     

一株耐盐甲基杆菌Methylobacterium sp.W-1的分离及促生潜能研究

[背景] 多种甲基杆菌属细菌对寄主植物有促生作用,其分布区域较广。筛选具有耐盐与促生特性的甲基杆菌属菌株可为微生物菌肥的开发提供依据。[目的] 从新疆乌尔禾地区盐渍土壤中筛选耐盐促生菌,对其培养基成分进行优化及促生能力进行研究,为微生物菌肥的开发提供依据。[方法] 采用阿须贝无氮培养基筛选耐盐菌株,对菌株进行基因序列分析及生理生化测定,采用平板试验法初步研究该菌对拟南芥的生长影响。[结果] 筛选出中度耐盐菌株W-1,经鉴定为甲基杆菌属（Methylobacteriumsp.）。菌株生长最佳无机盐为NaCl,最适浓度为1%–3%,最高耐受浓度达7%。最佳氮源为酸水解酪蛋白,产生长素最高达33.53 mg/L。溶磷能力达28.71 mg/L。菌株W-1接种拟南芥幼苗后叶绿素a和叶绿素b含量均高于对照组,同时对其根系发育有显著的促进作用。[结论] 菌株W-1促生性能显著,可为生物肥料制备提供菌种资源。     

Iron deficiency stress can induce MxNAS1 protein expression to facilitate iron redistribution in Malus xiaojinensis

• Iron (Fe) is a vital trace element in plants, and deficiency of this element in apple trees can reduce fruit quality. Nicotianamine (NA) is known to play an important role in Fe transport and endogenous hormone balance. In the present study, we investigated the role of a nicotianamine synthase 1 gene (MxNas1) in an apple species, Malus xiaojinensis, that has a more Fe‐efficient genotype than other apple species and ecotypes.
• To characterise the response of M. xiaojinensis to Fe deficiency, we used quantitative Q‐PCR to determine the level of expression of MxNas1 and Western blot to measure protein levels. Immunohistochemical staining and GFP fluorescence localisation of the MxNAS1 protein were also carried out. HPLC and polarised absorption spectrophotometry were performed to investigate the effects of overexpression of MxNas1 in order to elucidate the role of MxNAS1 in the cellular uptake of active Fe in tobacco suspension cells.
• We found that MxNas1 expression and protein levels were higher under Fe deficiency stress than under Fe sufficiency. Immunohistochemical staining showed that MxNAS1 was localised mainly in the epidermal and vascular tissues of the roots, vascular tissues of the stem and palisade cells of mature leaves, and in parenchyma cells of young leaves. MxNAS1 was mainly localised in the plasma membranes and vesicles of protoplasts. In addition, overexpression of MxNas1 in stable transgenic tobacco cells increased NA and active Fe content under Fe sufficiency.
• The results suggest that MxNas1 expression in M. xiaojinensis is induced in response to Fe deficiency stress, resulting in higher levels of the protein. MxNAS1 may be involved in the redistribution of Fe in M. xiaojinensis under Fe deficiency.

     

Identification of two phenotypes of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under <Emphasis Type="Italic">in vitro</Emphasis> salt stress conditions

This study describes two phenotypes of Arabidopsis thaliana (ecotype Columbia) developed in vitro under salt stress (75 mM NaCl). The phenotypes 01 and 02 appeared visibly distinguishable by rosette morphology and competence to produce flowers. Phenotype 01, sensible to salt stress, accumulated high quantities of Na⁺, showed a slight reduction in dry mass, and high protein and chlorophyll contents. Moreover, its anatomy exhibited some xeromorphic traits. Phenotype 02, clearly salt tolerant, showed a morphology similar to control plants, displaying typical phyllotactic rosette and flowering stalk production. Accumulation of Na⁺, protein and chlorophyll contents were close to control plants. Reversion experiments on NaCl free MS medium, showed a partially recovered phenotype 01. A threshold salt stress concentration that permits the simultaneous development of two phenotypes, was found.     

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

WRKY gene family evolution in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The Arabidopsis thaliana WRKY proteins are characterized by a sequence of 60 amino acids including WRKY domain. It is well established that these proteins are involved in the regulation of various physiological programs unique to plants including pathogen defense, senescence and response to environmental stresses, which attracts attention of the scientific community as to how this family might have evolved. We tried to satisfy this curiosity and analyze reasons for duplications of these gene sequences leading to their diversified gene actions. The WRKY sequences available in Arabidopsis thaliana were used to evaluate selection pressure following duplication events. A phylogenetic tree was constructed and the WRKY family was divided into five sub-families. After that, tests were conducted to decide whether positive or purified selection played key role in these events. Our results suggest that purifying selection played major role during the evolution of this family. Some amino acid changes were also detected in specific branches of phylogeny suggesting that relaxed constraints might also have contributed to functional divergence among sub-families. Sites relaxed from purifying selection were identified and mapped onto the structural and functional regions of the WRKY1 protein. These analyses will enhance our understanding of the precise role played by natural selection to create functional diversity in WRKY family.     

植物WRKY转录因子在应对盐胁迫中的作用研究进展

土壤中的高含盐量严重限制了植物的生长和作物的产量。植物的许多转录因子在植物逆境胁迫中发挥着重要的作用,但仍有很多转录因子的分子机制目前尚不清楚。WRKY转录因子作为高等植物中最大的转录因子家族之一,参与并影响着植物生长发育的多个方面,在盐胁迫的多种不同响应途径中发挥重要作用。WRKY蛋白对基因表达的调控主要是通过与DNA特定顺式调控元件——W-box元件（TTGACC）的结合来实现的。近年来,从模式植物拟南芥（Arabidopsis）到农作物,已经有许多研究揭示了WRKY家族成员的作用和机制。本文综述了WRKY转录因子在应对盐胁迫方面的最新研究进展,探讨了WRKY转录因子研究目前存在的问题和未来的展望。     

Interactions between iron nutrition and Verticillium wilt resistance in tomato

The relationship between Fe nutritional status and Verticillium wilt disease in tomatoes possessing single gene resistance to Race 1 of Verticillium dahliae was investigated using hydroponic culture media. Iron limiting conditions increased the sensitivity of resistant tomatoes to the pathogen as expressed by wilting and chlorosis. Distance of fungal vascular invasion was approximately the same in both Fe replete and Fe limited treatments. Comparison of near-isolines revealed that the magnitude of disease expressed in Fe deficient Pixie II (resistant) was considerably less than that expressed by the susceptible Pixie variety. Infection of tomato did not enhance the severity of low-Fe stress as quantified by root peroxidase activity and chlorophyll content of young leaves.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.