Drought Induces Distinct Growth Response, Protection, and Recovery Mechanisms in the Maize Leaf Growth Zone.

Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels.

BACKGROUND AND AIMS: Experimental evidence in the literature suggests that O(2)(*-) produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O(2)(*-). METHODS: Stress treatments were imposed using 150 mm NaCl or 300 mM sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O(2)(*-) was determined using nitro blue tetrazolium, and H(2)O(2) was determined using 2', 7'-dichlorofluorescin. KEY RESULTS: In non-stressed plants, the distribution of accelerating growth and highest O(2)(*-) levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O(2)(*-) levels increased in sorbitol-treated roots and decreased in NaCl-treated roots. CONCLUSIONS: The lack of association between apoplastic O(2)(*-) levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O(2)(*-) may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.

Salinity-induced decrease in NADPH oxidase activity in the maize leaf blade elongation zone.

We reported previously that salinity-induced elongation constraints in the expansion zone of maize leaves are associated with reduced reactive oxygen species (ROS) production and could be alleviated by the addition of ROS. The NaCl effect was salt-specific and not osmotic. This paper explores the causes for such reduction. The decrease in ROS levels under salinity was not accompanied by increases in soluble apoplastic antioxidant activities such as superoxide dismutase, peroxidases and ascorbate. In experimental systems devoid of cell walls (protoplasts and membrane fractions) superoxide anion (O(2)(-)) production was inhibited by 50 and 100 mM NaCl, 50 microM DPI, 10 mM EGTA, and 5mM verapamil, a Ca(2+) channel inhibitor. Inhibitory effects of NaCl and reduced Ca(2+) supply were also observed in in gel assessment of O(2)(-) -generating activity. The main activity band excised from the ND-PAGE was recognized by an antibody against the C-terminal portion of the tomato gp91(phox) homolog. These results indicate the *O(2)(-) -generating activity negatively affected by NaCl was compatible with that of plasma membrane NADPH oxidase.

Elongation growth in leaf blades of Chloris gayana under saline conditions.

In Chloris gayana, salinity-associated yield decreases are due mainly to leaf area reductions. To understand the physiological basis for such reduction, the effects of salinity were studied on the spatial and temporal distribution of extension in the intercalary meristem at the leaf base, and on hydraulic conductance in that zone. C. gayana plants were grown on sand irrigated with Hoagland solution with the addition of 0 or 200 mmol/L NaCl, and all measurements were performed on tiller leaf four. In salinised plants, that leaf was 20% shorter than in controls. Extension in the blade expansion zone was studied by pricking through the leaf sheaths and analysing the displacement of the pricks. In salt-treated plants, maximum growth rates were depressed by 53% and the growth zone was shorter by approximately 10 mm, nevertheless, extension proceeded for a longer period than in control plants. The analysis of specific leaf areas in the expansion zone suggests the rate of dry matter deposition was lowered by salinity and estimations of tissue displacement time within that zone suggest cell wall maturity was delayed. Hydraulic conductance was reduced by salinity and this may be the main cause for reduced growth under salinity in Chloris gayana.

Determination of reactive oxygen species in salt-stressed plant tissues.

Reactive oxygen species (ROS) participate in signaling events that regulate ion channel activity and gene expression. However, excess ROS exert adverse effects that stem from their interaction with macromolecules. Thus, the assessment of the effects of salinity on ROS changes are central to understanding how plants respond and cope with this stress. ROS determination in salt-stressed plants poses specific challenges. On the one hand, salinity comprises osmotic and ion-specific effects which may, in turn, have different effects on ROS production. On the other hand, changes in ROS production may happen when tissues from salinized plants are subject to water potential (Ψ) changes when incubated in non-isosmotic solutions. This chapter provides detailed accounts of methods for ROS detection in tissues from salt-stressed plants and includes suggestions for avoiding artifacts when dealing with such tissues.

Tipburn in salt-affected lettuce (Lactuca sativa L.) plants results from local oxidative stress.

Tipburn in lettuce is a physiological disorder expressed as a necrosis in the margins of young developing leaves and is commonly observed under saline conditions. Tipburn is usually attributed to Ca(2+) deficiencies, and there has very limited research on other mechanisms that may contribute to tipburn development. This work examines whether symptoms are mediated by increased reactive oxygen species (ROS) production. Two butter lettuce (Lactuca sativa L.) varieties, Sunstar (Su) and Pontina (Po), with contrasting tipburn susceptibility were grown in hydroponics with low Ca(2+) (0.5 mM), and with or without 50 mM NaCl. Tipburn symptoms were observed only in Su, and only in the saline treatment. Tipburn incidence in response to topical treatments with Ca(2+) scavengers, Ca(2+) transport inhibitors, and antioxidants was assessed. All treatments were applied before symptom expression, and evaluated later, when symptoms were expected to occur. Superoxide presence in tissues was determined with nitro blue tetrazolium (NBT) and oxidative damage as malondialdehyde (MDA) content. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities were assayed. Under control and saline conditions, tipburn could be induced in both varieties by topical treatments with a Ca(2+) scavenger (EGTA) and Ca(2+) transport inhibitors (verapamil, LaCl(3)) and reduced by supplying Ca(2+) along with a ionophore (A 23187). Tipburn symptoms were associated with locally produced ROS. O(2)(·-) and oxidative damage significantly increased in leaf margins before symptom expression, while topical antioxidant applications (Tiron, DPI) reduced symptoms in treated leaves, but not in the rest of the plant. Antioxidant enzyme activity was higher in Po, and increased more in response to EGTA treatments, and may contribute to mitigating oxidative damage and tipburn expression in this variety.

Are Sunflower chlorotic mottle virus infection symptoms modulated by early increases in leaf sugar concentration?

Symptom development in a susceptible sunflower line inoculated with Sunflower chlorotic mottle virus (SuCMoV) was followed in the second pair of leaves at different post-inoculation times: before symptom expression (BS), at early (ES) and late (LS) symptom expression. Sugar and starch increases and photoinhibition were observed as early effects BS, and were maintained or enhanced later on, however, chlorophyll loss was detected only at LS. Photoinhibition correlated with a drastic decrease in D1 protein level. The progress of infection was accompanied by decreasing levels of apoplastic reactive oxygen species (ROS). In infected leaves, higher antioxidant enzyme activities (superoxide dismutase, SOD; ascorbate peroxidase, APX; glutathione reductase, GR) were observed from BS. The purpose of this work was to evaluate whether the early increases in carbohydrate accumulation may participate in SuCMoV symptom expression. Similar effects on photoinhibition, apoplastic ROS generation and antioxidant activity were generated when healthy leaves were treated with sugars. These results suggest that photoinhibitory processes and lower apoplastic superoxide levels induced by SuCMoV infection may be modulated by sugar increases.

Leaf expansion in grasses under salt stress.

Restriction of leaf growth is among the earliest visible effects of many stress conditions, including salinity. Because leaves determine radiation interception and are the main photosynthetic organs, salinity effects on leaf expansion and function are directly related to yield constraints under saline conditions. The expanding zone of leaf blades spans from the meristem to the region in which cells reach their final length. Kinematic methods are used to describe cell division and cell expansion activities. Analyses of this type have indicated that the reduction in leaf expansion by salinity may be exerted through effects on both cell division and expansion. In turn, the components of vacuole-driven cell expansion may be differentially affected by salinity, and examination of salinity effects on osmotic and mechanical constraints to cell expansion have gradually led to the identification of the gene products involved in such control. The study of how reactive oxygen species affect cell expansion is an emerging topic in the study of salinity's regulation of leaf growth.

Why are Chloris gayana leaves shorter in salt-affected plants? Analyses in the elongation zone.

Reduced hydraulic conductance calculated from growth data was suggested to be the main reason for reduced leaf expansion in salt-stressed Chloris gayana (Rhodes grass). In this work, xylem vessel cross-sections and wall enzyme activities were analysed to re-examine the effects of salinity on leaf growth in this species. Maximal segmental growth rates were 20% lower and the growth zone was 23% shorter in leaves from salinized plants than in controls; however, growth rates between 0 mm and 15 mm from the ligule were similar in both types of leaves. Xylem cross-sectional areas in this region were about 65% smaller in leaves of salinized plants, suggesting that hydraulic restrictions in the leaves of salinized plants were much higher than overall growth reductions. Extractable xyloglucan endotransglucosylase activity in this zone was twice as high in leaves of salinized plants as in leaves of controls. Nevertheless, the activity of the extracted enzyme was not affected by up to 1 M NaCl added to the reaction medium. Therefore, increased xyloglucan endotransglucosylase activity under salinity may be due to a promotion of transcription of XTH (xyloglucan endotransglucosylase/hydrolases) genes and/or translation of preformed transcripts. These results suggest that, as in drought stress, increased activity of cell wall enzymes associated with wall loosening may contribute to the maintenance of growth under saline conditions despite hydraulic restrictions.

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension.

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity.

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.