Sap flow measurements in grapevines (Vitis vinifera L.) 2. Granier measurements

The Granier-system, a relatively simple and continuous method for measuring sap flux density, has been adapted and evaluated for its use in older, mature grapevines. The original calibration of Granier (1985, Annales Sciences Forestieres, 42, 193-200) could be extended to a sap flux density of up to 400 10^-6 m³ m^-2 s^-1 with only little error at high flux densities. A time lag of around 20 min was apparent between transpiration and calculated sap flow which was attributed to the thermal mass of the sensors themselves. The time lag and the consequently dampened response of the system caused a very low accuracy over short time periods thus reducing the value for detailed plant physiological investigations. However, when integrating over longer time intervals, much of the error cancelled out. For daily values the maximum error was within ±10% and after a period of 89 days only 1.5% error remained. This method is thus best suited for long term measurements of total water use. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of exogenous H2O2 on antioxidant enzymes of Brassica juncea L. seedlings in relation to 24-epibrassinolide under chilling stress

Hydrogen peroxide is most stable molecule among reactive oxygen species, which play a vital role in growth and development of plant as signaling molecule at low concentration in response to various abiotic and biotic stresses. Exogenous application of H2O2 is known to induce chilling tolerance in plants. Brassinosteroids are plant steroid hormones known for their anti-stress properties. In this study, effect of exogenous H2O2 on antioxidant defense system of Brassica juncea L. seedlings was investigated in 24-epibrassinolide (24-EBL) treated and untreated seedlings under chilling stress. The surface sterilized seeds of B. juncea L. were germinated in petriplates containing different concentrations of H2O2 alone and in combination with 10(-8) M 24-EBL. Chilling treatment (4 degrees C) was given to 10-days old seedlings grown in different treatments for 6 h daily up to 3 days. 24 h recovery period was given to chilling treated seedlings by placing at 25 degrees C + 2 degrees C and harvested for antioxidant enzymes on 14th day after sowing (DAS). Treatment of 24-EBL in combination with H2O2 (15 and 20 mM) helped in reducing the toxicity of seed and seedlings due to H2O2 exposure on their germination rate, shoot and root length respectively. 24-EBL treatment at seed and seedling stage helped in alleviating the toxic effect of H2O2 through antioxidant defense system by increasing the activities of various enzymes involved in antioxidant defense system such as catalase (CAT, E.C. 1.11.1.6), ascorbate peroxidase (APOX, E.C. 1.11.1.11), and superoxide dismutase (SOD, E.C. 1.15.1.1). In conclusion, exogenous pretreatment of H2O2 to seeds of B. juncea L. adapted the seedlings to tolerate chilling stress, which was further ameliorated in combination of H2O2 with 24-EBL.     

Microsatellite inferred genetic diversity and structure of Western Balkan grapevines (Vitis vinifera L.)

A collection of 196 grapevine samples from five countries of the Western Balkan region, representing local and traditional cultivars, was genotyped with 22 SSR markers. Identity analysis revealed 125 unique genotypes, which were further used for diversity assessment. The average number of alleles per locus detected was 11?±?3.53, ranging from 6 to 21. The low cumulative probability of identical genotypes (2.96?×?10^?20) shown in this study implies an even distribution of alleles within the analyzed set of grapevines and a sufficient number of loci. On the basis of the discriminatory power of each SSR, a set of five markers (VVMD5, VVMD7, VVMD28, VChr3a, and VChr8b) was determined as sufficient for high-throughput discrimination of the target cultivars. The maximum discriminating power was evidenced for loci VVMD28 and Vchr8b (0.96, 0.94, respectively). A core collection covering the entire genetic diversity resulted in a set of 60 genotypes representing approximately 50 % of the samples from each country. Structure clustering of Balkan and West European cultivars resulted in four well distinct groups identified according to the classification of Negrul (1946). The lowest level of admixed genotypes was assigned for grapevines from Bosnia and Herzegovina (61 %) and the highest for Serbian (87 %) grapevines. In terms of grape use, the wine cultivars were divided into three groups and the fourth group was intermixed, with half wine and half table grapes. The highest Nei’s genetic distance (0.22) was discovered between Slovenian and Macedonian cultivars, while the lowest (0.09) was between Slovenian and Serbian cultivars. Macedonian cultivars were genetically most distant from the others (0.17). A similar pattern of differentiation among populations is seen with distance-based clustering. Analysis of molecular variance revealed only 1 % of genetic variation among groups of different origin, while the variation among individuals within geographical groups and within individuals explained 13 and 86 % of the total variation, respectively.     

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.     

外源24-表油菜素内酯对弱光胁迫下番茄幼苗叶片形态及光合特性的影响

以弱光敏感型番茄品种‘基尔斯’为试验材料,采用营养液栽培,研究了外源24-表油菜素内酯对弱光胁迫下番茄幼苗叶片形态和光合特性的影响.结果表明：弱光胁迫下番茄幼苗叶片形态产生适应性变化,叶面积、比叶面积、茎叶夹角、茎叶垂角、垂度均显著提高,而叶片干质量显著降低;最大净光合速率、表观量子效率、暗呼吸速率、羧化效率、Rubisco大亚基含量均显著降低,而光补偿点和CO₂补偿点显著升高.弱光胁迫下叶面喷施24-表油菜素内酯后,叶面积、叶片干质量、茎叶夹角、茎叶垂角分别增加14.1%、57.1%、12.3%和7.7%,比叶面积、垂度分别减小30.5%和10.6%;表观量子效率、暗呼吸速率、羧化效率分别提高20.4%、17.9%和9.3%,光补偿点、CO₂补偿点分别降低21.9%和4.3%,差异均达到显著水平;Rubisco大亚基含量也显著升高.说明外源24 表油菜素内酯可以通过提高弱光下番茄幼苗叶片的表观量子效率、暗呼吸速率、羧化效率及Rubisco含量,降低光补偿点和CO₂补偿点,并维持叶片形态的稳定性,来改善光合性能,有效缓解弱光胁迫对番茄幼苗的伤害.     

Potassium nutrition of Cabernet Sauvignon grapevines (Vitis vinifera L.) as affected by shoot trimming

As potassium (K) requirement of grapevine (Vitis viniferaL.) berries is high and phloem translocation from mature leaves to developing organs is well established, it was posited that shoot trimming, a widely applied technique which alters the source-sink balance and the mature-to-immature foliage ratio of a canopy, may influence K deficiency. Six-year-old Cabernet Sauvignon grapevines were grown in 0.045 m³ pots (one plant per pot) filled with a soil sampled from a vineyard previously displaying K deficiency symptoms. Two levels of K supply (K₀, no K added; K_1, 25 g K per pot added in five splits from bloom to post veraison) were tested on vines that for each level were left (a) untrimmed and trimmed at ten main leaves with (b) or without (c) maintenance of lateral shoots in a split-plot design. Potassium concentration of leaf blades, berries and canes, vegetative growth and leaf gas exchange were recorded throughout the season; yield, grape quality and must characteristics were determined at fruit maturity. While adding potassium to the soil resulted in higher K concentration in blades of both main and lateral shoots and berries at harvest, trimming with removal of lateral shoots resulted in lower blade K concentration of the main leaves at harvest and more severe K deficiency symptoms regardless of K soil availability. Berry K concentration and the fraction of whole-plant K partitioned to clusters were not significantly affected by trimming. Gas-exchange, vegetative growth, yield and grape quality were not affected by the seasonal K fertilization, whereas the latter was impaired when trimming with excision of lateral shoots was applied. The results indicate that if shoot trimming is not followed by an adequate regrowth of secondary shoots an excessive depletion of potassium from the retained old basal leaves occurs during fruit maturity and increases the risk of leaf K deficiency, particularly in the K₀ treatment.     

The effect of 24-epibrassinolide and clotrimazole on the adaptation of Cajanus cajan (L.) Millsp. to salinity

The objective of this study was to evaluate the effects of one of brassinosteroids (24-epibrassinolide) and clotrimazole, (an inhibitor of brassinosteroid synthesis) on plant growth parameters, parameters related to leaf gas exchange (photosynthetic and transpiration rates; stomatal conductance; water use efficiency), photosynthetic pigment content and osmolyte (sugars and proline) content in Cajanus cajan exposed to salinity. Salt stress??caused by NaCl treatment??affected values of all parameters analyzed. The effects were ameliorated by 24-epibrassinolide and intensified by clotrimazole. The hormone increased fresh mass of the plant, shoot dry mass, leaf area, water content of leaves and roots, photosynthetic pigments, sugar concentration, photosynthetic rate, and water use efficiency. The effects of hormone were less evident in the absence of salt. However, under this condition the application of clotrimazole affected the values of parameters studied, indicating the importance of brassinosteroid synthesis for the normal development of the plant.     

Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

Effects of 24-epibrassinolide on the photosynthetic characteristics, antioxidant system, and chloroplast ultrastructure in Cucumis sativus L. under Ca(NO3)2 stress

The effects of 0.1?μM 24-epibrassinolide (EBL) on plant growth (plant height, leaf area, fresh weight, and dry weight), chlorophyll content, photosynthetic characteristics, antioxidant enzymes, and chloroplast ultrastructure were investigated using cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) with 80?mM Ca(NO(3))(2) to induce stress. The presence of Ca(NO(3))(2) caused significant reductions in net photosynthetic rate (P (N)), stomatal conductance (Gs), intercellular CO(2) concentration (Ci), and transpiration rate (Tr) of leaves. In addition, Ca(NO(3))(2) markedly reduced the chlorophyll content and inhibited photochemical activity, including the actual photochemical efficiency (ΦPSII). In contrast, EBL increased the chlorophyll content, especially chlorophyll b, and minimized the harmful effects on photosynthesis caused by the Ca(NO(3))(2). The application of EBL to the plants subjected to Ca(NO(3))(2)-enhanced photochemical activity. EBL protected the photosynthetic membrane system from oxidative damage due to up-regulating the capacity of the antioxidant systems. Microscopic analyses revealed that Ca(NO(3))(2) affected the structure of the photosynthetic apparatus and membrane system and induced damage of granal thylakoid layers, while EBL recovered the typical shape of chloroplasts and promoted the formation of grana. Taken together, EBL compensated for damage/losses by Ca(NO(3))(2) due to the regulation of photosynthetic characteristics and the antioxidant system.     

Sap flow measurements in grapevines (Vitis vinifera L.) 1. Stem morphology and use of the heat balance method

The heat balance method was evaluated in detail for its use in older, mature grapevines with stems of 35 – 45 mm in diameter. Dye colouring of the xylem vessels revealed that even 21 year old grapevines did not show any development of heartwood and that xylem vessels of that age still have the capacity to transport water. A comparison of weight loss of potted vines on a balance and sap flow measurements demonstrated that the heat balance system reflected rapid changes in flow rate without any time delay. However, since even 20 year old xylem vessels of grapevines have the capacity to conduct water, the heater band was not able to heat the sap in all year rings evenly. Apparently, at low flow rates this effect was small and sap flow was calculated correctly. With increasing flow rates large thermal heterogeneities developed upsetting the calculation of the heat balance and mass flow. Consequently, actual sap flow was overestimated by 50 to 100% at high flow rates. This could be attributed to thermal gradients in these relatively thick stems excluding the use of this technique for measurements of long term as well as short term water use patterns in older grapevines. This revised version was published online in June 2006 with corrections to the Cover Date.     

低氧胁迫下24-表油菜素内酯对黄瓜幼苗根系生长及其无氧呼吸同工酶表达的影响

采用营养液水培,研究了低氧胁迫下24-表油菜素内酯(EBR)对黄瓜幼苗根系生长及其无氧呼吸同工酶表达的影响.结果表明:低氧胁迫增强了黄瓜幼苗根系丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)、乳酸脱氢酶(LDH)同工酶的表达,低氧胁迫下施用外源EBR的第3天PDC、ADH同工酶的表达量分别提高了18.8％、28.8％,而第6、第9天PDC、ADH、LDH同工酶的表达减弱,比单纯低氧处理分别降低19.5％、25.6％、53.4％及26.4％、26.0％、28.4％;低氧胁迫至第9天,黄瓜幼苗根系的生长受到了显著抑制(P＜0.05),而低氧胁迫下施用EBR,黄瓜幼苗根系的生长受抑制程度减轻,其根系总长、干重、根尖数较单纯低氧处理显著增加(P＜0.05),低氧抑制了黄瓜幼苗根系的生长,低氧胁迫下营养液添加EBR可调节黄瓜根系无氧呼吸同工酶的表达,缓解低氧胁迫对黄瓜幼苗根系的伤害.     

低氧胁迫下24-表油菜素内酯对黄瓜幼苗叶片光合特性及多胺含量的影响

采用1/2 Hoagland营养液培养,研究了低氧胁迫下24-表油菜素内酯(EBR)对黄瓜幼苗叶片光合特性及多胺含量的影响.结果表明：低氧胁迫下黄瓜幼苗的净光合速率(P_n)、气孔导度(g_s)、蒸腾速率(T_r)、胞间CO₂浓度(C_i)显著下降,而叶绿素含量显著提高,幼苗生长受抑;低氧胁迫显著提高了黄瓜幼苗叶片的腐胺(Put)、亚精胺(Spd)、精胺(Spm)、多胺(PAs)含量和Put/PAs,但降低了(Spd+Spm) /Put.低氧胁迫下,外源EBR不仅显著提高了黄瓜幼苗的P_n、g_s、T_r及叶绿素含量,也显著提高了黄瓜幼苗叶片的游离态Spm、结合态Spd、Spm及束缚态Put、Spd、Spm含量,促进了PAs的进一步积累,且降低了Put/PAs,提高了(Spd+Spm)/Put．可见,外源EBR调节了黄瓜幼苗内源多胺含量及形态的变化,维持了较高的光合性能,促进了叶面积和干物质量的显著增加,缓解了低氧胁迫对黄瓜幼苗的伤害.     

Effect of 24-epibrassinolide on the photosynthetic activity of radish plants under cadmium stress

The present study was conducted to study the effect of 24-epibrassinolide (EBL) on changes of plant growth, net photosynthetic rate, carbonic anhydrase (E.C. 4.2.1.1) and nitrate reductase (E.C.1.6.6.1) activities in the leaves of Raphanus sativus L. under the influence of cadmium (Cd) stress. Cd reduced plant growth, photosynthetic pigment levels, net photosynthetic rate and the activities of carbonic anhydrase and nitrate reductase. However seed application of EBL reduced the toxic effect of Cd on plant growth, pigment content, photosynthesis and enzyme activities. The studies clearly demonstrated the ameliorating effect of 24-epibrassinolide in mitigating the toxicity of Cd in plants.     

Effects of 24-epibrassinolide and the synthetic brassinosteroid mimic on chili pepper under drought

Drought is major stress that severely reduces plant growth and productivity. To improve drought tolerance, an exogenous brassinosteroids (BRs) has been used effectively in the field condition. However, the application of BRs is expensive due to the scarcity of natural BRs and the multistep synthesis of BRs. In an attempt to reduce the cost, 7,8-dihydro-8α-20-hydroxyecdysone (DHECD) has been proposed to function as an imitation of 24-epibrassinolide (EBR). In this study, chili pepper plants (Capsicum annuum L. var. frutescens (L.) Kuntze) were sprayed with DHECD, EBR at 1 µM or distilled water (control). Plants were subjected to severe water stress (25% pot water capacity) for 5 days and their physiological effects and yield were investigated. The result showed that the applications of DHECD and EBR before the beginning of water stress could improve leaf water status determined by relative water content in plants grown under drought condition. The electrolyte leakage, lipid peroxidation level, and H₂O₂ production were significantly declined, while the accumulations of proline and total soluble sugar were increased in the treated plants. Moreover, the net photosynthesis (P_N) was elevated due to the increases of stomatal conductance (g_s) and intercellular CO₂ concentration (C_i) after BR pretreatments under drought. In addition, applications of DHECD and EBR maintained all chlorophyll fluorescence parameters; F_v/F_m, F_v′/F_m′, ΦPSII, qP, and ETR, to remain the photosynthesis. As a result, shoot biomass, fruit yield and capsaicin level were considerably enhanced in the treated plants. DHECD showed better performance to maintain membrane integrity; however, EBR had more effect on the osmotic maintenance. The result also showed that pretreatment with BRs had little or no effect on well-watered plants. The study concluded that DHECD and EBR alleviated the impact of drought on physiological responses and consequently minimized yield loss.     

Effects of mercury on ion and osmoregulation in the shore crab Carcinus maenas (L.)

The effects of lethal mercury concentrations on regulation of hemolymph electrolytes in the shore crab Carcinus maenas were investigated. In most experiments mercury exposure reduced hemolymph osmolality and Na+, Cl- and K+ levels to 55-90% of controls in 48 hr. Mercury exposure augmented calcium levels to 120-300% of controls. The effect of mercury on all of the electrolytes varied during the year, especially on magnesium levels which showed significant increases or decreases, or remained unaffected in different experiments.     

Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress

Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin.     

Effect of 24-epibrassinolide on growth, protein content and antioxidative defense system of Brassica juncea L. subjected to cobalt ion toxicity

Brassica juncea L. plants were subjected to cobalt (Co) ion (0, 5?×?10^?4, 10^?3, 1.5?×?10^?3 and 2?×?10^?3?M) toxicity and were sprayed with different concentrations of 24-epibrassinolide (24-EBL) (0, 10^?10, 10^?8 and 10^?6?M) at 15-day stage after sowing. They were sampled at 30 and 60?days after sowing and analyzed for growth parameters in terms of shoot length and number of leaves. Thereafter, leaves were excised and content of proteins and the activities of antioxidative enzymes (superoxide dismutase (SOD) (EC 1.15.1.1) catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APOX) (EC 1.11.1.11), guaiacol peroxidase (POD) (EC 1.11.1.7) glutathione reductase (GR) (EC 1.6.4.2), monodehydroascorbate reductase (MDHAR) (EC 1.1.5.4) and dehydroascorbate reductase (DHAR) (EC 1.8.5.1)) were analyzed. The plants exposed to cobalt ion exhibited a significant decline in growth in terms of shoot length and number of leaves. However, foliar spray treatment with 24-EBL was able to alleviate the stress generated by cobalt ion and significantly improved the above parameters. The activities of antioxidative enzymes (SOD, CAT, POD, GR, APOX, MDHAR and DHAR) and protein content were also regulated considerably in leaves of plants treated with 24-EBL alone, 10^?8?M concentration being the most effective. The activities of antioxidative enzymes also increased in leaves of B. juncea plants by the application of cobalt ion to soil and consequently sprayed with 24-EBL. Similarly, the protein content was also regulated in leaves of B. juncea plants treated with 24-EBL as compared to untreated control plants, thereby revealing stress-protective properties of 24-EBL.     

Protection of photosynthesis and antioxidative system by 24-epibrassinolide in Solanum melongena under cold stress

This study was carried out to understand the mechanism of protection of plants under cold stress by exogenous 24-epibrassinolide (EBR). The eggplant (Solanum melongena L.) seedlings were pretreated with five concentrations of EBR (0, 0.05, 0.1, 0.2 and 0.4 °M) and then exposed to day/night temperatures of 10/5 °C for 8 d. The results show that EBR, especially 0.1 °M EBR, dramatically alleviated growth suppression and a decrease in chlorophyll content and photosynthetic rate caused by the cold stress. In addition, EBR also decreased malondialdehyde content and O₂ ^·? production rate induced by the cold stress, and increased the activities of superoxide dismutase, guaiacol peroxidase, catalase, and ascorbate peroxidase, and proline content. The results of the present study suggest that exogenous EBR could improve cold tolerance of eggplant by regulating photosynthesis and antioxidative systems.     

In vitro root induction by 24-epibrassinolide on hypocotyl segments of soybean [Glycine max (L.) Merr.]

Soybean hypocotyl segments were treated in the dark with 24-epibrassinolide (BR) at a range of concentrations for different durations. The maximum effect on adventitious root induction, both in terms of number and length was obtained at very low concentration (0.0001 ppm) of BR applied for 8 h. Higher concentrations were supraoptimal unless applied for a shorter period (4 h). BR was ineffective when applied at low concentration in continuous light.