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 dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Oleic acid-dependent modulation of NITRIC OXIDE ASSOCIATED1 protein levels regulates nitric oxide-mediated defense signaling in Arabidopsis

The conserved cellular metabolites nitric oxide (NO) and oleic acid (18:1) are well-known regulators of disease physiologies in diverse organism. We show that NO production in plants is regulated via 18:1. Reduction in 18:1 levels, via a genetic mutation in the 18:1-synthesizing gene SUPPRESSOR OF SA INSENSITIVITY OF npr1-5 (SSI2) or exogenous application of glycerol, induced NO accumulation. Furthermore, both NO application and reduction in 18:1 induced the expression of similar sets of nuclear genes. The altered defense signaling in the ssi2 mutant was partially restored by a mutation in NITRIC OXIDE ASSOCIATED1 (NOA1) and completely restored by double mutations in NOA1 and either of the nitrate reductases. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1 synthesizing SSI2 proteins were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen-induced or low-18:1-induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggest that 18:1 levels regulate NO synthesis, and, thereby, NO-mediated signaling, by regulating NOA1 levels.     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

Enhanced sensitivity to oxidative stress in an Arabidopsis nitric oxide synthase mutant

The possible involvement of nitric oxide (NO) in oxidative stress tolerance was studied using Arabidopsis thaliana wild type (WT) and Atnos1 mutant plants, in which endogenous NO production is greatly diminished because 80% of nitric oxide synthase (NOS) activity is eliminated due to T-DNA insertion in the first exon of the NOS1 gene. Compared with WT, Atnos1 mutant plants showed increased hypersensitivity to salt stress and methyl viologen (MV) treatment. The maximal photochemical efficiency of photosystem II (F(v)/F(m)) and membrane integrity decreased in WT and Atnos1 mutant plants under stresses, but the extent was higher in the mutant. Treatment with sodium nitroprusside (SNP) (a NO donor) to Atnos1 mutant plants alleviated the damage. Instead, inhibition of nitric oxide accumulation in the WT plants produced opposite effects. Hydrogen peroxide and lipid peroxidation increased and the extent was higher in Atnos1 mutant plants than that in WT plants under MV stress. These results indicated that nitric oxide could protect the damage against NaCl and MV treatments.     

In vivo sucrose stimulation of colour change in citrus fruit epicarps: Interactions between nutritional and hormonal signals

During ripening, citrus fruit-peel undergoes 'colour break', a process characterized by the conversion of chloroplast to chromoplast. The process involves the progressive loss of chlorophylls and the gain of carotenoids, changing peel colour from green to orange. In the present work, the in vivo and in vitro effects of supplemented nutrients (sucrose and nitrogen) and phytohormones (gibberellins [GA] and ethylene) on colour change in fruit epicarp of Satsuma mandarin ( Citrus unshiu (Mak.) Marc., cv. Okitsu), were studied. The rate of colour break was correlated positively with sucrose content and negatively with nitrogen content. The removal of leaves blocked natural sucrose build-up and nitrogen reduction in the peel. Defoliation also inhibited chlorophyll disappearance and carotenoid accumulation, thereby preventing colour break. In vivo sucrose supplementation promoted sucrose accumulation and advanced colour break. In both in vivo and in vitro experiments, colour change promoted by sucrose was unaffected by ethylene but delayed by GA_3. In non-supplemented plants, ethylene accelerated colour break while GA₃ had no detectable effects. Ethylene inhibitors effectively counteracted the sucrose effects on colour change. Collectively, these results suggest that the chloroplast to chromoplast conversion in citrus fruit epicarps is stimulated by sucrose accumulation. The sugar regulation appears to operate via ethylene, whereas GA may act as a repressor of the sucrose-ethylene stimulation.     

马铃薯NOA基因的克隆及序列分析(英文)

以马铃薯(Solanum tuberosum)为实验材料,利用电子克隆和RACE技术,从马铃薯中克隆出NOA(nitric oxide associated factor)基因,命名为StNOA1,测序结果表明,其cDNA序列长度为1 929 bp,此片段包含一个长为1 632 bp的完整编码框.氨基酸序列比对分析表明,StNOA1与烟草(Nicotiana benthamiana),葡萄(Vitis vinifera),蓖麻(Ricinus communis),水稻(Oryza sativa),玉米(Zea mays)以及拟南芥(Arabidopsis thaliana)均有很高的同源性 (89.44%～63.56%).同AtNOA1一样,StNOA1也具有保守的GTP结合区.从结构分析结果推测,StNOA1和AtNOA1在功能上有一定的相关性,其也可能通过调节内源NO的释放参与到植物生长、发育、抗逆等过程中.     

Sucrose enhances phosphoenolpyruvate carboxylase activity of in vitro solanum tuberosum L. under non-limiting nitrogen conditions

Summary The effect of sucrose on in vitro potato (ev. Kennebec) metabolism was evaluated. Plants were grown in three different media: Murashige and Skoog basal medium containing high nitrogen concentration with 0 or 20 g l⁻¹ sucrose; or modified medium containing reduced nitrogen amount and 20 g l⁻¹ sucrose. Plants fed with 20 g l⁻¹ sucrose and high N exhibited higher phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase activities and high PEPC protein concentration at 7, 20 and 33 d of culture compared to those grown with 20 g l⁻¹ sucrose and low N, or with 0 g l⁻¹ sucrose and high nitrogen (control). The highest accumulation of starch and sucrose was found in plants grown with sucrose and low nitrogen. This accumulation occurred concomitantly with a reduced enzyme activity resulting from a low utilization of α-ketoglutarate by nitrogen assimilation, when plants were grown with reduced nitrogen. Our investigations on tricarboxylic acid cycle activity showed that sucrose led to the reduction of organic acid amounts in both leaves and roots when high nitrogen was supplied to plants. This was probably due to the intense exit of α-ketoglutarate, which was confirmed by measurements of cytosolic isocitrate dehydrogenase activity. The low leaf glutamine/glutamate ratio observed in plants grown with 20 g l⁻¹ sucrose and high nitrogen compared to their counterparts cultivated with low nitrogen might be due to glutamine conversion into proteins when nitrogen assimilation was intense. These results demonstrate that sucrose enhanced PEPC activity by increasing protein synthesis. They also suggest that sucrose metabolism is involved in the replenishment of the tricarboxylic acid cycle by providing carbon skeletons required to sustain phosphoenolpyruvate utilization during high nitrate assimilation.     

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild‐type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non‐photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild‐type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light‐regulated manner.     

Low-temperature damage and subsequent recovery of fab1 mutant Arabidopsis exposed to 2 degrees C.

The fab1 mutant of Arabidopsis thaliana, which contains increased levels of saturated fatty acids, was indistinguishable from the wild type when it was grown at 22 or 12 degrees C. During the first 7 to 10 d after transfer to 2 degrees C, the growth and photosynthetic characteristics of the fab1 plants remained indistinguishable from the wild type, with values for the potential quantum efficiency of photosystem II decreasing from 0.8 to 0.7 in plants of both lines. Whereas wild-type plants maintained quantum efficiency of photosystem II at approximately 0.7 for at least 35 d at 2 degrees C, this parameter declined rapidly in the mutant after 7 d and reached a value of less than 0.1 after 28 d at 2 degrees C. This decline in photosynthetic capacity was accompanied by reductions in chlorophyll content and the amount of chloroplast glycerolipids per gram of leaf. Electron microscopic examination of leaf samples revealed a rapid and extensive disruption of the thylakoid and chloroplast structure in the mutant, which is interpreted here as a form of selective autophagy. Despite the almost complete loss of photosynthetic function and the destruction of photosynthetic machinery, fab1 plants retained a substantial capacity for recovery following transfer to 22 degrees C. These results provide a further demonstration of the importance of chloroplast membrane unsaturation to the proper growth and development of plants at low temperature.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light-dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far-red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re-etiolated dicotyledonous plants, showing rapid P_fr destruction and rapid P_fr dark reversion. In contrast, the light-dependent expression of genes encoding the major chlorophyll a- and b-binding protein (CAB) and the re-accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re-induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV-A receptor are involved in the light-dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration-dependent and light-quality-dependent regulation of CAB mRNA accumulation. As the dark-grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark-grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light-dependent signal transduction in cells of higher plants.     

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid — host cell interaction

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - CEVd citrus exocortis viroid - CSVd chrysanthemum stunt viroid - PSTVd potato spindle tuber viroid - TMV tobacco mosaic virus - phc photoheterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture     

Regulation of synthesis of the photosystem I reaction center

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light- induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark- grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Effect of chloramphenicol and cycloheximide on synthesis of delta-aminolevulinate dehydratase in green and greening barley shoots

The synthesis of sigma-aminolevulinate dehydratase (EC 4.2.1.24) in green or greening barley shoots was shown to increase, when the plants were grown on chloramphenicol solutions of varying concentrations for 48 hrs upon illumination. This was evidenced from the increase in the enzyme activity of the chloroplast preparations isolated from the shoots as compared to the controls grown in aqueous media. Similar treatment by cycloheximide resulted in inhibition of the enzyme synthesis as observed in the experiments with green and greening shoots. The activity of porphobilinogenase (the porphobilinogene deaminase and uroporphirinogene III cosynthetase complex) showed similar dependence on the effect of the antibiotics. The results obtained are discussed in terms of localization of the chloroplast enzyme syntheses inside the cell.     

Effects of the herbicide san 9789 on photomorphogenic responses

The herbicide, 4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)- 3(2H)-pyridazinone (San 9789), an inhibitor that prevents both carotenoid and chlorophyll accumulation and normal chloroplast development in white light, does not affect the physiological effectiveness of phytochrome in dark-and light-grown plants. Red/far red reversibility of growth inhibition, stimulation of anthocyanin synthesis, and stimulation of phenylalanine ammonia-lyase synthesis are not significantly different in plants grown with and without San 9789. Despite the complete absence of photosynthesis, flowering could be induced in the long day plant Hordeum vulgare L. when sucrose was provided to the leaves. Since the nonphotochemical reactions of phytochrome also are not affected by the herbicide, San 9789 may be used as a tool to study the phytochrome system spectrophotometrically in plants grown for relatively long periods under high intensity white light.