Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato

Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease. InP. fluorescens-treated plants, the peroxidase and phenylalamine ammonia-lyase activity increased. Accumulation of phenolic compounds and lignin were shown to be increased in theP. fluorescens-treated plants. Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV.     

Comparison of spectrophotometric and radioisotopic methods for the assay of Rubisco in ozone-treated plants

Radioisotopic and spectrophotometric assays for ribulose-1,5-bisphosphate carboxy-lase/oxygenase (Rubisco) initial and final activities and Rubisco content were compared in plants chronically exposed to ozone (O₃) in a greenhouse and the field. In a greenhouse experiment, Glycine max was treated in exposure chambers with either charcoal-filtered air (CF air) or 100 nl O₃ 1⁻¹ for 6 h daily during vegetative growth. Samples were collected after 7 days of exposure. In a field experiment, G. max was treated in open-top chambers with either CF air or nonfiltered air with O₃ added at 1.5 times ambient O₃ for 12 h daily. Average daily O₃ concentrations were 21 and 92 nl T1 in the CF and O₃ treatments, respectively. Samples were collected during vegetative and reproductive growth. Both assays generally yielded comparable Rubisco initial and final activities for greenhouse-grown plants regardless of the O₃ treatment. However for field-grown plants, Rubisco initial and final activities averaged 15 and 23% lower when assayed by the spectrophotometric rather than the radioisotopic method. For Rubisco content estimated by the spectrophotometric method, lower r² values for the regression of Rubisco activity vs concentratio of carboxyarabinitol-1,5-bisphos-phate were observed in O₃ than in CF-treated plants. Both assays yielded comparable Rubisco contents in the greenhouse and in the field although the variation was larger with the spectrophotometric method in field-grown plants. Growth conditions, field vs greenhouse, were more critical to the spectrophotometric assay performance than the O₃ treatments for measurement of Rubisco activity and content.     

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.     

Tropospheric ozone-induced structural changes in leaf mesophyll cell walls in grapevine plants

The structural changes in leaves of grapevine plants (Vitis vinifera L.) exposed to different ozone concentrations were investigated. Ozone fumigations were performed in open-top chambers at four different ozone levels (charcoal-filtered air (F), ambient air (N), ambient air + 25 mm³m⁻³ ozone (O-25) and ambient air + 50 mm³m⁻³ ozone (O-50)). The leaves of plants from chambers with increased ozone concentrations (O-25 and O-50) were significantly thicker than the controls (F), owing to increased thickness of the mesophyll layer. Observing O-50 leaves, it was found that the mesophyll cell wall displayed structural changes. In some places cell wall thickness increased up to 1 μm. We found callose deposits on the inner side of the cell walls of mesophyll cells. These data are in accord with the concept that the mesophyll cell wall acts as a barrier against the penetration of tropospheric ozone into the cells.     

Effects of ozone on apoplast/cytoplasm partitioning of ascorbic acid in snap bean

Apoplast/cytoplasm partitioning of ascorbic acid (AA) was examined in four genotypes of snap bean ( Phaseolus vulgaris L.) known to differ in ozone sensitivity. Plants were grown in pots under field conditions using open-top chambers to establish charcoal-filtered (CF) air (36 nmol mol⁻¹ ozone) or elevated ozone (77 nmol mol⁻¹ ozone) treatments. AA in fully expanded leaves of 36-day-old plants was separated into apoplast and cytoplasm fractions by vacuum infiltration methods using glucose 6-phosphate as a marker for cytoplasm contamination. Apoplast ascorbate levels ranged from 30 to 150 nmol g⁻¹ fresh weight. Ozone-sensitive genotypes partitioned 1–2% of total AA into the apoplast under CF conditions and up to 7% following a 7-day ozone exposure. In contrast, an ozone-tolerant genotype partitioned 3–4% of total leaf AA into the leaf apoplast in both CF and ozone-treated plants. The results suggest that genetic background and ozone stress are factors that affect AA levels in the extracellular space. For all genotypes, the fraction of AA in the oxidized form was higher in the apoplast compared to the cytoplasm, indicative of a more oxidizing environment within the cell wall.     

Responses of gas exchange,cellular membrane integrity,and antioxidant enzymes activities of salinity-stressed winter wheat to ozone pollution

A sand-culture experiment was conducted in open-top chambers which were constructed in a greenhouse to investigate the responses of salt-stressed wheat (Triticum aestivum L.) to O₃. Plant seeding of JN17 (a popular winter wheat cultivar) was grown in saltless (−S) and saline (+S, 100 mM NaCl) conditions combined with charcoal-filtered air (CF, < 5 ppb O₃) and elevated O₃ (+O₃, 80 ± 5 ppb, 8 h day⁻¹) for 30 d. O₃ significantly reduced net photosynthetic rate (PN), stomatal conductance, chlorophyll contents and plant biomass in -S treatment, but no considerable differences were noted in those parameters between +O₃+S and CF+S treatments. O₃-induced loss in cellular membrane integrity was significant in -S plants, but not in +S plants evidenced by significant elevations being measured in electrolyte leakage (EL) and malondialdehyde (MDA) content in -S plants, but not in +S plants. Both O₃ and salinity increased proline content and stimulated antioxidant enzymes activities. Soluble protein increased by salinity but decreased by O₃. Abscisic acid (ABA) was significantly elevated by O₃ in -S plants but not in +S plants. The results of this study suggested that the specificity of different agricultural environments should be considered in order to develop reliable prediction models on O₃ damage to wheat plants.     

Influence of carbon dioxide enrichment, ozone and nitrogen fertilization on cotton (Gossypium hirsutum L.) leaf and root composition

The objective of this study was to test whether elevated [CO₂], [O₃] and nitrogen (N) fertility altered leaf mass per area (LMPA), non‐structural carbohydrate (TNC), N, lignin (LTGA) and proanthocyanidin (PA) concentrations in cotton (Gossypium hirsutum L.) leaves and roots. Cotton was grown in 14 dm³ pots with either sufficient (0·8 g N dm ^{? 3}) or deficient (0·4 and 0·2 g N dm ^{? 3}) N fertilization, and treated in open‐top chambers with either ambient or elevated ( + 175 and + 350 μ mol mol ^{? 1}) [CO₂] in combination with either charcoal‐filtered air (CF) or non‐filtered air plus 1·5 times ambient [O₃]. At about 50 d after planting, LMPA, starch and PA concentrations in canopy leaves were as much as 51–72% higher in plants treated with elevated [CO₂] compared with plants treated with ambient [CO₂], whereas leaf N concentration was 29% lower in elevated [CO₂]‐treated plants compared with controls. None of the treatments had a major effect on LTGA concentrations on a TNC‐free mass basis. LMPA and starch levels were up to 48% lower in plants treated with elevated [O₃] and ambient [CO₂] compared with CF controls, although the elevated [O₃] effect was diminished when plants were treated concurrently with elevated [CO₂]. On a total mass basis, leaf N and PA concentrations were higher in samples treated with elevated [O₃] in ambient [CO₂], but the difference was much reduced by elevated [CO₂]. On a TNC‐free basis, however, elevated [O₃] had little effect on tissue N and PA concentrations. Fertilization treatments resulted in higher PA and lower N concentrations in tissues from the deficient N fertility treatments. The experiment showed that suppression by elevated [O₃] of LMPA and starch was largely prevented by elevated [CO₂], and that interpretation of [CO₂] and [O₃] effects should include comparisons on a TNC‐free basis. Overall, the experiment indicated that allocation to starch and PA may be related to how environmental factors affect source–sink relationships in plants, although the effects of elevated [O₃] on secondary metabolites differed in this respect.     

Nutrient availability influences UV-B sensitivity of Plantago lanceolata

Seeds of Plantago lanceolata were collected in a dune grassland ecosystem in the Netherlands. Plants were grown in a greenhouse for 61 days under either low or high nutrient conditions and were exposed to four different levels of biologically effective UV-B radiation. The highest UV-B exposure level simulated 30% reduction of the stratospheric ozone layer during summertime in the Netherlands. Total biomass production of plants at low nutrient supply was 50% lower compared to plants grown at high nutrient supply, while net photosynthesis was decreased by only 12%. Increased levels of UV-B reduced biomass production under non-limiting nutrient conditions only. Biomass production of plants grown at limited nutrient supply was not affected by UV-B. This response was correlated to increased accumulation of carbohydrates under nutrient limitation, which agrees well with the carbon/nutrient balance hypothesis. It is concluded that the increased accumulation of carbon in nutrient-stressed plants, may lead to a reduction of UV-B induced damage because of increased foliar UV-B absorbance by enhanced accumulation of phenolic compounds and leaf thickening.     

臭氧对水稻根系活力、可溶性蛋白含量与抗氧化系统的影响

采用旋转布气法开顶式气室 (Open top chambers, OTCs) 装置, 研究4种臭氧 (O3) 浓度水平 (过滤大气, O3浓度20nl·L-1;环境大气, O3浓度40nl·L-1;中等O3浓度处理, O3浓度为75nl·L-1;高浓度处理, O3浓度为150nl·L-1) 下水稻 (Oryzasativa) 根系中根系活力、可溶性蛋白含量、膜脂过氧化程度与抗氧化系统的变化差异。主要结果表明与过滤大气处理相比, O3浓度升高 (75和150nl·L-1) 使植株根系活力显著降低, 根系大幅度、过早地衰退;根系可溶性蛋白质含量显著下降;根系MDA含量显著升高, 膜脂过氧化程度加剧;SOD活性呈先升高后下降的变化趋势根系中H2O2含量大幅度显著上升, 并随着O3处理浓度升高和暴露时间延长变化幅度增大;CAT与POD活性则表现出升高趋势, 但处理后期升高幅度略微降低;整个处理期间根系ASA含量无显著变化。环境大气处理与过滤大气处理植株各个指标变化趋势基本一致并略微下降, 随着处理时间延长根系活力与蛋白质含量出现显著下降, 其他指标无显著差异。试验结果表明O3浓度升高会对植物地下部分根系产生影响;随着O3胁迫时间的延长, 植物将面临着缺乏强有力的根系生理代谢活力支持。     

Influence of ozone on ribonuclease activity in wheat (Triticum aestivum) leaves

Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (P(i)) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O(3)) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O(3). Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O(3) mol(-1)) (control) or non-filtered air plus O(3) (90 nmol O(3) mol(-1)) (O(3)) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O(3)-sensitive cultivar in the O(3) treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [P(i)]. Soluble [P(i)] in leaf tissue extracts from the O(3) and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O(3) treatment. Isozymes stimulated in the O(3) treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [P(i)] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O(3) were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [P(i)] in the leaf tissue samples suggested that O(3)-induced injury and accelerated senescence was atypical of normal senescence processes in that P(i) export was not observed in O(3)-treated plants.     

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots

The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of ω-hydroxycarboxylic and 1,ω-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C₁₆ to C₂₄, whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C₁₆ to C₂₈) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C₁₆ and C₁₈ were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C₂₄ and C₂₆). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes. Received: 19 August 1998 / Accepted: 3 February 1999     

Cytochromes P450 in phenolic metabolism

Three independent cytochrome P450 enzyme families catalyze the three rate-limiting hydroxylation steps in the phenylpropanoid pathway leading to the biosynthesis of lignin and numerous other phenolic compounds in plants. Their characterization at the molecular and enzymatic level has revealed an unexpected complexity of phenolic metabolism as the major route involves shikimate/quinate esters and alcohol/aldehyde intermediates. Engineering expression of CYP73s (encoding cinnamate 4-hydroxylase), CYP98s (encoding 4-coumaroylshikimate 3′-hydroxylase) or CYP84s (encoding coniferaldehyde 5-hydroxylase) leads to modified lignin and seed phenolic composition. In particular CYP73s and CYP98s also play essential roles in plant growth and development, while CYP84 constitutes a check-point for the synthesis of syringyl lignin and sinapate esters. Although recent data shed new light on the main path for lignin synthesis, they also raised new questions. Mutants and engineered plants revealed the existence of (an) alternative pathway(s), which most likely involve(s) different precursors and oxygenases. On the other hand, phylogenetic analysis of plant genomes show the existence of P450 gene duplications in each family, which may have led to the acquisition of novel or additional physiological functions in planta. In addition to the main lignin pathway, P450s contribute to the biosynthesis of many bioactive phenolic derivatives, with potential applications in medicine and plant defense, including lignans, phenylethanoids, benzoic acids, xanthones or quinoid compounds. A very small proportion of these P450s have been characterized so far, and rarely at a molecular level. The possible involvement of P450s in salicylic acid is discussed.     

The influence of ozone on the stomatal and non-stomatal limitation of photosynthesis in Norway spruce,Picea abies (L.) Karst,exposed to soil moisture deficit

Summary Norway spruce, Picea abies (L.) Karst., was exposed to charcoal-filtered air (CF) and non-filtered air + ozone (NF+) and periods of soil moisture deficit from 1985 to 1988 in open-top chambers. Net photosynthesis, stomatal conductance, needle water potential and various shoot properties were measured on 1-year-old shoots during a period of soil moisture deficit. The gas exchange was measured at saturating photosynthetic photon flux density and across a range of CO₂ concentrations. The soil moisture deficit induced a mild drought stress in the plants, expressed by a pre-dawn needle water potential of approximately-0.9 MPa and a substantial reduction in net photosynthesis and gas phase conductance. In the CF treatment, intercellular CO₂ concentration was reduced, but was unaffected in the NF+ treatment. Furthermore, net photosynthesis declined more in response to the soil moisture deficit in the NF+ treatment than in the CF treatment. This is suggested to be attributed to the carboxylation efficiency at the operating point, which was decreased by 47% and 64% in shoots from the CF and the NF+ treatments, respectively. Stomatal limitation of net photosynthesis was increased by drought by 24–45% in the CF treatment, while it was unaffected in the NF+ treatment. Thus, our results imply that the coupling between the stomatal conductance and the photosynthetic rate was changed and that the marginal cost of water per given amount of carbon gain will increase in trees exposed to ozone, during periods of drought.     

Cell wall remodeling under salt stress: Insights into changes in polysaccharides,feruloylation, lignification,and phenolic metabolism in maize

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.