Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana

MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism.     

SUMO E3 Ligase AtMMS21 Regulates Drought Tolerance in Arabidopsis thaliana

Post-translational modifications of proteins by small ubiquitin-like modifiers (SUMOs) play crucial roles in plant growth and development, and in stress responses. The MMS21 is a newly-identified Arabidopsis thaliana L. SUMO E3 ligase gene aside from the SIZ1, and its function requires further elucidation. Here, we show that MMS21 deficient plants display improved drought tolerance, and constitutive expression of MMS21 reduces drought tolerance. The expression of MMS21 was reduced by abscisic acid (ABA), polyethylene glycol (PEG) or drought stress. Under drought conditions, mms21 mutants showed the highest survival rate and the slowest water loss, and accumulated a higher level of free proline compared to wild-type (WT) and MMS21 over-expression plants. Stomatal aperture, seed germination and cotyledon greening analysis indicated that mms21 was hypersensitive to ABA. Molecular genetic analysis revealed that MMS21 deficiency led to elevated expression of a series of ABA-mediated stress-responsive genes, including COR15A, RD22, and P5CS1 The ABA and drought-induced stress-responsive genes, including RAB18, RD29A and RD29B, were inhibited by constitutive expression of MMS21. Moreover, ABA-induced accumulation of SUMO-protein conjugates was blocked in the mms21 mutant. We thus conclude that MMS21 plays a role in the drought stress response, likely through regulation of gene expression in an ABA-dependent pathway.     

小拟南芥ApZFP基因异源超表达促进拟南芥开花并提高耐逆性

锌指蛋白(ZFP)是一类重要的转录因子, 广泛参与植物的生长发育和非生物胁迫应答。新疆小拟南芥(Arabidopsispumila)又名无苞芥, 是十字花科短命植物, 具有高光效、繁殖力强和适应干旱等生物学特征, 而且比模式植物拟南芥(A.thaliana)更耐高盐胁迫。将前期克隆的小拟南芥锌指蛋白基因ApZFP通过花滴法转化到哥伦比亚生态型拟南芥(Col-0)中,获得了独立表达的转基因株系。表型观察发现, 过量表达ApZFP基因可促使拟南芥在长短日照下均提前开花。实时荧光定量PCR结果显示, 转基因拟南芥株系中, 光周期途径中的CO基因和年龄途径中的SPL基因表达上调; 春化、环境温度和自主途径中的FLC基因表达下调; 编码成花素的基因FT及下游开花相关基因AP1和LFY的表达量均升高。进一步通过盐、干旱和ABA胁迫处理ApZFP转基因株系的种子和幼苗, 发现在胁迫处理下, 与对照相比, 转基因拟南芥种子萌发率较高, 幼苗主根较长。因此推测, ApZFP在植物发育过程中具有多种功能, 可能既参与植物的开花转变过程, 又同其它植物的锌指蛋白基因一样, 参与植物的耐逆过程。     

硫化氢信号能够通过生物钟调控拟南芥对冷胁迫的响应

硫化氢(hydrogen sulfide, H_2S)是继一氧化氮(nitric oxide, NO)与一氧化碳(carbon oxide, CO)之后的第3种气体信号分子,在动植物中均发挥着重要的生理功能。生物钟是生物体的内在计时器,对动植物适应环境和生长发育至关重要。鉴于H_2S与生物钟调控的生理过程有较大的相关性,本文以拟南芥(Arabidopsis thaliana)为实验材料,对二者之间的关系进行了探索。结果发现,外源NaHS(H_2S供体)处理能够上调生物钟相关基因CCA1(circadian clock associated 1)和PRR9(pseudo-response regulator 9)的表达,而且在H_2S生成关键酶编码基因缺失的双突变体lcd/des1中,CCA1与PRR9的峰值表达时间明显滞后。CBFs(c-repeat binding factors)是受CCA1调控的冷胁迫响应基因,其表达也受H_2S的调控。lcd/des1中CBF1和CBF3的峰值表达时间延迟,同时在lcd/des1中CBF1、CBF2和CBF3都下调表达。lcd/des1幼苗对冷胁迫表现出更高的敏感性。本文也对拟南芥内源H_2S生成关键酶L-半胱氨酸脱硫基酶(L-cysteine desulfhydrase, LCD)与脱硫基酶1(desulfhydrase 1, DES1)编码基因的转录水平节律性进行了初步的探索。LCD的表达在1 d内未见明显的变化,而DES1的表达有明显的节律性,在早上8:00达到峰值。综上所述,H_2S能够通过调节CCA1与PRR9基因的表达调控生物钟,进而影响下游靶标CBFs基因的表达以增加拟南芥对冷胁迫的耐受性。     

eATP位于H2S下游参与乙烯诱导的拟南芥气孔关闭过程

以拟南芥(Arabidopsis thaliana)野生型、H₂S合成突变体(Atl-cdes和Atd-cdes)和ABC转运体突变体(Atmrp4、Atmrp5 和Atmrp4/5)为材料, 探讨乙烯诱导气孔关闭过程中eATP与H₂S之间的关系。结果显示, ABC转运体阻断剂格列本脲(Gli)、P2受体抑制剂磷酸吡哆醛-6-偶氮苯基-2',4'-二硫酸(PPADS)和三磷酸腺苷双磷酸酶(Apyrase)可抑制乙烯诱导的气孔关闭乙烯可提高拟南芥幼苗叶片eATP含量及AtMRP4和AtMRP5相对表达量, 但对Atmrp4、Atmrp5和Atmrp4/5突变体幼苗叶片eATP含量和气孔运动没有显著作用。实验结果表明, eATP是乙烯诱导拟南芥气孔关闭过程的重要信号分子, AtMRP4和AtMRP5参与胞内ATP的分泌H₂S清除剂次牛磺酸(HT)能阻遏乙烯诱导的拟南芥幼苗叶片eATP含量的升高乙烯对Atl-cdes、Atd-cdes幼苗叶片eATP含量及AtMRP4和AtMRP5相对表达量无显著影响。据此推测eATP位于H₂S下游参与乙烯诱导的拟南芥气孔关闭过程。     

拟南芥耐盐、耐旱和抗寒的分子机理及其利用

非生物胁迫,尤其是盐分、干旱和寒冷是导致全球作物减产的主要原因.植物对环境胁迫的适应,依赖于与胁迫感知、信号转导、基因表达相关的分子级联网络的激活.因此,保护和维持细胞成分的结构和功能的基因工程可以增强植物对胁迫的耐受性.综述拟南芥对盐分、干旱和寒冷3种主要非生物胁迫因子耐受性的分子机理,以及相关机理和耐逆基因在改良作物耐逆性方面的应用.     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

Hydrogen peroxide-induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.     

过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性

干旱和低温是影响农作物生长发育的重要因素,培育转基因作物是解决此问题的有效途径。冷激蛋白（Cold Shock Proteins,CSPs）是一类高度保守的核酸结合蛋白,参与非生物胁迫应答等细胞生理活动,转CSP基因可增强作物抗逆能力。以海洋微生物宏基因组DNA为模板,采用锚定PCR的方法克隆得到了MbCSP基因,其ORF为216 bp,编码一个由71个氨基酸构成的蛋白;对其进行同源性分析,显示该氨基酸序列与EcCSPG、EcCSPA（大肠杆菌 Escherichia coli）,BsCspB（枯草芽孢杆菌Bacillus subtilis）和BcCspA（蜡样芽孢杆菌 Bacillus cereus）等冷休克蛋白氨基酸序列同源性在60%~90%;对该氨基酸序列进行多重序列比对和系统发育树分析,结果发现MbCSP蛋白包含RNP1（KGFGFI）和RNP2（VFVHF）等CSP蛋白经典的保守结构域,其与EcCspG（大肠杆菌）和CmCspG、CmCspB（堆肥宏基因组）等生物的冷休克蛋白亲缘关系较近。为进一步探讨冷休克蛋白MbCSP的功能,构建了植物表达载体pTF101-MbCSP,采用花序浸染法转化拟南芥,经过除草剂筛选和PCR检测,获得转基因植株。进行半定量RT-PCR分析,选择表达量最高的阳性株系进行后续的生理检测。结果表明：在干旱胁迫及低温胁迫下,转基因拟南芥的生长状况明显优于野生型,其生物量显著高于野生型植株;转基因拟南芥的叶片相对含水量、叶绿素含量和超氧化物歧化酶活性均高于野生型拟南芥,而丙二醛含量则低于野生型拟南芥。上述结果表明,过表达海洋微生物宏基因组MbCSP能够提高转基因拟南芥的抗旱和耐寒能力,为培育转基因作物新品种奠定了基础。     

Biochar Application Improves the Drought Tolerance in Maize Seedlings

Application of biochar to agricultural soils is mostly used to improve soil fertility. Experimental treatments were comprised of two factors: i) drought at two level, i.e., 80% and 40% water holding capacity (WHC) which was maintained on gravimetric basis ii) three levels of biochar i.e., control, 2 t ha^-1 and 4 t ha^-1 added to soil. Experimentation was done to examine potential of biochar application to enhance the growth attributes, water relations, photosynthetic pigments and antioxidants activities in maize (Zea mays L.) seedlings. Results of study revealed that biochar application increased the growth qualities (total seedlings biomass, dry weight of shoot and root, shoot length and root length). In addition; contents of photosynthetic pigments (chlorophyll a, b, a + b and a/b), water relation (relative water contents, turgor potential, osmotic potential and water potential) were improved significantly due to addition of biochar. Addition of 4 t ha^-1 biochar led to significant rise activity of enzymatic antioxidant catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) in leaf of maize seedling sunder drought as well as well watered circumstances. However, biochar applied at the rate 4 t ha^-1 improved the all the physiological and biochemical attributes in maize seedlings under drought. From the results it was concluded that biochar application is an efficient way to alleviate adverse effect of drought stress on maize. In drought prone areas, long term impacts of biochar on production of maize and properties of soil could be recommended as upcoming shove.     

Hydrogen sulfide improves drought resistance in Arabidopsis thaliana

Hydrogen sulfide (H₂S) plays a crucial role in human and animal physiology. Its ubiquity and versatile properties have recently caught the attention of plant physiologists and biochemists. Two cysteine desulfhydrases (CDes), l-cysteine desulfhydrase and d-cysteine desulfhydrase, were identified as being mainly responsible for the degradation of cysteine in order to generate H₂S. This study investigated the expression regulation of these genes and their relationship to drought tolerance in Arabidopsis. First, the expression pattern of CDes in Arabidopsis was investigated. The expression levels of CDes gradually increased in an age-dependent manner. The expression of CDes was significantly higher in stems and cauline leaves than in roots, rosette leaves and flowers. Second, the protective effect of H₂S against drought was evaluated. The expression pattern of CDes was similar to the drought associated genes induced by dehydration, and H₂S fumigation was found to stimulate further the expression of drought associated genes. Drought also significantly induced increased H₂S production, a process that was reversed by re-watering. In addition, seedlings after treatment with NaHS (a H₂S donor) showed a higher survival rate and displayed a significant reduction in the size of the stomatal aperture compared to the control. These findings provide evidence that H₂S, as a gasotransmitter, improves drought resistance in Arabidopsis.     

Large Root Cortical Cell Size Improves Drought Tolerance in Maize

The objective of this study was to test the hypothesis that large cortical cell size (CCS) would improve drought tolerance by reducing root metabolic costs. Maize (Zea mays) lines contrasting in root CCS measured as cross-sectional area were grown under well-watered and water-stressed conditions in greenhouse mesocosms and in the field in the United States and Malawi. CCS varied among genotypes, ranging from 101 to 533 µm². In mesocosms, large CCS reduced respiration per unit of root length by 59%. Under water stress in mesocosms, lines with large CCS had between 21% and 27% deeper rooting (depth above which 95% of total root length is located in the soil profile), 50% greater stomatal conductance, 59% greater leaf CO₂ assimilation, and between 34% and 44% greater shoot biomass than lines with small CCS. Under water stress in the field, lines with large CCS had between 32% and 41% deeper rooting (depth above which 95% of total root length is located in the soil profile), 32% lighter stem water isotopic ratio of ¹⁸O to ¹⁶O signature, signifying deeper water capture, between 22% and 30% greater leaf relative water content, between 51% and 100% greater shoot biomass at flowering, and between 99% and 145% greater yield than lines with small cells. Our results are consistent with the hypothesis that large CCS improves drought tolerance by reducing the metabolic cost of soil exploration, enabling deeper soil exploration, greater water acquisition, and improved growth and yield under water stress. These results, coupled with the substantial genetic variation for CCS in diverse maize germplasm, suggest that CCS merits attention as a potential breeding target to improve the drought tolerance of maize and possibly other cereal crops.Suboptimal water availability is a primary constraint for terrestrial plants and a primary limitation to crop production. In developing countries, the problem of yield loss due to drought is most severe (Edmeades, 2008, 2013), and the problem will be further exacerbated in the future due to climate change (Burke et al., 2009; Schlenker and Lobell, 2010; Lobell et al., 2011a; IPCC, 2014; St. Clair and Lynch, 2010). The development of drought-tolerant crops is therefore an important goal for global agriculture. Breeding for drought adaptation using yield as a selection criterion is generally not efficient, since yield is an integration of complex mechanisms at different levels of organization affected by many elements of the phenotype and the environment interacting in complex and often unknown ways. Trait-based selection or ideotype breeding is generally a more efficient selection strategy, permitting the identification of useful sources of variation among lines that have poor agronomic adaptation, elucidation of genotype-environment interactions, and informed trait stacking (Lynch, 2007; Araus et al., 2008; Richards et al., 2010; Wasson et al., 2012; York et al., 2013; Lynch, 2014).Under drought stress, plants allocate more resources to root growth relative to shoot growth, which can enhance water acquisition (Sharp and Davies, 1979; Palta and Gregory, 1997; Lynch and Ho, 2005). The metabolic costs of soil exploration by root systems are significant and can exceed 50% of daily photosynthesis (Lambers et al., 2002). With a large root system, each unit of leaf area has more nonphotosynthetic tissue to sustain, which may reduce productivity by diverting resources from shoot and reproductive growth (Smucker, 1993; Nielsen et al., 2001; Boyer and Westgate, 2004). Genotypes with less costly root tissue could develop the extensive, deep root systems required to fully utilize soil water resources in drying soil without as much yield penalty. Therefore, root phenes that reduce the metabolic costs of soil exploration, thereby improving water acquisition, are likely to be valuable for improving drought tolerance (Lynch and Ho, 2005; Zhu et al., 2010; Lynch, 2011; Richardson et al., 2011; Jaramillo et al., 2013; Lynch 2014).Maize (Zea mays) is the principal global cereal. Maize production is facing major challenges as a result of the increasing frequency and intensity of drought (Tuberosa and Salvi, 2006), and this problem will likely be exacerbated by climate change (Lobell et al., 2011b). The Steep, Cheap, and Deep ideotype has been proposed for improving water and nitrogen acquisition by maize when these resources are limited (Lynch, 2013). This ideotype consists of root architectural, anatomical, and physiological traits that may increase rooting depth and thereby improve water acquisition from drying soils. Anatomical phenes could influence the metabolic cost of soil exploration by changing the proportion of respiring and nonrespiring root tissue and affecting the metabolic cost of tissue construction and maintenance, which is an important limitation to root growth and plant development under edaphic stress. Specific anatomical phenes that may contribute to rooting depth by reducing root metabolic costs include components of living cortical area (LCA; Jaramillo et al., 2013), including root cortical aerenchyma (RCA), cortical cell size (CCS), and cortical cell file number (Lynch, 2013).RCA consists of large air-filled lacunae that replace living cortical cells as a result of programmed cell death (Evans, 2004). Previous studies have demonstrated that RCA improves crop adaptation to edaphic stress by reducing the metabolic cost of soil exploration and exploitation (Fan et al., 2003; Zhu et al., 2010; Postma and Lynch, 2011a, Saengwilai et al., 2014a). RCA is associated with a disproportionate reduction of root respiration, thereby permitting greater root growth and acquisition of soil resources (Fan et al., 2003; Zhu et al., 2010). SimRoot modeling indicated that RCA can substantially increase the acquisition of nitrogen, phosphorus, and potassium in maize by reducing respiration and the nutrient content of root tissue (Postma and Lynch, 2011b). Under water stress in the field, maize genotypes with more RCA had deeper roots, better leaf water status, and 800% greater yield than genotypes with less RCA (Zhu et al., 2010). Under nitrogen stress in the field and in greenhouse mesocosms, maize genotypes with more RCA had greater rooting depth, greater nitrogen capture from deep soil strata, greater nitrogen content, greater leaf photosynthesis, greater biomass, and greater yield (Saengwilai et al., 2014a).LCA refers to the living portion of the cortex that remains after the formation of aerenchyma (Jaramillo et al., 2013). Recently, we reported that LCA is an important determinant of root metabolic cost and a better predictor of root respiration than RCA (Jaramillo et al., 2013). In that study, maize lines contrasting in LCA were grown under well-watered or water-stressed conditions in soil mesocosms, and LCA was associated with a reduction of specific root respiration. These results provided the impetus to investigate the relative contribution of each component of LCA to metabolic cost. Our focus here is on root CCS.Plant cell size varies substantially both among and within species (Sugimoto-Shirasu and Roberts, 2003). Cell size in a given species and tissue is under genetic control and results from the coordinated control of cell growth and cell division (Sablowski and Carnier Dornelas, 2014). The increased volume of individual cells is attributable to cytoplasmic growth and cell expansion (Marshall et al., 2012; Chevalier et al., 2014). Cytoplasmic growth is the net accumulation of macromolecules and cellular organelles, while cell expansion refers to increased cell volume caused by enlargement of the vacuole (Taiz, 1992; Sablowski and Carnier Dornelas, 2014). Lynch (2013) proposed that large CCS would decrease the metabolic costs of root growth and maintenance, both in terms of the carbon cost of root respiration and the nutrient content of living tissue, by increasing the ratio of vacuolar to cytoplasmic volume.The objective of this study was to test the hypothesis that large CCS would reduce specific root respiration (i.e. respiration per unit of root length), which under water stress would result in greater root growth, greater acquisition of subsoil water, better plant water status, and improved plant growth and yield. Diverse sets of genotypes (including landraces and recombinant inbred lines [RILs]) contrasting for CCS were evaluated under water stress and well-watered conditions in soil mesocosms in controlled environments, in the field in the United States using automated rainout shelters, and in the field in Malawi. Our results demonstrate that substantial variation for CCS exists in maize and that this variation has substantial effects on the metabolic cost of soil exploration and thereby water acquisition under drought.     

拟南芥乙烯突变体在干旱胁迫下的形态学差异

为了揭示乙烯在植物与环境相互作用过程中的生物学功能,以拟南芥（Arabidopsis thaliana）的ein2-5、ein3-1、EIN3ox、EIL1ox 4种乙烯突变体与Col-0野生型为材料,对比研究它们在干旱胁迫条件下的生长和形态学变化。研究发现,干旱胁迫导致莲座叶直径、叶片面积、花序、水势等指标发生显著变化,同时不同突变体的形态适应特点呈现显著差异。这些结果表明,乙烯积极参与了植物形态塑造过程,与植物的抗旱性具有紧密相关性。