破坏光敏色素A 改变生长素反应因子8 的表达谱

拟南芥生长素反应因子8(auxin response factor 8, ARF8)受光诱导表达, 涉及光信号转导。为阐明光信号通过光受体传递给ARF8的过程和机理, 首先利用半定量RT-PCR分析发现, PhyA的突变促进幼苗中ARF8的表达, 而PhyB、Cry1和Cry2的突变对ARF8的表达没有明显影响。进而, 利用GUS染色以及半定量和定量RT-PCR方法, 系统分析PhyA基因的突变对ARF8基因表达的影响。结果表明, 在黑暗、白光和远红光条件下, PhyA突变均明显提高拟南芥(Arabidopsis thaliana)幼苗子叶和叶片中ARF8的表达水平, 并且明显降低其在幼苗下胚轴、茎尖和根尖中的表达水平。上述结果说明,PhyA基因的突变组成型地改变了ARF8基因的表达谱。然而, ARF8的突变并未明显改变PhyA的表达。说明在远红光信号通路中, ARF8位于PhyA的下游。     

拟南芥光敏色素A反义RNA载体的构建及转化

光敏色素A是植物远红光信号的关键受体,在植物光信号转导途径中起重要作用。一些具有重要功能的基因(如生长素反应因子8:auxin response factor 8,ARF8)受到PhyA调控。本实验拟通过构建PhyA基因反义株系,研究PhyA调控ARF8等重要功能基因表达的机理。以培养7 d的拟南芥幼苗为材料提取RNA,利用RT-PCR技术扩增出PhyA的全长CDS,将其反向插入表达载体pMD1得到反义表达载体pMD1-PhyA CDSR,并通过农杆菌介导转化拟南芥,经卡那霉素抗性平板筛选和PCR鉴定得到13个PhyA转基因株系。pMD1-PhyA CDSR及其转基因株系的获得为研究PhyA调控ARF8等基因表达的机理奠定了材料基础。     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

拟南芥F-box基因At3g16740的表达分析

拟南芥At3g16740基因为F-box基因家族成员,其功能尚不清楚.通过连续和瞬时光照处理分析,发现蓝光、红光和远红光都诱导At3g16740基因的表达,其中远红光的诱导作用最明显.蓝光受体cry1、cry2,红光受体phyB或远红光受体phyA突变导致At3g16740基因表达的光诱导作用减弱或者消失,表明该基因为光信号通路相关基因.通过实时荧光定量PCR分析At3g16740基因在拟南芥不同组织器官中的表达,发现其在拟南芥根、茎、叶、花和果荚中都有表达,花和果荚中的表达量最高,推测该基因可能参与植物花和/或果荚的发育.酵母双杂交分析发现,At3g16740蛋白通过F-box结构域与拟南芥ASK(arabidopsis-SKP1-like)家族成员ASK1、ASK2和ASK11相互作用,表明At3g16740是SCF(Skp、Cullin、F-box)复合物的成员.     

胁迫相关基因CIPK14在PHYA介导抑制拟南芥远红光黄化苗转绿过程中的作用

本文对CBL互作蛋白激酶, CIPK14参与拟南芥光敏色素A介导的, 远红光黄化苗转绿抑制调控进行了研究. 结果发现, 拟南芥光敏色素A功能缺失突变体(phyA)远红光黄化苗(4天)转入白光处理后, 仅0.5 h叶片迅速转绿; 相同条件下, CIPK14基因插入失活突变体(cipk14)远红光黄化苗, 经过15 h白光处理之后叶片才开始转绿; 野生型(Col-4)远红光黄化苗转绿时间介于突变体phyA与cipk14之间. 基因表达分析表明, 上述不同基因型拟南芥远红光黄化苗转绿的快慢, 与原叶绿素酸酯还原酶基因表达量存在正相关性. 结合研究发现——CIPK14基因受到远红光调节, 并且表达具有时钟节律性认为, Ca²⁺调节蛋白CIPK14,可能在PhyA信号传导途径的上游分支介入PhyA介导的远红光黄化苗转绿抑制调控.     

GA2ox8基因过量表达诱导蓝光下拟南芥光形态建成

检测不同光照强度蓝光下,过量表达GA2ox8基因的转基因植株光形态建成表型的结果表明,突变体比各自的母本的下胚轴短,茎尖角度和子叶张开度较大,花青素和叶绿素的含量较高,并且其差异与GA2ox8基因的表达量呈正相关.RT-PCR检测光调节基因表达水平的结果显示,暗培养条件下其突变体幼苗中的水平比各自的母本高,蓝光下35S::GFP-GA2ox8-1.35S::GFP-GA2ox8-8与母本co1-4之间差异不明显,但scc7-D中的水平比母本crylcry2高.这似乎说明,GA2ox8基因过量表达可诱导蓝光下拟南芥幼苗光形态建成.     

红光下拟南芥光敏素调节基因的蛋白质鉴定与mRNA分析

光敏素是植物体内一种重要的光受体家族,它们可介导植物对外界红光和远红光的应答,在植物的生理发育过程中发挥重要作用.基因芯片分析结果表明,PHYA和PHYB在转导红光信号至光应答基因的过程中起关键作用.为了获得与PHYA,PHYB相关的光应答基因,本研究采用双向电泳技术比较分析了在持续红光下生长7天的拟南芥光敏素双突变体phyAphyB和野生型col-4幼苗的全蛋白图谱.采用基质辅助激光解吸飞行时间串联质谱（MALDI-TOF-TOF）进行肽质谱指纹图谱分析,成功鉴定到了32个差异蛋白点.选取其中的10个蛋白点,对应于10个不同的基因,进行了RT-PCR分析,并用Q-PCR对其中的2个基因表达情况进行了验证.结果表明,光敏素可能从mRNA水平或蛋白质水平来调节基因的表达.蛋白质组学分析为寻找光敏素依赖基因提供了新的途径。     

拟南芥QUA1基因在光信号途径中的表达与功能分析

光信号在植物生长发育过程中具有非常重要的作用。不同的光信号通过调节植物下游基因的表达,进而影响细胞分化、结构和功能的改变,以及组织和器官的形成,参与植物光形态建成。QUA1 (QUASIMODO1)是拟南芥糖基转移酶家族中的一个成员,参与植物细胞壁中果胶的合成。本文以拟南芥qua1-1/cry1以及qua1-1/phyB双突变体为材料,对QUA1基因在光信号途径中的功能进行了分析。结果显示,qua1-1突变体在暗、蓝光、红光以及远红外光培养条件下下胚轴的伸长均受到抑制,QUA1基因的表达同样受到光信号的调节,而且突变体中多种光信号调节基因的表达也受到了影响。通过对qua1-1突变体下胚轴的观察发现,突变体下胚轴表皮细胞长度明显变短。与cry1以及phyB突变体相比,qua1-1/cry1和qua1-1/phyB双突变体下胚轴长度明显变短,而且双突变体中光信号调节基因的表达也有明显变化,表明QUA1可能参与了CRY1以及PHYB介导的蓝光及红光信号传导。以上结果表明QUA1影响了下胚轴细胞的伸长以及光信号调节基因的表达,并参与调控多种光信号传导途径。     

拟南芥CKL3基因表达对非生物胁迫的响应

以拟南芥为材料,采用PCR和RT-PCR技术在DNA和RNA水平上鉴定出了与CKL3基因对应的T-DNA插入纯合突变体,并对其表型变化进行了观察.半定量RT-PCR检测CKL3基因在拟南芥不同器官和非生物胁迫响应中表达的结果表明,CKL3基因在根、花、叶中表达较高,在茎、叶柄中表达较弱;盐胁迫下CKL3基因表达下降,蓝光下CKL3基因表达升高,但热激和红光对此基因表达量的影响不大.     

GA20x8基因过量表达诱导蓝光下拟南芥光形态建成

检测不同光照强度蓝光下,过量表达GA20x8基因的转基因植株光形态建成表型的结果表明,突变体比各自的母本的下胚轴短,茎尖角度和子叶张开度较大,花青素和叶绿素的含量较高,并且其差异与GA20x8基因的表达量呈正相关。RT—PCR检测光调节基因表达水平的结果显示,暗培养条件下其突变体幼苗中的水平比各自的母本高,蓝光下35S：：GFP—GA20x8-1,35S：：GFP—GA20x8—8与母本col-4之间差异不明显,但scc7-D中的水平比母本crylcry2高。这似乎说明,GA20x8基因过量表达可诱导蓝光下拟南芥幼苗光形态建成。     

Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-red-absorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.     

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana

Phytochrome A (PhyA) mediates most, if not all various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana . The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2–1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2–1 mutant was comparable to that in wild type. Seedling of the fin2–1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2–1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.     

The Histidine Kinase-Related Domain of Arabidopsis Phytochrome A Controls the Spectral Sensitivity and the Subcellular Distribution of the Photoreceptor

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.     

SPA1: a new genetic locus involved in phytochrome A-specific signal transduction.

To identify mutants potentially defective in signaling intermediates specific to phytochrome A (phyA), we screened for extragenic mutations that suppress the morphological phenotype exhibited by a weak phyA mutant (phyA-105) of Arabidopsis. A new recessive mutant, designated spa1 (for suppressor of phyA-105), was isolated and mapped to the bottom of chromosome 2. spa1 phyA-105 double mutants exhibit restoration of several responses to limiting fluence rates of continuous far-red light that are absent in the parental phyA-105 mutant, such as deetiolation, anthocyanin accumulation, and a far-red light-induced inability of seedlings to green upon subsequent transfer to continuous white light. spa1 mutations do not cause a phenotype in darkness, indicating that the suppression phenotype is light dependent. Enhanced photoresponsiveness was observed in spa1 seedlings in a wild-type PHYA background as well as in the mutant phyA-105 background but not in a mutant phyA null background. These results indicate that phyA is necessary in a non-allele-specific fashion for the expression of the spa1 mutant phenotype and that phyB to phyE are not sufficient for this effect. Taken together, the data suggest that spa1 mutations specifically amplify phyA signaling and therefore that the SPA1 locus encodes a component that acts negatively early in the phyA-specific signaling pathway.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

RED AND FAR-RED INSENSITIVE 2, a RING-domain zinc finger protein, mediates phytochrome-controlled seedling deetiolation responses

Light is arguably the most important resource for plants, and an array of photosensory pigments enables plants to develop optimally in a broad range of ambient-light conditions. The red- and far-red-light-absorbing photosensory pigments or phytochromes (phy) regulate seedling deetiolation responses, photoperiodic flowering, and circadian rhythm. We have identified a long hypocotyl mutant under red and far-red light, rfi2-1 (red and far-red insensitive 2 to 1). rfi2-1 was also impaired in phytochrome-mediated end-of-day far-red light response, cotyledon expansion, far-red light block of greening, and light-induced expression of CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE. Introduction of rfi2-1 mutation into phyB-9 or phyA-211 did not enhance or suppress the long hypocotyl phenotype of phyB-9 or phyA-211 under red or far-red light, respectively, and RFI2 likely functions downstream of phyB or phyA. RFI2 was identified through the segregation of two T-DNA insertions into different recombinant lines, genetic rescue, and phenotypic characterization of a second mutant allele rfi2-2. RFI2 encodes a protein with a C₃H₂C₃-type zinc finger or RING domain known to mediate protein-protein or protein-DNA interactions, and RFI2 is localized to the nucleus. RFI2 therefore reveals a signaling step that mediates phytochrome control of seedling deetiolation.     

Effects of missense mutation on structure and function of photoreceptor

Phytochromes (PHYs) are photoreceptors of the red (R ~660  nm) and far-red (FR ~730 nm) light, and they control a wide range of responses affecting crucial aspects of plant life. There are five genes PHYA-PHYE encoding for phytochromes of different but overlapping function. One of these, PHYA has the unique function controlling specific responses in high irradiance far-red, as well as in very weak light. Appropriate PHYA functioning requires not only the photoreversibility of molecule but also the proper nuclear localization and degradation of receptor. Recently, we identified and described a mutant PHYA allele (phyA-5) in Arabidopsis thaliana, which showed reduced binding affinity to FHY1/FHL, the proteins regulating its nuclear transport, resulting in impaired nuclear localization and altered signaling under certain conditions. We present here a hypothesis to explain how the identified amino acid substitution may lead to structural changes manifested as altered signaling and phenotype displayed by the phyA-5 mutant.