An Arabidopsis ctpA homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease (CtpA) homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

An <Emphasis Type="Italic">Arabidopsis ctpA</Emphasis> homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease （CtpA） homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

拟南芥SnRK2.2/2.3激酶参与调节镉胁迫响应的机制探究

为探究拟南芥SnRK2.2和SnRK2.3基因对Cd胁迫响应的分子机制. 以野生型（WT）、双突变体SnRK2.2/2.3、过表达SnRK2.2和过表达SnRK2.3的转基因植物为材料,研究SnRK2.2和SnRK2.3基因与Cd胁迫响应的关系.发现过表达两个基因可以提高拟南芥对Cd的耐受性,表现为可以减少Cd、丙二醛（MDA）及活性氧（ROS）的累积量,增加抗氧化酶CAT、POD和SOD的活性. qRT PCR结果显示在Cd胁迫下,两种过表达植株中铁转运蛋白IRT1和转录因子FIT、bHLH038和bHLH039表达水平受到明显抑制,ABA合成相关基因AAO3和NCED3的表达量显著上调.在Cd胁迫下,两种过表达植株中ABA含量显著高于WT和双突变体. 以上结果表明：拟南芥遭受Cd胁迫时,SnRK2.2和SnRK2.3基因通过下调IRT1基因表达从而减少植物对Cd的吸收,同时通过增加内源ABA含量来缓解Cd对植物的毒害.     

An on-line multi-parameter analyzing optical biosensor for real-time and non-invasive monitoring of plant stress responses in vivo

Photosynthetic dysfunction and reactive oxygen species （ROS） production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence （DF） emission, which is an excellent marker for evaluating photosynthesis, an on-line multiparameter analyzing optical biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type （WT） Arabidopsis and heat shock protein （Hsp） 101 T-DNA knockout mutant （hsp101） plants with different thermotolerance. Results demonstrated that DF intensity correlates with net photosynthesis rate （Pn） in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both the WT Arabidopsis and hsp101 mutant plants after heat stress （HS, 40℃ for 30 min）, respectively. In all experiments discussed above, the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor. In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery following HS. Nevertheless, pre-infiltration with catalase （CAT） reduced ROS production and prevented the declines of the DF intensity. Therefore, HS-caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference.     

Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein,calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum (ER) of plants.Here,we identified three loss-of-function mutants ofcalreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses.There are three genes encoding calreticulin in Arabidopsis,and they are named AtCRT1,2,and 3,respectively.We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis.Moreover,crt3 mutant showed more sensitivity to salt treatment at germination stage,but tolerance to salt stress at later stage compared with wild-type plant.However,there was no obvious growth difference in the mutant crt1 and crt2 compared with wild-type Arabidopsis under calcium and salt stresses.These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein, calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum （ER） of plants. Here, we identified three loss-of-function mutants of calreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses. There are three genes encoding calreticulin in Arabidopsis, and they are named AtCRT1, 2, and 3, respectively. We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis. Moreover, crt3 mutant showed more sensitivity to salt treatment at germination stage, but tolerance to salt stress at later stage compared with wild-type plant. However, there was no obvious growth difference in the mutant crtl and crt2 compared with wild-type Arabidopsis under calcium and salt stresses. These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover, whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems. In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry. In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively. Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.     

抗坏血酸对暗诱导拟南芥叶片衰老的影响

分析黑暗诱导衰老条件下3种内源抗坏血酸含量不同的拟南芥（GLDH基因超表达植株gldh236OE、哥伦比亚野生型Col和拟南芥抗坏血酸缺乏突变体vtc2-1）表型、抗坏血酸含量及叶绿素荧光参数等生理指标的变化,研究抗坏血酸对暗诱导拟南芥叶片衰老的影响.结果表明,黑暗胁迫后3种拟南芥抗坏血酸含量均呈下降趋势,与Col相比,vtc2-1的抗坏血酸含量显著降低（P005）,叶片黄化速度明显较快;gldh236OE叶片黄化速度较慢;黑暗均引起了PSII最大光能转化效率（Fv/Fm）和PSII实际光化学量子产量（Yield）降低,PSII的光合效率受到阻碍;也减少了vtc2-1非光化学猝灭（NPQ）,外施抗坏血酸对植株热耗散能力显著增强（P005）.研究表明,内源抗坏血酸含量的缺失会导致植株对黑暗胁迫的适应性降低,外施1 mmol/L抗坏血酸能部分缓解植物的黑暗胁迫.     

拟南芥AtHHR3响应热胁迫应答的初步分析

拟南芥基因AtHHR3编码一个RING结构域的E3连接酶,通过生物信息学分析发现其可能参与植物热胁迫相关的应答.为了探索其具体的功能,构建了AtHHR3互补株系,并在DNA水平和转录水平分别鉴定了AtHHR3互补株系,用RT-PCR技术分析了AtHHR3在热处理条件下基因表达的变化情况.在热胁迫下分析了野生型、突变体athhr3、回复株系幼苗存活以及种子萌发的表型变化情况,发现突变体athhr3表现出对热胁迫的耐受性,并检测了热胁迫下不同株系的HSF、HSP等热相关基因的转录水平的变化,初步的研究表明拟南芥基因AtHHR3负调控植物对热胁迫的耐受性.     

Involvement of a cytoplasmic glyceraldehyde-3- phosphate dehydrogenase GapC-2 in low-phosphate- induced anthocyanin accumulation in Arabidopsis

Phosphorous is one of the essential mineral elements for plant growth and development.Typically, the shoots of plant seedlings usually turn a dark-brown or purple colour under low-Pi stress. Using protein 2-D gel and peptide mass fingerprinting mapping (PMF) methods, a cytoplasmic glyceralde-hyde-3-phosphate dehydrogenase GapC-2 was identified as a low-Pi responsive protein in Arabidopsisplants. Expression of AtGapC-2 protein was significantly decreased after 4 d of low-Pi stress. Two in-dependent T-DNA insertion lines of GapC-2 gene (At1g13440) showed a hypersensitive phenotype inresponse to low-Pi stress compared with wild type plants, while the transgenic complementation linesof the mutants showed a similar phenotype to the wild type. These results indicate that AtGapC-2 mayplay an important role in Arabidopsis responses to low-Pi stress, possibly by regulation of glycoly-sis-associated "Pi-pool" and accumulation of anthocyanin pigments in plants.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation

Touch sensitivity in animals relies on nerve endings in the skin that convert mechanical force into electrical signals. In the nematode Caenorhabditis elegans, gentle touch to the body wall is sensed by six mechanosensory neurons that express two amiloride-sensitive Na+ channel proteins (DEG/ENaC). These proteins, MEC-4 and MEC-10, are required for touch sensation and can mutate to cause neuronal degeneration. Here we show that these mutant or 'd' forms of MEC-4 and MEC-10 produce a constitutively active, amiloride-sensitive ionic current when co-expressed in Xenopus oocytes, but not on their own. MEC-2, a stomatin-related protein needed for touch sensitivity, increased the activity of mutant channels about 40-fold and allowed currents to be detected with wild-type MEC-4 and MEC-10. Whereas neither the central, stomatin-like domain of MEC-2 nor human stomatin retained the activity of full-length MEC-2, both produced amiloride-sensitive currents with MEC-4d. Our findings indicate that MEC-2 regulates MEC-4/MEC-10 ion channels and raise the possibility that similar ion channels may be formed by stomatin-like proteins and DEG/ENaC proteins that are co-expressed in both vertebrates and invertebrates. Some of these channels may mediate mechanosensory responses.     

HEMT中二维电子气的电子密度的研究

考虑到界面态影响，通过求解泊松方程，推导出了HEMT中二维电子气的电子密度表达式，对文献未考虑界面态影响的HEMT中二维电子气的电子密度表达式作了重要修正。     

Osmotically stress-regulated the expression of glutathione peroxidase 3 in Arabidopsis

Gene expression of glutathione peroxidase 3 (ATGPX3) in response to osmotic stress was analyzed in Arabidopsis using ATGPX3 promoter-glucuronidase (GUS) transgenic plants. High levels of GUS ex- pression were detected under osmotic stress in ATGPX3 promoter-GUS transgenic plants. Compared with the wild type, the growth and development of ATGPX3 mutants (atgpx3-1) were more sensitive to mannitol. In addition, the expression of RD29A, ABI1, ABI2 and RbohD in atgpx3-1 was induced by ABA stress. These results suggest that ATGPX3 might be involved in the signal transduction of osmotic stress.     

Deg1 is involved in the degradation of the PsbO oxygen-evolving protein of photosystem II in Arabidopsis

Deg1, a thylakoid lumen-localized protease, retains both chaperone and protease activities. The in vivo function of Deg1 has been shown to be involved not only in PSII assembly but also in the degradation of PSII reaction center protein D1. Here we used the transgenic plants with reduced Deg1 to examine whether the lumen-localized proteins are also the substrates of Deg1 in vivo. Our results showed that the transgenic plants accumulated degradation products of the PsbO protein while the levels of full-length PsbO were not affected. The PsbO degradation products could be efficiently degraded by the recombinant Deg1. These results suggest that Deg1 is involved in the degradation of the PsbO degradation fragments, but not in the initial cleavage event itself.     

小麦耐甘露醇变异系生理特性分析

目的对二次离体筛选获得的小麦耐甘露醇变异细胞系进行生理生化及分子生物学鉴定。方法检测该变异细胞系在甘露醇,NaCl和聚乙二醇(PEG-6000)模拟的渗透胁迫环境中的耐受生长能力,并测定变异系在甘露醇胁迫下游离脯氨酸含量、可溶性糖含量、可溶性蛋白含量、Na /K 含量等生理生化特性及可溶性蛋白质组成和基因组DNA多态性等分子特征。结果通过愈伤组织在甘露醇,NaCl和聚乙二醇(PEG-6000)模拟的渗透胁迫条件下的生长实验,发现变异系细胞系可以在对照细胞系不能生长的20%甘露醇,1.5%NaCl和20%PEG-6000胁迫条件下,分别表现出14.5%(见图1),12.8%(见图2),41.8%(见图3)的相对生长量;在20%甘露醇胁迫条件下变异系细胞系游离脯氨酸积累量为对照系的80%(见图4),可溶性糖积累量为对照系的1.2倍(见图5),可溶性蛋白含量为对照系的1.3倍(见表1)。在相同浓度的甘露醇模拟的渗透胁迫环境中变异系再生植株比对照植株相能维持较高的K /Na 比值(见表2)。与对照相比,耐甘露醇变异细胞系再生植株可溶性蛋白SDS-PAGE发生显著变化:6条新可溶性蛋白谱带出现在变异系再生植株中,同时对照系中的1条可溶性蛋白谱带在突变株中缺失(见图6)。变异系植株与对照株RAPD带型呈现一定的多态性(见图7),表明变异系基因组DNA与对照相比发生了突变。结论所研究的小麦耐甘露醇变异细胞系是一个具有较强渗透胁迫耐受能力,可用于进一步育种工作的良好突变体材料。     

Construction and characterization of double mutants in nitrogenase of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Two mutants in nitrogenase of Klebsiella pneumoniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for α-Glutamine 190 and α-Histidine 194 respectively (Kp-Q α190 K and Kp-Hα194 Q). The above two substitutions are respectively introduced into a nifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are obtained (Kp-Q α190 K-nifV and Kp-H α194 Q-nifV). All four mutants exhibit strict Nif phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered nitrogneases are effectively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q α190 K-nifV abolish cell C2H2 reduction activity, but Kp-H α194 Q-nifV cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mutants in comparison to the wild type and nifV mutant is also detected. The results show that only single α-Gln^194 substitution does not perturb the stereospecificity of protonation of C2D2. These results indicate that the α-Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that α-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.     

拟南芥CARK家族响应脱落酸信号的研究

植物激素脱落酸(Abscisic acid,ABA)在植物应对生物和非生物胁迫中起着重要作用.本研究利用以carks单基因突变体为亲本,构建双重突变体来检测CARKs在ABA信号途径中的功能.然后,分析多重突变体在ABA处理下,种子萌发率和子叶变绿的响应.结果显示:单基因突变体和双重变体与野生型相比,萌发率更高,双重突变体的子叶绿芽率高于单基因突变体.以上结果表明,CARKs家族基因在ABA信号途径中起正调控作用,而且它们的功能是冗余的.     

低温胁迫对拟南芥G蛋白突变体SOD、POD酶活性和丙二醛含量的影响

采用分光光度法测定了低温胁迫下两种拟南芥G蛋白突变体的超氧化物歧化酶（SOD）、过氧化物酶（POD）的活性及丙二醛（MDA）含量的变化。结果表明：在低温12h,野生型（Ws）和突变体gpal-1的SOD酶活性迅速升高,分别为15．501U／mgprotein和9．453U／mgpro—tein,突变体gpal-2则在低温24h升至最高点,酶活性为6．325U／mgprotein。低温胁迫下,三种材料的POD酶活性都呈现降低趋势,而MDA含量呈上升趋势。初步证明外界低温刺激与植物细胞内G蛋白有一定的关联。