Species- and age-dependent sensitivity to ozone in young plants of pea,wheat and spinach: Effects on acyl lipid and pigment content and metabolism

Acyl lipids and pigments were analyzed in young plants of garden pea, spring wheat and spinach exposed to < 5 or 65 nl l^?1 ozone 12 h per day for 6 days. In one set of experiments, the plants were exposed to ¹⁴CO₂ for 2 h 3 days prior to ozone exposure. The plants responded differently to the moderately enhanced level of ozone used Spinach was not at all sensitive while in both pea and wheat, leaves of different ages differed in ozone sensitivity. In pea, ozone sensitivity increased with leaf age. In the second and third oldest leaves, the amounts of galactolipids per leaf area and the proportions of 18:3 of the total lipid extract and of phosphatidylglycerol decreased. In the second oldest leaf, ozone also caused a decreased proportion of 18:3 of monogalactosyldiacylglycerol. In the fourth oldest leaf, lipid composition and galactolipid unsaturation was unaffected, but ozone caused decreased leaf expansion resulting in increased acyl lipid content per leaf area. In both the first and second leaves of wheat, ozone fumigation caused a marked decrease in the content of monogalactosyldiacylglycerol and in the first leaf, the contents of phosphatidylcholine and phosphatidylethanolamine increased. The proportion of 18:3 in phosphatidylcholine was larger in ozone-fumigated than in control plants, while the reverse applied for phosphatidylglycerol. In the oldest sampled leaves of pea and wheat, ozone caused an increase in the radioactivity associated with β-carotene, indicating increased turnover. Thus, while spinach was unaffected, in both pea and wheat ozone caused a decrease in the proportion of chloroplast membrane lipids to non-chloroplast membrane lipids in older leaves while younger leaves were less sensitive.     

Ozone stress‐induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage

Considerable soybean yield losses caused by ozone (O₃) stress have been demonstrated by large‐scale meta‐analyses of free‐gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O₃ target structures such as leaf and chloroplasts of soybean seedlings. Acute O₃ exposure (120 parts‐per‐billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O₃‐induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O₃‐treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O₃. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O₃, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O₃ stress.     

Whole Plant and Leaf Steady State Gas Exchange during Ethylene Exposure in Xanthium strumarium L

The effects of ethylene evolved from ethephon on leaf and whole plant photosynthesis in Xanthium strumarium L. were examined. Ethylene-induced epinasty reduced light interception by the leaves of ethephon treated plants by up to 60%. Gas exchange values of individual, attached leaves under identical assay conditions were not inhibited even after 36 hours of ethylene exposure, although treated leaves required a longer induction period to achieve steady state photosynthesis. The speed of translocation of recently fixed ¹¹C-assimilate movement was not seriously impaired following ethephon treatment; however, a greater proportion of the assimilate was partitioned downward toward the roots. Within 24 hours of ethephon treatment, the whole plant net carbon exchange rate expressed on a per plant basis or a leaf area basis had dropped by 35%. The apparent inhibition of net carbon exchange rate was reversed by physically repositioning the leaves with respect to the light source. Ethylene exposure also inhibited expansion of young leaves which was partially reversed when the leaves were repositioned. The data indicated that ethylene indirectly affected net C gain and plant growth through modification of light interception and altered sink demand without directly inhibiting leaf photosynthesis.     

EFFECTS OF SINGLE AND MULTIPLE EXPOSURES OF FERULIC ACID ON THE VEGETATIVE AND REPRODUCTIVE GROWTH OF PHASEOLUS VULGARIS BBL-290

Phaseolus vulgaris BBL-290 plants were grown in growth chambers in the Southeastern Plant Environment Laboratory and exposed to either single (at seedling, flower, or podfill) or multiple (biweekly or weekly) treatments of ferulic acid (FA). In the first experiment, plants were harvested one week after FA treatment (0, 1.0, 2.0 mM) and at final harvest (56 days old). FA delayed leaf expansion during the seedling and flowering stages. The total plant leaf area and the plant dry weight of plants treated with 1.0 and 2.0 mM FA as seedlings were reduced one week after treatment by 38–48%. The total plant leaf area and the plant dry weight of plants treated at flowering with 2.0 mM FA were reduced by 25% one week after treatment. Treatment with 2.0 mM FA at podfill caused the senescence and abscission of older leaves and reduced total plant leaf area, plant dry weight and mean pod dry weight by 54, 40, and 48%, respectively, one week after treatment. The plants treated at the seedling and flowering stages recovered by final harvest. In a subsequent experiment, FA (0, 0.50, 1.0, 1.5 mM) reduced total plant leaf area at the seedling and flowering stages but not at podfill. The youngest expanding leaves were most sensitive to FA at flowering. The leaf area of these leaves was reduced by 35 and 25%, one and two weeks after treatment, respectively. Their absolute growth rates were reduced from 31 to 56% one week after treatment at flowering. Their relative growth rates were reduced by 50% one week after treatment. Growth rates then recovered within two weeks after treatment. In the final experiment, biweekly exposures of FA (0.25, 0.50, 0.75, 1.0) reduced total plant leaf area but did not affect any other growth parameters. Weekly exposures of FA (0.25, 0.50, 0.75, 1.0) reduced total plant leaf area up to 34%, absolute growth rate up to 58%, leaf number up to 31% and pod number up to 58%. As the frequency of exposure to FA increased, the concentration necessary to affect bean plant growth and development decreased.     

Does elevated ozone have differing effects in flowering and deblossomed strawberry?

Fruiting and deblossomed plants of strawberry ( Fragaria × ananassa ) were exposed to 92 ppb ozone or filtered air in open-top chambers for 69 d. Flower and fruit production, relative growth rate of leaf area, leaf gas exchange and plant biomass were investigated. Ozone caused an initial acceleration in inflorescence production, which was followed by a reduction in inflorescence production, fruit set, and, later, individual fruit weight, although total fruit yield was not affected before the end of the fumigation period. Ozone accelerated leaf senescence and had a greater negative effect on the rate of photosynthesis in older than in younger leaves in fruiting and deblossomed plants, but the response of net photosynthesis to ozone did not differ between the two groups of plants. Relative growth rate of leaf area was the first parameter to be reduced by ozone fumigation, with the effect being significant in fruiting, but not in deblossomed, plants. Final above-ground biomass was also significantly decreased by ozone in fruiting plants, but not in deblossomed plants. Root and crown biomass were not significantly affected by ozone fumigation in either fruiting or deblossomed plants.     

S-Nitroso-Proteome in Poplar Leaves in Response to Acute Ozone Stress

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = −3.6) and caffeic acid O-methyltransferase (−3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.     

EVIDENCE FOR THE REPAIR OF OZONE-INDUCED MEMBRANE INJURY

The increase in [¹⁴C]-2-deoxy-D-glucose uptake by leaf discs 24 hr after fumigation was used as a measure of ozone injury to pinto bean leaves (Phaseolus vulgaris). This method showed that the primary leaves were most affected by ozone when plants were 10 to 12 days old, which coincided with the time of maximum leaf necrosis. However, 8-day-old plants, which had no visible leaf injury, still showed a higher uptake rate than controls, indicating that injury occurred at the cellular level. In these younger plants, uptake was shown to return to normal over a 5 day period. Moreover, the rate of this “repair” was retarded by cold or continuous darkness, enhanced by continuous light, and very markedly increased by glucose applied to the leaves. The timing of the glucose application was not critical, nor did H₂O or mannitol have an effect. The results suggest that ozone injury at the cellular level can be repaired by energy-dependent processes so that necrosis of the leaf tissue does not occur. These experiments also show that conditions and treatments after ozone exposure can alter the degree of ozone injury.     

Photosynthetic Determinants of Growth in Maize Plants: Effects of Nitrogen Nutrition on Growth, Carbon Fixation and Photochemical Features

Maize(Zea mays L.) plants were grown in a greenhouse with differentlevels of nitrate-N (2 to 20 millimolar). Nitrogen nutritionhad dramatic effects on plant growth and photosynthetic characteristicsof mature leaves. Increasing nitrogen resulted in greater biomassproduction, shoot/root ratios, and rates of leaf expansion duringthe day. The elongating zone of high-N plants had higher activities(per gram fresh weight) of sucrose synthase and neutral invertasethan low-N plants, suggesting that increased leaf growth wasrelated to a greater biochemical capacity for sucrose metabolism. Mature leaves of high-N plants had higher rates of photosynthesisand assimilate export (sucrose formation), and partitioned morecarbon into sucrose relative to starch. Increased photosyntheticrates (leaf area basis) were associated with higher levels ofribulose-l,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylaseand pyruvate, phosphate dikinase (determined immunochemically).In addition, N-nutrition affected the functional organizationof chlorophyll in the leaves. Large increases in the numberof PS I reaction centers were observed which fully accountedfor increases in leaf chlorophyll content with increasing nitratesupply. Collectively, the results suggest that increased growth of maizeplants at high light and optimal nitrogen nutrition is relatedto greater capacity for photosynthesis and translocation inmature leaves, and possibly increased capacity for sucrose metabolismin expanding leaves. (Received May 22, 1989; Accepted August 28, 1989)     

Response of subterranean clover (Trifolium subterraneum) to ozone in relation to plant age and light conditions during exposure

Potted subterranean clover ( Trifolium subterraneum ) plants of different ages were exposed to 70 nl l⁻¹ ozone for 6 h, either during the light or during the dark period in a laboratory-based climate chamber. There was limited visible leaf injury on plants which were 14–20 and 28–34 d old and no significant decrease in biomass after daytime ozone exposure. The oldest leaves of 22–26 d old plants exhibited severe visible injury, which was associated with a significant reduction in biomass in 24–26 d old plants. Thus, ozone-induced visible injury of different magnitude developed in all plants, but was associated with biomass reduction only during a limited period of the plant's life-span. Apart from modifying ozone uptake by plants, climatic conditions are important as growth modifiers. It is suggested that subterranean clover plants of defined developmental stages should be used in bioindication of ozone. Night ozone exposure injured significantly fewer leaves than day exposure. However, some leaves developed visible injury even after night ozone exposure. Night uptake of ozone may be of more importance in northern than in central and southern Europe, because summer nights are short and, for a certain period, never completely dark.     

Effect of tropospheric ozone on morphological characteristics of Nicotiana tabacum L., Phaseolus vulgaris L. and Petunia×Hybrida L. in ambient air conditions

The cumulative ozone effect on morphological parameters (visible leaf injury, plant height and leaf growth, number of bean pods, petunia flowers and stalks) was examined in this study. Well-known ozonesensitive (Bel W3) and ozone-resistant (Bel B) tobacco cultivars as well as bean cv. Nerina and petunia cv. White cascade, both recognized as ozone sensitive, were used in the experiment. Investigations were carried out at two exposure sites varying in tropospheric ozone levels. Ozone negatively affected the leaf growth of both tobacco cultivars and bean. A negative relation was also found for ozone concentration and tobacco plant height. Number of petunia flowers and stalks and bean pods was positively correlated with ozone concentration. This could have been connected with earlier plant maturation due to faster generative development of plants in ozone-stress conditions.     

Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2]

While exposure of C3 plants to elevated [CO2] would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the present study suggest that exposure of plants to elevated [CO2] can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated [CO2]. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated [CO2]. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated [CO2] or [O3] revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated [CO2].     

Effects of ozone and Phytophthora citricola on non-structural carbohydrates of European beech (Fagus sylvatica) saplings

A lysimeter study was performed to monitor long term effects of chronic ozone enrichment on saplings of European beech (Fagus sylvatica L). After 3 years of ozone exposure a root infection with Phytophthora citricola Swada was established in the fourth year to study the interaction between elevated ozone and the root infection on the carbon budget of beech saplings. By using quantitative PCR no differences in root infection with P. citricola were observed between the ozone treatments. In contrast to the first 3 years of ozone exposure, sucrose and starch concentrations in leaves were diminished in ozone treated plants in the fourth year. The root infection reduced sucrose concentrations in leaves. Starch reserves of the heterotrophic biomass were not affected by any treatments. Thus 4 years of ozone exposure and 1 year of P. citricola root infection had only limited effect on carbohydrate metabolism in beech saplings.     

Influence of Potassium Deficiency on Photosynthesis,Chlorophyll Content,and Chloroplast Ultrastructure of Cotton Plants

In cotton (Gossypium hirsutum L.) grown in controlled-environment growth chamber the effects of K deficiency during floral bud development on leaf photosynthesis, contents of chlorophyll (Chl) and nonstructural saccharides, leaf anatomy, chloroplast ultrastructure, and plant dry matter accumulation were studied. After cotton plants received 35-d K-free nutrient solution at the early square stage, net photosynthetic rate (P _N) of the uppermost fully expanded main-stem leaves was only 23 % of the control plants receiving a full K supply. Decreased leaf P _N of K-deficient cotton was mainly associated with dramatically low Chl content, poor chloroplast ultrastructure, and restricted saccharide translocation, rather than limited stomata conductance in K-deficient leaves. Accumulation of sucrose in leaves of K-deficient plants might be associated with reduced entry of sucrose into the transport pool or decreased phloem loading. K deficiency during squaring also dramatically reduced leaf area and dry matter accumulation, and affected assimilate partitioning among plant tissues.     

Effect of solar ultraviolet-B radiation during springtime ozone depletion on photosynthesis and biomass production of Antarctic vascular plants

We assessed the influence of springtime solar UV-B radiation that was naturally enhanced during several days due to ozone depletion on biomass production and photosynthesis of vascular plants along the Antarctic Peninsula. Naturally growing plants of Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarctica Desv. were potted and grown under filters that absorbed or transmitted most solar UV-B. Plants exposed to solar UV-B from mid-October to early January produced 11% to 22% less total, as well as above ground biomass, and 24% to 31% less total leaf area. These growth reductions did not appear to be associated with reductions in photosynthesis per se: Although rates of photosynthetic O(2) evolution were reduced on a chlorophyll and a dry-mass basis, on a leaf area basis they were not affected by UV-B exposure. Leaves on plants exposed to UV-B were denser, probably thicker, and had higher concentrations of photosynthetic and UV-B absorbing pigments. We suspect that the development of thicker leaves containing more photosynthetic and screening pigments allowed these plants to maintain their photosynthetic rates per unit leaf area. Exposure to UV-B led to reductions in quantum yield of photosystem II, based on fluorescence measurements of adaxial leaf surfaces, and we suspect that UV-B impaired photosynthesis in the upper mesophyll of leaves. Because the ratio of variable to maximal fluorescence, as well as the initial slope of the photosynthetic light response, were unaffected by UV-B exposure, we suggest that impairments in photosynthesis in the upper mesophyll were associated with light-independent enzymatic, rather than photosystem II, limitations.     

Poplar under drought: comparison of leaf and cambial proteomic responses

The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.     

Effects of Abscisic Acid on Growth of Wheat (Triticum aestivum L)

Daily application of abscisic acid (ABA) to growing wheat plants,although initially inhibiting growth, resulted, after a shortlag, in an increase in the number of leaves and tillers. Thismay have been due to reduced apical dominance. At 84 days thetotal dry weight and area of all leaves produced up to thistime was less for the plants treated with ABA than for the controlplants. However, the area of green, living leaves and the dryweight were not significantly affected by the ABA treatment.Further effects of the daily ABA treatment were the inhibitionof transpiration, especially on the abaxial surface, the reductionof leaf size, the promotion of flowering and the stimulationof trichome formation on the leaf surfaces. ABA did not promoteleaf senescence in whole plants and actually increased leaflongevity. Triticum aestivum L., wheat, leaf senescence, transpiration, growth, flowering, abscisic acid