拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

ATMYB123和ATKOR1基因参与拟南芥根发育的调控

从拟南芥T-DNA插入突变体库中筛选到2个根发育相关基因ATMYB123和ATKOR1表达缺失的突变体atmyb123和atkor1,通过杂交构建这两个基因表达缺失的双突变体atmyb123/atkor1,以明确这两个基因在根发育中的作用。结果显示:(1)ATMYB123表达缺失突变体atmyb123植株地上部分发育减缓,种皮颜色变黄,而ATKOR1表达缺失突变体atkor1植株在这两方面与其野生型没有明显差异;两基因缺失均显著影响了拟南芥根的发育,根生长受到了严重抑制。(2)双突变体atmyb123/atkor1在植株形态和种皮颜色方面表现出单突变体AT-MYB123的特点,而其根长却介于两单突变体的中间。(3)进一步研究发现,培养基pH改变、NaCl处理、外源GA施用均没有改变突变体根生长趋势,显示这3种因素与两基因缺失突变引起的根发育抑制无关。研究表明,AT-MYB123和ATKOR1基因参与拟南芥根的发育调控,转录因子ATMYB123可能作为主调控因子参与ATKOR1对拟南芥根发育的调控。     

拟南芥株型突变体zpr1的性状特征与基因鉴定分析

对本研究室经T-DNA插入法获得的拟南芥株型突变株系——隐性突变体zpr1植株进行植物学性状调查和遗传分析,并对该突变基因进行鉴定、表达定位和调控元件分析。结果显示:(1)性状分析表明,与野生型拟南芥Ws-2相比,突变体zpr1的茎生叶分枝数量增加,茎生叶分枝发生于拟南芥顶端花序部位;野生型拟南芥茎生叶为披针形,而突变体zpr1没有出现分枝的茎生叶呈倒卵形,出现分枝的茎生叶呈披针型;突变体zpr1的主花序高度、株高、分枝高度和分枝长度都高于野生型,且分枝数多于野生型。(2)利用质粒挽救和反向PCR法(IPCR)确定了ZPR1基因突变发生位置是该基因起始密码子上游426bp处,证明T-DNA插入破坏了ZPR1基因的启动子区域,导致该基因在拟南芥内不能正常表达。(3)基因转录调控区域的顺式作用元件分析发现在ZPR1基因的转录调控区有多个与植物激素相关的调控元件,还有与光周期调节相关的调控元件。(4)亚细胞定位发现,ZPR1基因在所有细胞中的细胞膜中表达,而在部分细胞的细胞膜、细胞质和细胞核中均有表达。研究表明,ZPR1基因的表达对植物株型发育有重要的调控作用,该基因的表达水平受植物激素和光照的调节,最终导致了植物株型的变化。     

拟南芥ANAC055基因突变体对高盐和渗透胁迫的响应

高盐和渗透等非生物胁迫是影响农作物产量和品质的重要因素,非生物胁迫发生时,植物通过体内各类转录因子启动胁迫应答反应,进而降低非生物胁迫对植物的损伤。本研究筛选出植物特异性转录因子ANAC055编码基因的纯合T-DNA插入突变体SALK_152738,测序分析发现T-DNA插在ANAC055基因的3'UTR区域。实时荧光定量PCR结果表明叶中ANAC055基因表达量最高;与野生型相比,突变体叶、茎和花中ANAC055基因表达量分别下降了40%、50%和70%。高盐胁迫后,野生型和突变体叶中ANAC055基因表达量分别比对照上升了320%和55.4%;而渗透胁迫时,该基因叶中的表达量分别比对照下降了47.7%和56.3%;电子表达谱分析发现该基因根中的表达可受高盐和渗透等多种非生物胁迫的诱导表达。高盐和渗透胁迫时野生型和突变体幼根的生长均受到明显抑制,但高盐胁迫对突变体根生长的抑制作用比对野生型根生长的抑制作用更大。上述分析表明拟南芥ANAC055基因可受高盐和渗透等非生物胁迫的诱导表达,并且其在拟南芥幼根的生长发育过程中具有一定的作用,本研究有助于进一步明确其在非生物胁迫过程中的作用。     

拟南芥SPL1基因参与调节低磷条件下的根际酸化反应

根际酸化是植物适应低磷胁迫的重要策略, 但植物是如何感知和转导低磷信号, 进而促进根际酸化的分子机制至今还不十分清楚。利用pH指示剂(溴甲酚紫)显色法从拟南芥(Arabidopsis thaliana) T-DNA插入突变体库中分离得到了1株低磷诱导根际酸化缺失突变体spl1。在含溴甲酚紫的低磷培养基上培养8小时, 野生型拟南芥根际培养基的颜色变为黄色, 而突变体spl1根际培养基的颜色没有明显变化, 表明spl1的低磷根际酸化反应能力降低。当低磷胁迫处理延长20天, spl1叶片的花青素积累明显高于野生型。同时也出现, 即使在磷营养正常条件下, spl1突变体也表现出根毛数量与长度增加的特征。进一步的研究表明, 在低磷条件下, spl1突变体根部的磷含量略高于野生型, 与磷转运相关基因的表达量明显高于野生型。分子遗传学分析结果表明, SPL1基因受低磷胁迫诱导, 主要在拟南芥的叶片和花等组织中表达, 其编码的蛋白广泛分布在细胞的各个部位。以上结果表明, SPL1参与介导低磷诱导的拟南芥根际酸化反应, 调节多种低磷胁迫反应及低磷条件下磷饥饿诱导基因的表达。     

细胞周期后期促进因子DIG9对植物侧根发生的调控研究

在植物体内,细胞周期对于植物的萌发、生长、开花、结实等各个生长发育阶段具有重要作用。细胞周期正常运转需要依赖一些细胞周期蛋白,但是目前关于细胞周期蛋白调控根发育的分子机制还不清楚。通过筛选模式植物拟南芥的根发育异常突变体,分离鉴定了1个突变体dig9（drought inhibition of lateral root growth）,该突变体表现为主根短、侧根少、发育迟缓、顶端分生组织变小、叶片扭曲、无主茎等表型。通过图位克隆,成功定位并克隆了DIG9基因,该基因编码一个细胞周期蛋白,是有丝分裂后期促进复合体的一个亚基APC8 （anaphase-promoting complex）。通过亚细胞定位发现DIG9定位于细胞核;qRT-PCR检测发现DIG9基因在根中有较高的表达量,进一步通过启动子-GUS报告系统发现DIG9在根尖、侧根和顶端分生组织等细胞分裂旺盛区域表达。外施IAA能恢复dig9突变体的侧根表型但不能恢复根短表型。dig9突变体对干旱及盐胁迫反应不敏感。研究结果表明DIG9基因可能通过影响IAA的产生来调控植物的侧根发育。     

ST273是拟南芥绒毡层和小孢子发育的必需基因

绒毡层在拟南芥花药花粉发育过程中具有重要作用,包括分泌降解胼胝质的胼胝质酶、为花粉壁的形成提供原料以及为小孢子发育提供营养物质.本文通过对拟南芥雄性不育突变体st273的分析,研究了ST273基因在花药花粉发育过程中的功能.st273是通过T-DNA插入诱变野生型拟南芥得到的一株突变体,遗传分析表明st273是单隐性核基因控制的.利用图位克隆的方法对不育基因ST273进行了定位,结果表明ST273基因与拟南芥第三条染色体上分子标记CIW11连锁.生物信息学分析发现该分子标记附近有一个调控花粉发育的基因TDF1.测序分析结果表明在st273突变体中,TDF1基因第三个外显子上459位的碱基发生了由G459变成了A459的单碱基变化,导致ST273基因该位点提前终止突变.等位分析结果表明st273与tdf1是等位突变体.st273突变体营养生长期发育正常,但生殖生长发育出现异常.亚历山大染色结果显示st273突变体花药中没有花粉.组织切片观察结果表明,突变体花药绒毡层异常肥大且空泡化,四分体不能正常释放小孢子,最终无法形成花粉.这些结果揭示了ST273蛋白质参与调控了绒毡层和小孢子发育过程.     

拟南芥钾转运突变体atkup12鉴定及非生物胁迫的敏感性检测

[目的]鉴定获得拟南芥HAK/KUP/KT高亲和钾离子转运突变体atkup12,通过检测种萌期atkup12突变体在低钾、盐及氧化胁迫下的生长指标以初步明确拟南芥AtKUP12基因是否参与植物对非生物胁迫的响应。[方法]以拟南芥atkup12突变体基因组和总RNA反转后的cDNA为模板,通过PCR扩增确定AtKUP12基因T-DNA插入失活的纯合突变体。将野生型和atkup12突变体点种于0.5μmol/L低钾、不同NaCl浓度和1μmol/L甲基紫精的胁迫培养基,测定并比较突变体与野生型拟南芥在根长、种子萌发率及子叶绿化率间的差异。[结果]利用双引物法,结合反转录PCR,在DNA及RNA水平鉴定获得了AtKUP12基因的纯合突变体。低钾胁迫下,atkup12突变体种苗的根长比较野生型拟南芥短40%左右,较野生型受到了显著抑制;不同NaCl浓度胁迫下,atkup12突变体种子的萌发率较野生型显著降低;1μmol/L甲基紫精显著的抑制了突变体种苗的子叶绿化率。[结论]成功鉴定获得了AtKUP12基因T-DNA插入失活的纯合突变体,且AtKUP12基因的缺失会增加植物对盐、低钾及氧化等非生物胁迫的敏感性。     

光信号转导因子HY5调控拟南芥分枝的功能研究

光是影响植物分枝的重要外在环境因素,但光信号因子HY5（ELONGATED HYPOCOTYL5）是否调控植物分枝目前尚不清楚。创制了HY5转基因超表达植株,并获得商业化T-DNA插入突变体纯合植株。通过比较野生型（WT）、超表达植株（HY5-OE）、突变体（hy5-215）的分枝数目发现,与野生型相比,超表达植株分枝数目显著增加,而突变体分枝数目则显著减少。进一步比较这些遗传材料的拟南芥植株分枝的负调控关键因子BRC1（BRANCHED1）转录本水平差异,发现与野生型相比,超表达植株中BRC1转录本显著下调、突变体中显著上调。研究结果表明,HY5通过抑制拟南芥分枝关键负调控因子BRC1的转录水平,进而促进拟南芥的分枝。研究结果为阐明HY5调控分枝的生物学功能提供了一定的理论依据。     

WD40重复蛋白家族基因Atlg65030调控拟南芥种子的重量与体积

植物中WD40-repeat蛋白在细胞周期调控等方面具有重要作用。本研究鉴定了一株拟南芥WD40-repeat蛋白基因突变体atlg65030。与野生型植株相比种子重量增重体积变大,营养生长长势较弱,角果种子结实率较低。以突变体作为母本／父本与野生型父本／．母本杂交,前者杂交后代未显示有母本的突变表型,后者部分杂交后代显示出父本的突变表型,统计突变体后代分离比符合l：1。用苯胺兰（DAB）、4,6．二氨基．2．苯基吲哚（DAPI）、2,3,5．氯化三苯基四氮唑（TTc）、碘．碘化钾花粉染色,发现花粉部分败育且主要为核败育。爱氏苏木精花粉染色结果显示可观察到正常减数分裂各时期形态。采取热不对称交错PCR（thermal asymmetric interlaced PCR,TAIL—PcR）方法确认突变基因位于第一条染色体65030位置,生物信息学分析表明该基因含有DWD基序。半定量RT-PCR分析发现在拟南芥发育晚期该基因在花器官中大量表达,过表达该基因使种子重量减轻。推测Atlg65030影响了拟南芥花粉发育细胞核有丝分裂过程,该研究增加了人们对调控拟南芥花粉发育分子机制的认识。     

Somatic Embryogenesis Receptor Kinases Control Root Development Mainly via Brassinosteroid-Independent Actions in Arabidopsis thaliana

Brassinosteroids(BRs),a group of plant steroidal hormones,play critical roles in many aspects of plant growth and development.Previous studies showed that BRI1-mediated BR signaling regulates cell division and differentiation during Arabidopsis root development via interplaying with auxin and other phytohormones.Arabidopsis somatic embryogenesis receptor-like kinases(SERKs),as co-receptors of BRI1,were found to play a fundamental role in an early activation step of BR signaling pathway.Here we report a novel function of SERKs in regulating Arabidopsis root development.Genetic analyses indicated that SERKs control root growth mainly via a BR-independent pathway.Although BR signaling pathway is completely disrupted in the serk1 bak1 bkk1 triple mutant,the root growth of the triple mutant is much severely damaged than the BR deficiency or signaling null mutants.More detailed analyses indicated that the triple mutant exhibited drastically reduced expression of a number of genes critical to polar auxin transport,cell cycle,endodermis development and root meristem differentiation,which were not observed in null BR biosynthesis mutant cpd and null BR signaling mutant bri1-701.     

Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.     

OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice.     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root

Cytokinin (CK) influences many aspects of plant growth and development, and its function often involves intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood. Here we describe the isolation and characterization of the Arabidopsis CK-induced root curling 1 (ckrc1) mutant. CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. The ckrc1 mutant exhibits a defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin or IPA. Furthermore, we show that CK up-regulates CKRC1/TAA1 expression but inhibits polar auxin transport in roots in an AHK3/ARR1/12-dependent and ethylene-independent manner. Our results suggest that CK regulates root growth and development not only by down-regulating polar auxin transport, but also by stimulating local auxin biosynthesis.     

Arabidopsis actin gene ACT7 plays an essential role in germination and root growth

Arabidopsis contains eight actin genes. Of these ACT7 is the most strongly expressed in young plant tissues and shows the greatest response to physiological cues. Adult plants homozygous for the act7 mutant alleles show no obvious above-ground phenotypes, which suggests a high degree of functional redundancy among plant actins. However, act7-1 mutant plants are at a strong selective disadvantage when grown in competition with wild-type plants and therefore must have undetected physical defects. The act7-1 and act7-4 alleles contain T-DNA insertions just after the stop codon and within the first intron, respectively. Homozygous mutant seedlings of both alleles showed less than 7% of normal ACT7 protein levels. Mutants displayed delayed and less efficient germination, increased root twisting and waving, and retarded root growth. The act7-4 mutant showed the most dramatic reduction in root growth. The act7-4 root apical cells were not in straight files and contained oblique junctions between cells suggesting a possible role for ACT7 in determining cell polarity. Wild-type root growth was fully restored to the act7-1 mutant by the addition of an exogenous copy of the ACT7 gene. T-DNA insertions just downstream of the major polyadenylation sites (act7-2, act7-3) appeared fully wild type. The act7 mutant phenotypes demonstrate a significant requirement for functional ACT7 protein during root development and explain the strong negative selection component seen for the act7-1 mutant.     

The hpa1 mutant of Arabidopsis reveals a crucial role of histidine homeostasis in root meristem maintenance

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.     

Nitric oxide is essential for vesicle formation and trafficking in Arabidopsis root hair growth

The functions of nitric oxide (NO) in processes associated with root hair growth in Arabidopsis were analysed. NO is located at high concentrations in the root hair cell files at any stage of development. NO is detected inside of the vacuole in immature actively growing root hairs and, later, NO is localized in the cytoplasm when they become mature. Experiments performed by depleting NO in Arabidopsis root hairs indicate that NO is required for endocytosis, vesicle formation, and trafficking and it is not involved in nucleus migration, vacuolar development, and transvacuolar strands. The Arabidopsis G'4,3 mutant (double mutant nia1/nia2) is severely impaired in NO production and generates smaller root hairs than the wild type (WT). Root hairs from the Arabidopsis G'4,3 mutant show altered vesicular trafficking and are reminiscent of NO-depleted root hairs from the Arabidopsis WT. Interestingly, normal vesicle formation and trafficking as well as root hair growth is restored by exogenous NO application in the Arabidopsis G'4,3 mutant. All together, these results firmly support the essential role played by NO in the Arabidopsis root-hair-growing process.     

AtCYT-INV1, a neutral invertase,is involved in osmotic stress-induced inhibition on lateral root growth in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Neutral/Alkaline invertases are unique to plant and photosynthetic bacteria. The function of Neutral/Alkaline invertases in plant development is not clear so far. In this study, we isolated an Arabidopsis (Col-0) mutant insensitive to osmotic stress-induced inhibition on lateral root growth. Map-based cloning reveals that a neutral invertase gene (AtCYT-INV1) was point-mutated. The mutant Atcyt-inv1 showed short primary root, smaller size of leaves and siliques, and promotion of the reproductive compared to the wild type (WT). Carbohydrate measurement showed that sucrose is accumulated and glucose is reduced in the mutant Atcyt-inv1 under normal and 3% mannitol treatments. Taken together, AtCYT-INV1 plays multiple roles in plant development and is involved in osmotic stress-induced inhibition on lateral root growth by controlling the concentration of hexose in cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.