Phospholipids, free sterols and adenosine triphosphatase of plasma membrane-enriched preparations from roots of citrus genotypes differing in chloride exclusion ability

Plasma membrane-enriched preparations from fibrous roots of three citrus genotypes differing in their abilities for chloride exclusion, and grown in the presence of 0,50 or 100 mM NaCl for 4 weeks, were analysed for phospholipid and free sterol content and vanadate-sensitive adenosine triphosphatase (ATPase) activity over a range of temperatures. The best chloride excluder, Rangpur lime (Citrus reticulaia Blanco var. austera hyb.?), had significantly higher maximal ATPase activity than both the moderate chloride excluder. Kharna khatta (Citrus kharna Raf.), and the worst chloride excluder, Etrog citron (Citrus medico L.), at all assay temperatures below 28°C. Salt treatment had no effect on maximal ATPase activity of either Rangpur lime or Etrog citron but resulted in increased activity of the enzyme in Kharna khatta at temperatures below 28°C. Arrhenius plots of ATPase activity from the three citrus genotypes showed that, in controls, the activation energy (E.,) of Rangpur lime ATPase was significantly lower than that of both Kharna khatta and Etrog citron. The thermotropic phase transition temperature (T_f) for Rangpur lime (27°C) was also lower than for the other citrus genotypes (31°C). Salt treatment resulted in increases in both Ea and T, for Rangpur lime, decreases in both parameters for Kharna khatta and no change of either parameter for Etrog citron. An inverse relationship between E_a and the phospholipid to free sterol ratio was evident for plasma membrane preparations from all three citrus genotypes in the presence and absence of salt treatment suggesting that changes in membrane fluidity, particularly those induced by free sterols, have the potential to influence active as well as passive ion transport processes and thus may play a significant role in the chloride exclusion mechanism.     

Activation of abscisic acid biosynthesis in the leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> in response to water deficit

It is well known that endogenous abscisic acid (ABA) levels increase rapidly in response to drought stress and that this induces stomatal closure. In Arabidopsis thaliana, ABA levels increased rapidly in the leaves and roots when intact wild-type whole plants were exposed to drought stress. However, if the leaves and roots were separated and exposed to drought independently, the ABA level increased only in the leaves. These results suggest that, under our experimental conditions, ABA is synthesized mainly in the leaves in response to drought stress and that some of the ABA accumulated in the leaves is transported to the roots. Tracer experiments using isotope-labeled ABA indicate that the movement of ABA from leaves to roots is activated by water deficit in the roots. We also demonstrate that the endogenous ABA level in the leaves increased only when the leaves themselves were exposed to drought stress, suggesting that leaves play a major role in the production of ABA in response to acute water shortage.     

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

5个毛果杨PtrZFP基因的鉴定和表达分析

ZFP转录因子是植物中的一类具有指环结构域的转录因子。从毛果杨中鉴定出5条ZFP基因（命名为PtrZFP1-5）,对其特性和表达模式进行了分析,以期初步了解这些基因是否能对胁迫做出应答。对PtrZFP1-5基因进行生物学分析,进一步利用qRT-PCR技术分析NaCl、PEG₆₀₀₀和ABA胁迫处理后毛果杨根、茎和叶中5条基因的表达情况。PtrZFP1-5基因编码蛋白氨基酸残基数为258~338 aa,编码蛋白的分子量为27.7~37.3 kDa,理论等电点为4.87~8.61,5个基因不均等的分布在毛果杨基因组的3条染色体上。qRT-PCR结果显示,0.2 mol·L^-1 NaCl、15%（w/v）PEG6000和100 μmol·L^-1 ABA胁迫处理后,5个PtrZFP基因在毛果杨根、茎和叶中的表达模式明显不同。PtrZFP1基因在3种胁迫后毛果杨中均被明显的上调表达;PtrZFP2基因在盐、渗透和ABA胁迫处理后,叶中的表达都明显被抑制;PtrZFP3基因受到干旱胁迫时在根中的响应最为明显;而叶和茎中,表达量在大部分胁迫的大部分时间点无明显改变。PtrZFP4基因也能在根和茎中对干旱胁迫做出明显应答。PtrZFP5基因在经受盐和ABA胁迫后,在叶中的表达受到明显抑制。PtrZFP1-5这5个基因至少能在一种器官中对一种胁迫处理做出应答,但参与的胁迫应答类型和机制可能不同。     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

Gas exchange is related to the hormone balance in mycorrhizal or nitrogen-fixing alfalfa subjected to drought

The beneficial effect of mycorrhization on photosynthetic gas exchange of host plants under drought conditions could be related to factors other than changes in phosphorus nutrition and water uptake. Our objective was to study the influence of drought on phytohormones and gas exchange parameters in Medicago sativa L. cv. Aragón associated with or in the absence of arbuscular mycorrhizal (AM) fungi and/or nitrogen-fixing bacteria. Four treatments were used: (1) plants inoculated with Glomus fasciculatum (Taxter sensu Gerd.) Gerdemann and Trappe and Rhizobium meliloti 102 F51 strain (MR); (2) plants inoculated with only Rhizobium (R); (3) plants inoculated with only mycorrhizae (M); and (4) non-inoculated plants (N). When endophytes were well established, treatments received different levels of phosphorus and nitrogen in the nutrient solution in order to obtain plants similar in size. Sixty days after planting, plants were subjected to two cycles of drought and recovery. Midday leaf water potential (Ψ), CO₂ exchange rate (CER), leaf conductance (g_w) and transpiration (T), as well as leaf and root abscisic acid (ABA) and cytokinin concentrations were measured after the second drought period. Gas exchange parameters were determined by infrared gas analysis. Cytokinins and ABA levels in tissues were analysed by ELISA and HPLC, respectively. Nodulated R and MR plants had the lowest ABA concentrations in roots under well-watered conditions. Water stress increased ABA concentrations in leaves of N, R and MR plants, while ABA concentration in M plants did not change. The highest production of ABA under water deficit was in the roots of non-mycorrhizal plants. The ratio of ABA to cytokinin concentration strongly increased in leaves and roots of non-mycorrhizal plants under drought. By contrast, this ratio was lowered in roots of M plants and remained unchanged in leaves and roots of MR plants when stress was imposed. The highest leaf conductances and transpirational fluxes under well-watered conditions were those of nitrogen-fixing R and MR plants, but these results were not impaired with increased CO₂ exchange rates. Photosynthesis, leaf conductance and transpiration rates decreased in all treatments when stress was imposed, with the strongest decrease occurring in non-mycorrhizal plants. The relationships found between these gas exchange parameters and the hormone concentrations in stressed alfalfa tissues suggest that microsymbionts have an important role in the control of gas exchange of the host plant through hormone production in roots and the ABA/cytokinin balance in leaves. The most relevant effect of mycorrhizal fungi was observed under drought conditions.     

Cloning of Ten Peroxidase (<Emphasis Type="Italic">POD</Emphasis>) Genes from <Emphasis Type="Italic">Tamarix Hispida</Emphasis> and Characterization of their Responses to Abiotic Stress

Plant peroxidases (PODs) have been ascribed a variety of biological functions, including hydrogen peroxide detoxification, lignin biosynthesis, hormonal signaling, and stress response. In the present study, ten POD genes, including three ascorbate peroxidases (class I PODs) and seven secretory peroxidases (class III PODs), were cloned from Tamarix hispida. The roles of the ten POD genes were addressed under different abiotic stress conditions, and gene expression profiles in roots, stems, and leaves were evaluated using real-time quantitative reverse-transcribed polymerase chain reaction. Our results showed that the relative abundance of the PODs was markedly different in roots, stems, and leaves, indicating that POD activity differs in these three organs. ThPOD1 and ThPOD8 were the most and least abundant, respectively, in all organs. The expression profiles in response to abiotic stresses were organ specific. All of the genes were highly induced by drought, salt, salt–alkaline, CdCl₂, and abscisic acid (ABA) treatments in at least one organ. Five ThPOD genes were induced in roots, stems, and leaves under all of the studied stress conditions, indicating that they are closely associated with abiotic stress. Our results demonstrate that the ten plant peroxidases are all expressed in leaves, stems, and roots, that they are involved in different abiotic stress responses, and that they are controlled by an ABA-dependent stress signaling pathway.     

Characterization of potential ABA receptors in Vitis vinifera

Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. The phytohormone abscisic acid (ABA) is a key endogenous messenger in a plant’s response to such stresses. A novel ABA binding mechanism which plays a key role in plant cell signaling cascades has recently been uncovered. In the absence of ABA, a type 2C protein phosphatase (PP2C) interacts and inhibits the kinase SnRK2. Binding of ABA to the PYR/PYLs receptors enables interaction between the ABA receptor and the PP2C protein, and abrogates the SnRK2 inactivation. The active SnRK2 is then free to activate the ABA-responsive element Binding Factors which target ABA-dependent gene expression. We used the grape as a model to study the ABA perception mechanism in fruit trees. The grape ABA signaling cascade consists of at least seven ABA receptors and six PP2Cs. We used a yeast two-hybrid system to examine physical interaction in vitro between the grape ABA receptors and their interacting partners, and found that twenty-two receptor-PP2C interactions can occur. Moreover, quantifying these affinities by the use of the LacZ reporter enables us to show that VvPP2C4 and VvPP2C9 are the major binding partners of the ABA receptor. We also tested in vivo the root and leaf gene expression of the various ABA receptors and PP2Cs in the presence of exogenic ABA and under different abiotic stresses such as high salt concentration, cold and drought, and found that many of these genes are regulated by such abiotic environmental factors. Our results indicate organ specificity in the ABA receptor genes and stress specificity in the VvPP2Cs. We suggest that VvPP2C4 is the major PP2C involved in ABA perception in leaves and roots, and VvRCAR6 and VvRCAR5 respectively, are the major receptors involved in ABA perception in these organs. Identification, characterization and manipulation of the central players in the ABA signaling cascades in fruit trees is likely to prove essential for improving their performance in the future.     

Exogenous 5-aminolevulinic acid improves strawberry tolerance to osmotic stress and its possible mechanisms

Cultivated strawberry, one of the major fruit crops worldwide, is an evergreen plant with shallow root system, and thus sensitive to environmental changes, including drought stress. To investigate the effect of 5-aminolevulinic acid (ALA), a new environment-friendly plant growth regulator, on strawberry drought tolerance and its possible mechanisms, we treated strawberry (Fragaria × annanasa Duch. cv. ‘Benihoppe’) with 15% polyethylene glycol 6000 to simulate osmotic stress with or without 10 mg l⁻¹ ALA. We found that ALA significantly alleviated PEG-inhibited plant growth and improved water absorption and xylem sap flux, indicating ALA mitigates the adverse effect of osmotic stress on strawberry plants. Gas exchange and chlorophyll fluorescence analysis showed that ALA mitigated PEG-induced decreases of P_n, G_s, T_r, P_n/C_i, photosystem I and II reaction center activities, electron transport activity, and photosynthetic performance indexes. Equally important, ALA promoted PEG-increased antioxidant enzyme activities and repressed PEG-increased malondialdehyde and superoxide anion in both leaves and roots. Specially, ALA repressed H₂O₂ increase in leaves, but stimulated it in roots. Furthermore, ALA repressed abscisic acid (ABA) biosynthesis and signaling gene expressions in leaves, but promoted those in roots. In addition, ALA blocked PEG-downregulated expressions of plasmalemma and tonoplast aquaporin genes PIP and TIP in both leaves and roots. Taken together, ALA effectively enhances strawberry drought tolerance and the mechanism is related to the improvement of water absorption and conductivity. The tissue-specific responses of ABA biosynthesis, ABA signaling, and H₂O₂ accumulation to ALA in leaves and roots play key roles in ALA-improved strawberry tolerance to osmotic stress.     

西葫芦NCED基因家族鉴定及其响应干旱胁迫分析

NCED基因家族成员在调节植物响应干旱胁迫中发挥着关键作用,该研究通过生物信息学技术分析NCED在西葫芦基因组中的分布、结构及进化,研究家族成员在不同组织中的表达特异性及其对10%PEG 6000模拟干旱、0.1 mmol·L-1ABA激素和自然干旱胁迫的响应,以解析NCED基因家族的生物学功能。结果表明:(1)从西葫芦全基因组中鉴定出6个NCED家族基因(CpNCED1~6),且6个基因均不含内含子、分别分布于西葫芦的1、10、12、14、19和20号共6条染色体上。(2)理化性质分析发现,CpNCED1~6蛋白长度为569~590 aa,理论分子量在62.64~65.54 kD之间。(3)蛋白保守元件分析显示,除CpNCED3蛋白在遗传进化过程中出现3个基序(motif 12、motif 13和motif 15)的缺失外,其余5个蛋白都有完整的16个motif保守基序,且分布在600个氨基酸以内,同时大部分NCED蛋白序列保守性较高。(4)顺式作用元件分析显示,西葫芦CpNCED1~6基因均含ABRE、W box、MBS、P-box、TCA-element、CGTCA-motif、TGA-element和TGA-box等潜在的干旱胁迫响应元件。(5)qRT-PCR分析表明,CpNCED1~6基因在西葫芦不同组织中的表达具有组织特异性,其中,CpNCED4和CpNCED1在茎中的表达量显著高于其他4个基因,CpNCED2、CpNCED4、CpNCED6在花中的表达显著高于其余3个基因且CpNCED2表达量最高,CpNCED1~6在果实和叶中的表达量均相对较低;与对照组相比,CpNCED1~6受模拟干旱、ABA激素和自然干旱胁迫均上调表达;伴随干旱胁迫的产生,叶片中脱落酸(ABA)含量逐渐升高,暗示CpNCEDs在西葫芦干旱胁迫响应与ABA的生物合成过程中发挥着正向调控作用。研究发现,6个CpNCED1~6基因与西葫芦干旱胁迫响应密切相关,且对西葫芦干旱胁迫的响应以及ABA生物合成具有重要作用,尤其以CpNCED2和CpNCED4基因的作用更为明显。