Nitric Oxide Regulates Dark-Induced Leaf Senescence Through EIN2 in Arabidopsis

The nitric oxide (NO)-deficient mutant nos1/noa1 exhibited an early leaf senescence phenotype. ETHY-LENE INSENSITIVE 2 (EIN2) was previously reported to function as a positive regulator of ethylene-induced senescence. The aim of this study was to address the question of how NO interacts with ethylene to regulate leaf senescence by characterizing the double mutant ein2-1 nos1/noa1 (Arabidopsis thaliana). Double mutant analysis revealed that the nos1/noa1-mediated, dark-induced early senescence phenotype was suppressed by mutations in EIN2, suggesting that EIN2 is involved in nitric oxide signaling in the regulation of leaf senescence. The results showed that chlorophyll degradation in the double mutant leaves was significantly delayed. In addition, nos1/noa1-mediated impairment in photochemical efficiency and integrity of thylakoid membranes was reverted by EIN2 mutations. The rapid upregulation of the known senescence marker genes in the nos1/noa1 mutant was severely inhibited in the double mutant during leaf senescence. Interestingly, the response of dark-grown nos1/noa1 mutant seedlings to ethylene was similar to that of wild type seedlings. Taken together, our findings suggest that EIN2 is involved in the regulation of early leaf senescence caused by NO deficiency, but NO deficiency caused by NOS1/NOA1 mutations does not affect ethylene signaling.     

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.     

冬小麦叶片持绿能力及其衰老特征研究

以12个冬小麦(Triticum aestivum L.)品种为供试材料,通过田间实验连续2年于开花后定期测定各品种的绿叶数目、绿叶面积、叶绿素和MDA含量以及SOD和CAT活性等指标,并以生理成熟时的保绿度、衰老启动时间为指标进行Hierarchical聚类分析,对小麦品种持绿能力进行分级.结果表明,参试冬小麦品种可分为持绿和非持绿两种类型,‘潍麦8号'(WM8)和‘豫麦66'(YM66)两年均表现为持绿型小麦.在整个灌浆期,持绿型小麦品种绿叶数目、面积、叶绿素含量明显高于非持绿型品种,叶片保护酶SOD与CAT活性也较非持绿小麦强,而其MDA含量明显低于非持绿型小麦品种.持绿型小麦叶片衰老启动时间延迟,生育后期绿叶面积较大,光合作用时间延长,具有较高的产量.本研究结果为冬小麦的品种选育、布局等相关研究奠定基础.     

异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老

蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在Pro_OsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。     

Arabidopsis Thylakoid Formation 1 Is a Critical Regulator for Dynamics of PSII-LHCII Complexes in Leaf Senescence and Excess Light

In higher plants, photosystem II （PSII） is a large pigment-protein supramolecular complex composed of the PSII core complex and the plant-specific peripheral light-harvesting complexes （LHCil）. PSli-LHCII complexes are highly dynamic in their quantity and macro-organization to various environmental conditions. In this study, we reported a critical factor, the Arabidopsis Thylakoid Formation 1 （THF1） protein, which controls PSII-LHCII dynamics during dark- induced senescence and light acclimation. Loss-of-function mutations in THF1 lead to a stay-green phenotype in path- ogen-infected and senescent leaves. Both LHCII and PSll core subunits are retained in dark-induced senescent leaves of thfl, indicative of the presence of PSII-LHCII complexes. Blue native （BN）-polyacrylamide gel electrophoresis （PAGE） and immunoblot analysis showed that, in dark- and high-light-treated thfl leaves, a type of PSII-LHCII megacomplex is selec- tively retained while the stability of PSII-LHCII supercomplexes significantly decreased, suggesting a dual role of THF1 in dynamics of PSII-LHCII complexes. We showed further that THF1 interacts with Lhcb proteins in a pH-dependent manner and that the stay-green phenotype of thfl relies on the presence of LHCII complexes. Taken together, the data suggest that THF1 is required for dynamics of PSII-LHCII supramolecular organization in higher plants.     

Defects in a proteolytic step of light-harvesting complex II in an<Emphasis Type="Italic">Arabidopsis</Emphasis> stay-green mutant,<Emphasis Type="Italic">ore10</Emphasis>, during dark-induced leaf senescence

During dark-induced leaf senescence (DIS), the non-functional stay-green mutantore10 showed delayed chlorophyll (Chl) degradation and increased stability in its light-harvesting complex II (LHCII). These phenomena were closely related to the formation of aggregates that mainly consisted of terminal-truncated LHCII (Oh et al., 2003). Theore10 mutant apparently lacks the protease needed to degrade the truncated LHCII. In wild-type (WT) plants, protease was found in the thylakoid fraction, but not the soluble fraction. A similar experiment using dansylated LHCII revealed that the protease degraded both WT andore10 LHCII, indicating that its stability inore10 perhaps did not result from a defect in the LHCII polypeptides themselves. Although protease activity was not present in non-senesced WT leaves, it was induced during DIS. It also was possible to diminish the high level of protease present in the thylakoids through high-salt washing, suggesting that this enzyme is extrinsically bound to the outer surface of the stroma-exposed thylakoid regions.     

拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥 (Arabidopsisthaliana (L .)Heynh .)叶发育研究中 ,as2是一个经典突变体。as2典型的表型是叶片开裂或形成一种小叶状结构。遗传学和分子生物学实验证明 ,AS2基因具有抑制KNOX基因在叶中表达的功能。在本文中 ,我们着重研究了新得到的在Landsbergerecta (Ler)遗传背景下的as2突变体。除了前人报道过的as2表型外 ,新as2突变体的部分叶柄长在叶片的下方 ,形成一种荷叶状结构 ,更严重的甚至长成花丝状叶结构。这两种结构都反映了不正常的叶腹背轴极性分化。在我们所收集到的as2等位突变体中 ,只有在Ler背景下这两种结构才以高频率出现。我们通过图位克隆方法分离了AS2基因。该基因编码一个含有亮氨酸拉链结构的蛋白。在拟南芥中 ,AS2同源基因共 4 3个 ,除AS2外 ,其他基因的功能都不清楚。AS2在叶和花中表达 ,在茎中无表达 ,这种表达模式和as2突变体的表型是吻合的。     

硫氮配施对持绿型小麦氮素运转及叶片衰老的影响

以持绿型小麦品种‘豫麦66号’、‘潍麦8号’及非持绿型品种‘小偃6号’为材料,采用以氮肥为主区硫肥为副区的田间裂区试验,研究了2个施氮水平[纯氮120kg/hm2(N120)、220kg/hm2(N220)]和3个施硫水平[纯硫0kg/hm2(S0)、20kg/hm2(S20)、60kg/hm2(S60)]下植株各部位的含氮量、叶片干鲜重及叶片叶绿素含量的变化,探讨硫氮配施对不同类型小麦氮吸收及衰老的影响。结果表明:在相同处理下,持绿型小麦植株的总含氮量、氮素转运量、叶绿素含量、叶片含水量及穗粒数和千粒重均高于非持绿型小麦。N120处理条件下,不同硫肥处理时持绿型小麦与非持绿型小麦变化趋势相同,开花期茎和叶含氮量及叶绿素含量在S60处理下均低于其他2个硫肥处理,生育后期叶片含水量下降幅度也明显高于其他处理;在N220处理条件下,3个品种开花期叶含氮量、收获期总氮累积量、氮收获指数、叶绿素含量及叶片含水量在S60处理下均高于其他2个处理,其中非持绿型小麦在高硫处理条件下灌浆期的叶绿素含量的增长率明显高于持绿型小麦,而灌浆中后期叶片含水量的下降幅度则明显低于持绿型小麦。研究发现,施用硫肥在氮肥不足时会对小麦植株氮素的吸收利用及叶片衰老等方面产生负面影响,但在氮肥充足时却在氮素的吸收利用、延缓叶片衰老及最终籽粒产量和总生物量等方面表现出正面效应;本实验条件下,220kg/hm2左右施氮量和60kg/hm2左右施硫量有利于各品种小麦生长发育和产量提高;高N和高S水平对于延缓小麦的衰老而言,非持绿性小麦比持绿型小麦更明显。     

不同绿豆品种生育后期叶片衰老的研究

以夏播绿豆品种‘冀绿2号’和‘泰来’为材料,研究其开花至成熟期间主茎开花节叶片的叶绿素含量、净光合速率、酶促防御系统的保护酶SOD、CAT、POD活性和MDA积累量的动态变化规律。结果表明,两品种各功能叶片的叶绿素含量、净光合速率、SOD、CAT活性均在其开花15 d后逐渐下降,但POD活性和MDA含量随着叶片的衰老而升高。虽然两品种叶片衰老的总体变化趋势一致,但衰老进程存在着显著差异。与‘泰来’绿豆相比,‘冀绿2号’各功能叶片衰老过程中SOD、CAT活性下降较慢,叶片功能期持续时间长,生育后期仍能保持相对较高的净光合速率,籽粒产量显著高于‘泰来’绿豆。综合分析表明,在绿豆开花结荚期间,有效控制或延缓开花节叶片的衰老进程,维持叶片生理功能,对籽粒产量形成具有重要作用。     

AtCLH2, a Typical but Possibly Distinctive Chlorophyllase Gene in Arabidopsis

Chlorophyllase （EC 3.1.1.14） is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thaliana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here, we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly, ＂activation＂ of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.     

Conversion of chlorophyll b to chlorophyll a precedes magnesium dechelation for protection against necrosis in Arabidopsis

Chlorophyll is a deleterious molecule that generates reactive oxygen species and must be converted to non‐toxic molecules during plant senescence. The degradation pathway of chlorophyll a has been determined; however, that of chlorophyll b is poorly understood, and multiple pathways of chlorophyll b degradation have been proposed. In this study, we found that chlorophyll b is degraded by a single pathway, and elucidated the importance of this pathway in avoiding cell death. In order to determine the chlorophyll degradation pathway, we first examined the substrate specificity of 7‐hydroxymethyl chlorophyll a reductase. 7‐hydroxymethyl chlorophyll a reductase reduces 7‐hydroxymethyl chlorophyll a but not 7‐hydroxymethyl pheophytin a or 7‐hydroxymethyl pheophorbide a. These results indicate that the first step of chlorophyll b degradation is its conversion to 7‐hydroxymethyl chlorophyll a by chlorophyll b reductase, although chlorophyll b reductase has broad substrate specificity. In vitro experiments showed that chlorophyll b reductase converted all of the chlorophyll b in the light‐harvesting chlorophyll a/b protein complex to 7‐hydroxymethyl chlorophyll a, but did not completely convert chlorophyll b in the core antenna complexes. When plants whose core antennae contained chlorophyll b were incubated in the dark, chlorophyll b was not properly degraded, and the accumulation of 7‐hydroxymethyl pheophorbide a and pheophorbide b resulted in cell death. This result indicates that chlorophyll b is not properly degraded when it exists in core antenna complexes. Based on these results, we discuss the importance of the proper degradation of chlorophyll b.     

拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥(Arabidopsis thaliana (L.) Heynh.)叶发育研究中,as2是一个经典突变体.as2典型的表型是叶片开裂或形成一种小叶状结构.遗传学和分子生物学实验证明,AS2基因具有抑制KNOX基因在叶中表达的功能.在本文中,我们着重研究了新得到的在Landsberg erecta (Ler)遗传背景下的as2突变体.除了前人报道过的as2表型外,新as2突变体的部分叶柄长在叶片的下方,形成一种荷叶状结构,更严重的甚至长成花丝状叶结构.这两种结构都反映了不正常的叶腹背轴极性分化.在我们所收集到的as2等位突变体中,只有在Ler背景下这两种结构才以高频率出现.我们通过图位克隆方法分离了AS2基因.该基因编码一个含有亮氨酸拉链结构的蛋白.在拟南芥中,AS2同源基因共43个,除AS2外,其他基因的功能都不清楚.AS2在叶和花中表达,在茎中无表达,这种表达模式和as2突变体的表型是吻合的.     

分析拟南芥叶片在激素促进的衰老中内源激素变化来解析抑制磷脂酶Dα1延缓激素促进衰老的机制

采后衰老进程在很大程度上受到内源和外源激素的影响。抑制拟南芥中磷脂酶Dα1 (phospholipase Dα1, PLDα1)的表达后,使得外源脱落酸（abscisic acid,ABA）和乙烯加速的离体叶片衰老过程在一定程度上得到了缓解。然而,内源激素在这个过程中的作用尚不清楚。本研究对比分析了野生型和PLDα1缺失型两种基因型拟南芥叶片在3种不同人工老化过程中（离体诱导的、外源ABA和乙烯促进的衰老过程）,内源ABA,茉莉酸甲酯（methyl jasmonate,MeJA）、 吲哚乙酸（indole 3 acetic acid,IAA）、玉米素核苷（zeatin riboside,ZR）和赤霉素（gibberellic acid,GA3）的含量变化。这5种激素对3种不同衰老处理方式的响应模式表明了人工老化过程存在着两个不同阶段,并且在衰老早期每种激素的变化模式相同。PLDα1功能缺失使得激素加速的衰老过程得以延缓,这与内源ABA、MeJA、ZR和IAA的含量变化有关,而与GA3的含量变化无关。同时,ZR和IAA的变化模式也说明了这两种激素的变化可能是缺失PLDα1延缓激素加速的衰老过程这一事件的原因而非结果。     

The G4 Gene of Arabidopsis thaliana Encodes a Chlorophyll Synthase of Etiolated Plants

The gene coding for a putative chlorophyll synthase gene (C4) from Arabidopsis thaliana was amplified by the polymerase chain reaction and cloned into the expression vector pQE- 31. Lysates of bacteria (E.coli) that had been transformed with this construct were used for in vitro enzymatic assays. The chlorophyll synthase catalyzed esterification of chlorophyllides a and b at the same rate but preferred geranylgeranyl-PP over phytyl-PP. This corresponds to the enzyme specificity previously described for etiolated plants and differed from that of green plants.