定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因.它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性.免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达,在长日照豌豆同时期非正常叶绿体中丰度非常低.对转基因拟南芥和野生型植株的叶片衰老进程比较发现, PPF1在拟南芥中的过量表达可以延缓叶片的衰老,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度.对转基因拟南芥的超微结构分析显示,PPF1在拟南芥中过量表达时,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜;相反,反义PPF1表达抑制其在拟南芥中的同源物时,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统.这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关.我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育.     

转P_(SAG12-ipt)基因水稻叶片中叶绿体超微结构的变化

研究了转PSAG12-ipt基因水稻和对照植株发育过程中叶片中的叶绿体结构的变化。发现水稻发育到乳熟期,转基因植株叶片中的叶绿体与对照植株开始出现明显的差别。对照叶绿体中嗜锇体体积增大,数目增多,大部分基粒的类囊体膜膨胀、裂解,片层结构解体。而转基因植株叶片中的叶绿体结构变化不大,嗜锇体相对有所增加,但体积较小,大部分基粒类囊体片层结构仍然排列整齐,少数类囊体垛叠化丧失。     

拟南芥ASL25/LBD28基因过量表达对叶片形态建成的影响

为研究ASL25/LBD28基因在植物发育过程中的作用,该研究构建了拟南芥ASL25/LBD28的过量表达载体并将其转入野生型拟南芥中,结果发现,ASL25/LBD28基因的过量表达可导致转基因拟南芥的叶片变得狭长;在叶极性发育突变体as2中,ASL25/LBD28基因过量表达导致部分转基因植株在形成1～3片畸形叶后顶端分生组织的发育会终止;而许多转基因植株则会形成许多"针状"叶.扫描电镜观察表明,不正常的叶片近轴面或"针状"叶的表皮细胞具有远轴面化的长条形细胞,说明在as2突变体中过量表达ASL25/LBD28基因影响叶片的极性发育.     

拟南芥热激转录因子耐高温功能分析

从拟南芥基因组中克隆了热激转录因子(At Hsf A6a),构建了过量表达(over-expression,OE)和反义(anti-sense,AS)植物表达载体并转化拟南芥,获得了拟南芥纯合转基因株系。对其进行耐高温处理,结果显示:43℃处理2 h,过量表达转基因植株存活率(86%)远高于野生型(59%);而反义转基因植株存活率则只有43%,显著低于野生型。43℃处理0.5 h,过量表达转基因植株的离子渗漏水平显著低于野生型,而反义转基因植株则大幅度升高。基因表达分析证明,AtHsfA6a的表达受热胁迫诱导,并且Hsp70是受AtHsfA6a调控的下游靶基因。上述结果表明,拟南芥AtHsfA6a可能通过调节Hsp70表达,提高植物耐受高温胁迫的能力。     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花(英文)

AtLH基因是BcpLH基因在拟南芥(Arabidopsis thealiana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域。在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关。为研究AtLH基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥。与野生型比较而言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量表达的植株。这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腋处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强;转基因植株对激素的敏感性改变,IAA刺激根生长的作用增强,ABA抑制根生长的作用减弱。由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲。     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花

AtLH基因是BcpLH基因在拟南芥(Arapsis thaliana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域.在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关.为研究AtLH的基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥.与野生型比较页言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量的植株.这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腑处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强,转基因植株对激素的敏感性改变,IAA剌激根生长的作用增强,ABA抑制根生长的作用减弱.由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲.     

大豆GmcpSecA基因的克隆及表达特异性

Sec途径是将核编码的叶绿体蛋白输入到类囊体腔的蛋白分选途径之一,对叶绿体正确行使其功能有重要作用。前期研究获得了拟南芥AtcpSecA功能缺失的突变体agyl,其叶片呈黄白色,叶绿体发育缺陷,内部缺少类囊体片层结构。我们从大豆中克隆了拟南芥AtepSecA的同源基因GmcpSecA基因的全长cDNA序列和5’端ATG上游1．5kb的启动子序列,通过RT-PCR的方法对GmcpSecA基因表达的器官特异性进行了初步分析;并构建了GmcpSecA：：GUS和35S：：GmcpSecA融合基因,以农杆菌介导的转化方法获得转基因拟南芥。GUS组织化学染色结果表明：在转基因拟南芥的子叶、叶片、花萼等绿色组织中都有较强的GUS表达,而在非绿色组织中没有GUS表达。通过将过表达载体p35S：：GmcpSecA转化agyl,结果表明GmcpSecA能够部分回补拟南芥agyl突变体的表型。推测GmcpSecA基因具有与AtcpSecA基因相似的功能,在叶绿体发育过程中发挥重要作用。     

过表达大豆类受体蛋白激酶基因RLPK2促进转基因拟南芥叶片的衰老北大核心CSCD

大豆RLPK2基因(GenBank登录号:AY687391)是一个编码N-末端富含亮氨酸重复序列的类受体蛋白激酶基因。为分析大豆RLPK2基因的功能,该研究以野生型拟南芥和大豆RLPK2基因过表达拟南芥植株为材料,通过农杆菌介导法转化野生型拟南芥,构建了大豆RLPK2基因过表达载体,分析了叶片衰老过程中叶绿素荧光参数、抗氧化酶活性及衰老相关基因表达量的变化。结果表明:(1)无论是野生型还是转基因拟南芥,随着叶片衰老进程的进行,光系统Ⅱ(PSⅡ)的最大光化学效率(F_(v)/F_(m))、PSⅡ实际光化学效率(Φ_(PSⅡ))、光化学淬灭系数(qP)和光合电子传递速率(ETR)均呈下降趋势,但后者下降趋势更明显;(2)激发压(1-qP)在叶片衰老前期的变化较为平稳,后期则急剧增加,且转基因型比野生型拟南芥增加更明显;(3)在叶片衰老的各个时期,转基因拟南芥叶片丙二醛(MDA)含量均显著高于野生型,而超氧化物岐化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著低于野生型;(4)实时荧光定量PCR检测结果表明,RLPK2转基因拟南芥中衰老标志基因ATSAG12,衰老关键转录因子ATNAP、ATWRKY6和叶绿素降解关键酶编码基因ATACD1表达量显著上调。综上认为,大豆类受体蛋白激酶基因RLPK2参与调控植物叶片衰老进程,其表达对叶片衰老具有促进作用。     

高粱ATP合成酶E亚基基因(SbATPase-E)的克隆及其抗逆功能研究

高粱是一种抗旱性较强的禾谷类作物。本研究在高粱中克隆到一个全长为693 bp的编码ATP合成酶E亚基的基因(SbATPase-E)。在高粱幼苗期,SbATPase-E基因受Na Cl和脱落酸(ABA)处理诱导上调表达。该基因在拟南芥中过量表达可提高转基因植株的耐旱性和耐盐性,在逆境胁迫条件下转基因拟南芥植株较野生型植株根系发达,可能是转基因植株耐旱性和耐盐性提高的主要原因。在干旱胁迫条件下,转基因植株中DREB2A、P5CS1、RD29A、RAB18和ABI1基因的表达量相对于野生型植株中的表达量提高更为显著;在高盐处理条件下,转基因植株中SOS1和SOS2基因的表达量也较野生型植株中的表达量明显提高。这些抗逆相关基因的上调表达可能是转基因植株抗逆性提高的主要分子机制。     

Developmentally regulated association of plastid division protein FtsZ1 with thylakoid membranes in Arabidopsis thaliana

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

THE EFFECT OF LIGHT INTENSITY AND TEMPERATURE ON PLANT GROWTH AND CHLOROPLAST ULTRASTRUCTURE IN SOYBEAN

Soybean plants grown in controlled environment cabinets under light intensities of 220 w/m² or 90 w/m² (400–700 nm) and day to night temperatures of 27.5–22.5 C or 20.0–12.5 C in all combinations, exhibited differences in growth rate, leaf anatomy, chloroplast ultrastructure, and leaf starch, chlorophyll, and chloroplast lipid contents. Leaves grown under the lower light intensity at both temperatures had palisade mesophyll chloroplasts containing well-formed grana. The corresponding leaves developed under the higher light intensity had very rudimentary grana. Chloroplasts from high temperature and high light had grana consisting of two or three appressed thylakoids, while grana from the low temperature were confined to occasional thylakoid overlap. Spongy mesophyll chloroplasts were less sensitive to growth conditions. Transfer experiments showed that the ultrastructure of chloroplasts from mature leaves could be modified by changing the conditions, though the effect was less marked than when the leaf was growing.     

An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.     

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.     

PPF1 inhibits programmed cell death in apical meristems of both G2 pea and transgenic Arabidopsis plants possibly by delaying cytosolic Ca2+ elevation

PPF1 encodes a putative calcium ion carrier that affects the flowering time of transgenic Arabidopsis by modulating Ca(2+) storage capacities in chloroplasts of a plant cell. In the current work, we found that differential expression of PPF1 might affect processes of programmed cell death (PCD) since DNA fragmentation was detected in senescencing apical buds of long day-grown G2 pea (Pisum sativum L.) plants, but was not in non-senescencing short day-grown counterparts at all growth stages. An animal inhibitor of caspase-activated DNase (ICAD) homologue was detected in short day-grown plant continuously throughout the whole experiment and only in early stages of long day-grown pre-floral G2 pea apical buds. DNA fragmentation was significantly inhibited in apical meristems of transgenic Arabidopsis that over-expressed the PPF1 gene when compared to that of either wild-type control or to PPF1 (-) plants. The expression of ICAD-like protein decreased to undetectable level at 45 dpg in apical tissues of PPF1 (-) Arabidopsis, which was much earlier than that found in PPF1 (+) or wild-type controls. In epidermal cells of PPF1 (-) plants, we recorded significantly earlier calcium transient prior to PCD. We suggest that the expression of PPF1, a chloroplast localized Ca(2+) ion channel may inhibit programmed cell death in apical meristems of flowering plants by keeping a low cytoplasmic calcium content that might inhibit DNA fragmentation in plant cells.     

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.