大豆耐铝毒候选基因GmSTOP1的克隆与表达分析

酸性土壤中的铝毒害是限制作物生长和产量的主要因素之一。拟南芥中的At STOP1(Arabidopsis thaliana sensitive to proton rhizotoxicity 1)是一个调控多种铝毒耐受机制相关基因表达的转录因子,在拟南芥耐铝毒中发挥重要作用。为研究大豆中STOP1-like基因的表达特性,本研究利用RT-PCR从耐铝毒大豆品种科丰1号中克隆了一个位于第16染色体的STOP1-like基因,命名为Gm STOP1。该基因的编码区(coding DNA sequence,CDS)序列长度为1566 bp,编码521个氨基酸。在Gm STOP1起始密码子上游1500 bp的核苷酸序列区间预测到多种顺式作用元件,包括与激素、热、逆境响应等相关的应答元件,如ABRE、HSE、TC-rich重复序列等。蛋白质结构预测表明Gm STOP1不具有跨膜结构和信号肽,含有4个保守的Cys-2-His-2锌指蛋白结构域。系统进化分析显示Gm STOP1与菜豆(Phaseolus vulgaris)中的STOP1-like蛋白亲缘关系较近。亚细胞定位结果显示Gm STOP1定位于细胞核,说明Gm STOP1蛋白可能在细胞核中发挥其功能。Gm STOP1基因在种子中的相对表达量最高,在根、茎尖分生组织、茎、叶、花、荚等多种组织中也均有表达。用25μmol L–1 Al Cl3溶液处理大豆幼苗,Gm STOP1基因在根中上调表达,24 h达到最高相对表达量,约为对照(0μmol L–1 Al Cl3)的9.2倍,表明该基因的表达受铝离子的诱导。此外,ABA、Na Cl和PEG等胁迫也能诱导大豆根和叶中Gm STOP1基因的上调表达。由此推测Gm STOP1基因可能参与大豆对铝毒、高盐和渗透等非生物胁迫的应答过程。

葡萄耐铝毒基因STOP1的克隆与表达分析

【目的】STOP1是植物耐铝毒的重要基因,为探明葡萄STOP1基因的结构特征及其表达量与其耐铝性的关系。【方法】以耐铝性弱的山葡萄(Vitis amurensis)和耐铝性强的小叶葡萄(V.sinocinerea)的叶片cDNA为模板,采用PCR和TA克隆技术获得VaSTOP1和VsSTOP1两基因序列,对其进行生物信息学分析和铝胁迫下的表达分析,并测定铝胁迫下的膜脂过氧化指标,对2个种STOP1基因的表达和膜脂过氧化物质的变化与耐铝性的关系进行比较。【结果】克隆获得的VaSTOP1和VsSTOP1基因序列长度分别为1782和1800 bp,分别编码594和600个氨基酸,相对分子量分别为67.03和67.48 kD,理论等电点为5.21和6.42;二者的二级结构中均有无规则卷曲>α-螺旋>折叠延伸链的规律,由α-螺旋、β-折叠片、β-转角和锌指结构等组成三级空间结构;进化树研究得出,VaSTOP1与VsSTOP1的同源性高达96%,二者与CcSTOP1、PnSTOP1、NtSTOP1、ErSTOP1的同源性均在80%以上。VaSTOP1基因表达量在铝胁迫28 d内呈先上升后下降的规律,较早出现峰值,而VsSTOP1基因表达量则稳定上升,这与二者的膜脂过氧化物质的变化规律基本一致。【结论】葡萄的耐铝性与其STOP1基因的表达量呈正相关,研究为进一步探明葡萄耐铝毒的分子调控机制提供了基因资源。

The MEKK1-MKK1/2-MPK4 cascade phosphorylates and stabilizes STOP1 to confer aluminum resistance in Arabidopsis

Aluminum(Al)toxicity can seriously restrict crop production on acidic soils,which comprise 40%of the world’s potentially arable land.The zinc finger transcription factor STOP1 has a conserved and essential function in mediating plant Al resistance.Al stress induces STOP1 accumulation via post-transcriptional regulatory mechanisms.However,the upstream signaling pathway involved in Al-triggered STOP1 accumulation remains unclear.Here,we report that the MEKK1-MKK1/2-MPK4 cascade positively regulates STOP1 phosphorylation and stability.Mutations of MEKK1,MKK1/2,or MPK4 lead to decreased STOP1 stability and Al resistance.Al stress induces the kinase activity of MPK4,which interacts with and phosphorylates STOP1.The phosphorylation of STOP1 reduces its interaction with the F-box protein RAE1 that mediates STOP1 degradation,thereby leading to enhanced STOP1 stability and Al resistance.Taken together,our results suggest that the MEKK1-MKK1/2-MPK4 cascade is important for Al signaling and confers Al resistance through phosphorylation-mediated enhancement of STOP1 accumulation in Arabidopsis.

STOP1介导多种逆境响应分子机制的研究进展

STOP1 (sensitive to proton rhizotoxicity 1)是一个在植物多种胁迫耐受机制中发挥重要调控作用的转录因子。酸性土壤条件下,STOP1能调控植物根系对质子和铝毒害的耐受性。在营养逆境下,STOP1参与调控植物根系对土壤磷和氮的利用效率。在土壤盐碱条件下,STOP1能赋予根系耐盐性;然而在水分胁迫下,STOP1则会抑制植物耐旱性。此外,STOP1通过调控地上部生理响应促进植物对缺氧胁迫的耐受能力。因此,探究STOP1调控不同胁迫下植物性状的分子机制,对于提高作物同时抗多种逆境能力具有重要意义。

STOP2 Activates Transcription of Several Genesfor AI- and Low pH-Tolerance that Are Regulatedby STOP1 in Arabidopsis

The zinc-finger protein STOP1 （sensitive to proton rhizotoxicity 1） regulates transcription of multiple genescritical for tolerance to aluminum （AI） and low pH in Arabidopsis. We evaluated the contributions of genes that are sup-pressed in the stop1 mutant to AI- and low pH-tolerance using T-DNA-inserted disruptants, and transgenic stop1 mutantsexpressing each of the suppressed genes. STOP2, a STOP1 homolog, partially recovered AI- and low pH-tolerance byrecovering the expression of genes regulated by STOP1. Growth and root tip viability under proton stress were partiallyrescued in the STOP2-complemented line. STOP2 localized in the nucleus and regulated transcription of two genes （PGIP1and PGIP2） associated with cell wall stabilization at low pH. GUS assays revealed that STOP1 and STOP2 showed similarcellular expression in the root. However, the expression level of STOP2 was much lower than that of STOP1. In a STOP1promoter：：STOP2-complemented line, AI tolerance was slightly recovered, concomitant with the recovery of expressionof ALS3 （aluminum sensitive 3） and AtMATE （Arabidopsis thaliana multidrug and toxic compound extrusion）, while theexpression of AtALMT1 （aluminum-activated malate transporter 1） was not recovered. These analyses indicated thatSTOP2 is a physiologically minor isoform of STOP1, but it can activate expression of some genes regulated by STOP1.

A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis

Phosphorus(P)is an indispensable macronutrient required for plant growth and development.Natural phosphate(Pi)reserves are finite,and a better understanding of Pi utilization by crops is therefore vital for worldwide food security.Ammonium has long been known to enhance Pi acquisition efficiency in agriculture;however,the molecular mechanisms coordinating Pi nutrition and ammonium remains unclear.Here,we reveal that ammonium is a novel initiator that stimulates the accumulation of a key regulatory protein,STOP1,in the nuclei of Arabidopsis root cells under Pi deficiency.We show that Pi deficiency promotes ammonium uptake mediated by AMT1 transporters and causes rapid acidification of the root surface.Rhizosphere acidification-triggered STOP1 accumulation activates the excretion of organic acids,which help to solubilize Pi from insoluble iron or calcium phosphates.Ammonium uptake by AMT1 transporters is downregulated by a CIPK23 protein kinase whose expression is directly modulated by STOP1 when ammonium reaches toxic levels.Taken together,we have identified a STOP1-centered regulatory network that links external ammonium with efficient Pi acquisition from insoluble phosphate sources.These findings provide a framework for developing possible strategies to improve crop production by enhancing the utilization of non-bioavailable nutrients in soil.

SIZ1 negatively regulates aluminum resistance by mediating the STOP1–ALMT1 pathway in Arabidopsis

Sensitive to proton rhizotoxicity 1(STOP1) functions as a crucial regulator of root growth during aluminum(Al) stress. However, how this transcription factor is regulated by Al stress to affect downstream genes expression is not well understood. To explore the underlying mechanisms of the function and regulation of STOP1, we employed a yeast two hybrid screen to identify STOP1-interacting proteins. The SUMO E3 ligase SIZ1, was found to interact with STOP1 and mainly facilitate its SUMO modification at K40 and K212 residues. Simultaneous introduction of K40 R and K212 R substitutions in STOP1 enhances its transactivation activity to upregulate the expression of aluminum-activated malate transporter 1(ALMT1)via increasing the association with mediator 16(MED16) transcriptional co-activator. Loss of function of SIZ1 causes highly increased expression of ALMT1, thus enhancing Al-induced malate exudation and Al tolerance. Also, we found that the protein level of SIZ1 is reduced in response to Al stress. Genetic evidence demonstrates that STOP1/ALMT1 is epistatic to SIZ1 in regulating root growth response to Al stress. This study suggests a mechanism about how the SIZ1–STOP1–ALMT1 signaling module is involved in root growth response to Al stress.

STOP1 regulatory system: Centered on multiple stress tolerance and cellular nutrientmanagement

Transcriptional regulation plays a crucial role in plant adaptation to diverse environments.Several transcription factors(TFs),the so-called master switch TFs or hub TFs,regulate various genes critical for adaptation to different stresses.STOP1(SENSITIVE TO PROTON RHIZOTOXICITY 1),a zinc-finger TF of Arabidopsis(Arabidopsis thaliana),is one such master/hub TF that transcriptionally regulates multiple stress tolerance(Sadhukhan et al.,2021).STOP1 plays a critical role in tolerance to acid soil syndrome(i.e.,H+and Al3+tolerance)(Iuchi et al.,2007)and hypoxia tolerance(Enomoto et al.,2019),and negatively regulates drought tolerance(Sadhukhan et al.,2019).In addition,Tian et al.

Involvement of cytokinins in STOP1-mediated resistance to proton toxicity

STOP1(sensitive to proton rhizotoxicity1)is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH(i.e.,proton)stresses in Arabidopsis.However,the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear.Here,we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities.Interestingly,the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type.Significantly,low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses.Moreover,lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins.Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root.Furthermore,the expression of critical genes involved in cytokinin biosynthesis,biodegradation,and signaling is altered in the stop1 mutant in response to low-pH stresses.In conclusion,our results reveal a complex network of resistance to low-pH stresses,which involves coordinated actions of STOP1,cytokinins,and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.

Brassinosteroid signaling regulates phosphate starvation-induced malate secretion in plants

Inorganic phosphate(Pi)is often limited in soils due to precipitation with iron(Fe)and aluminum(Al).To scavenge heterogeneously distributed phosphorus(P)resources,plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe-P or Al-P oxides.In this study,we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE-RESISTANT 1(BZR1)expression in Arabidopsis thaliana.Exogenous 2,4-epibrassinolide treatment or constitutive activation of BZR1(in the bzr1-D mutant)significantly reduced primary root growth inhibition under Pi-starvation conditions by downregulating ALUMINUM-ACTIVATED MALATE TRANSPORTER 1(ALMT1)expression and malate secretion.Furthermore,At BZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1(STOP1)on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis.The ratio of nuclear-localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis.In addition,BZR1-inhibited malate secretion is conserved in rice(Oryza sativa).Our findings provide insight into plant mechanisms for optimizing the secretion of malate,an important carbon resource,to adapt to Pi-deficiency stress.

Plant adaptation to low phosphorus availability:Core signaling,crosstalks,and applied implications

Phosphorus(P)is an essential nutrient for plant growth and reproduction.Plants preferentially absorb P as orthophosphate(Pi),an ion that displays low solubility and that is readily fixed in the soil,making P limita-tion a condition common to many soils and Pi fertilization an inefficient practice.To cope with Pi limitation,plants have evolved a series of developmental and physiological responses,collectively known as the Pi starvation rescue system(PSR),aimed to improve Pi acquisition and use efficiency(PUE)and protect from Pi-starvation-induced stress.Intensive research has been carried out during the last 20 years to un-ravel the mechanisms underlying the control of the PSR in plants.Here we review the results of this research effort that have led to the identification and characterization of several core Pi starvation signaling components,including sensors,transcription factors,microRNAs(miRNAs)and miRNA inhibitors,kinases,phosphatases,and components of the proteostasis machinery.We also refer to recent results revealing the existence of intricate signaling interplays between Pi and other nutrients and antagonists,N,Fe,Zn,and As,that have changed the initial single-nutrient-centric view to a more integrated view of nutrient homeostasis.Finally,we discuss advances toward improving PUE and future research priorities.