Root Morphology and Gene Expression Analysis in Response to Drought Stress in Maize (Zea mays)

Water-deficit stress tolerance is a complex trait, and water deficit results in various physiological and chemical changes in maize (Zea mays L.) and exacerbates pre-harvest aflatoxin contamination. The objective of this study was to characterize the variations in morphology, physiology, and gene expression in two contrasting inbred lines, Lo964 and Lo1016, in order to understand the differences in response to water-deficit stress. The results revealed that Lo964 was less sensitive to water-deficit stress, and had a strong lateral root system and a higher root/shoot ratio in comparison to Lo1016. In response to water-deficit stress by comparing stressed versus well-watered conditions, abscisic acid syntheses were increased in leaves, roots, and kernels of both Lo964 and Lo1016, but by different magnitudes. Indole-3-acetic acid (IAA) was undetectable in the leaves and roots of either genotype regardless of treatments, but increases of 58% and 8% in IAA concentration were observed in 20 DAP kernels, in response to water-deficit stress, respectively. The expression of the MIPS was up-regulated 7-fold in leaf tissues of Lo964 compared to Lo1016 at watered conditions, but decreased significantly to similar levels in both genotypes at water-deficit conditions. ZmPR10 and ZmFer1 expressions tended to up-regulate although ZmPR10 was expressed higher in root tissue while ZmFer1 was expressed higher in leaf tissue. Further study is needed to confirm if Lo964 has reduced aflatoxin contamination associated with the drought tolerance in the field in order to utilize the resistant trait in breeding.     

Effect of Exogenous Abscisic Acid on Proline Dehydrogenase Activity in Maize (Zea mays L.)

Plant responses to drought stress include proline and abscisic acid (ABA) accumulation. Proline dehydrogenase (PDH) (EC 1.4.3) is the first enzyme in the proline oxidation pathway, and its activity has been shown to decline in response to water stress (PJ Rayapati, CR Stewart [1991] Plant Physiol 95: 787-791). In this investigation, we determined whether ABA treatment affects PDH activity in a manner similar to drought stress in maize (Zea mays L.) seedlings. Four exogenous ABA treatments (0, 11, 33, and 100 micromolar ABA) were applied to well-watered maize seedlings. Mitochondria were isolated and PDH was solubilized using Nonidet P-40. PDH activity was measured by the reduction of iodonitrotetrazolium violet under proline-dependent conditions. There was no effect of ABA on PDH activity at 33 and 100 micromolar ABA, but there was a 38% decline at 11 micromolar. This decline was less than the 69% reduction in activity under drought stress. Endogenous ABA determinations and plant growth rate showed that ABA entered the plant and was affecting metabolic processes. ABA treatments had a small effect on shoot and root proline concentration, whereas drought stress caused a 220% increase in root tissues. We conclude that ABA is not part of the pathway linking drought stress and decreased PDH activity.     

Effect of Abscisic Acid on the Transpiration of Zea mays

Intact plants of Zea mays L. were treated with foliar sprays of cis-trans-abscisic acid (ABA) at concentrations from 10⁻⁹ to 10⁻⁴M. Even the lowest concentration caused a reduction of the transpiration rate as measured between 1 and 33 h after spraying. With increasing ABA concentrations, there was a nearly linear relationship between the logarithm of the ABA concentration and the (decreasing) transpiration rate within that period. Subsequently a partial recovery of the transpiration rate set in, beginning progressively later as the ABA concentration was increased. After 5 1/2 days the transpiration rate of plants treated with 10⁻⁹ and 10⁻⁸M was nearly back to normal, whereas plants treated with 10⁻⁴M transpiration at only about 2/3 their normal rate. In experiments with detached maize leaves supplied with water or ABA solutions (10⁻⁸ to 10⁻⁵M) through their cut bases, the transpiration of control leaves decreased gradually to a low level in 24 h. ABA caused a marked and rapid reduction of the transpiration rate compared to that of the controls. After a few hours, the transpiration of the treated leaves decreased at a slower rate than that of the controls, thus approaching the control values. After 35 h, the transpiration of leaves treated with 10⁻⁵M ABA was nearly the same as in untreated leaves. Exchanging the ABA solution for distilled water after 24 h had little effect on the subsequent course of the transpiration rate.     

Abscisic Acid Biosynthesis in Isolated Embryos of Zea mays L

Previous labeling experiments with ¹⁸O₂ have supported the hypothesis that stress-induced abscisic acid (ABA) is synthesized through an indirect pathway involving an oxygenated carotenoid (xanthophyll) as a precursor. To investigate ABA formation under nonstress conditions, an ¹⁸O₂ labeling experiment was conducted with isolated embryos from in vitro grown maize (Zea mays L.) kernels. Of the ABA produced during the incubation in ¹⁸O₂, three-fourths contained a single ¹⁸O atom located in the carboxyl group. Approximately one-fourth of the ABA synthesized during the experiment contained two ¹⁸O atoms. These results suggest that ABA synthesized in maize embryos under nonstress conditions also proceeds via the indirect pathway, requiring a xanthophyll precursor. It was also found that the newly synthesized ABA was preferentially released into the surrounding medium.     

Physiological Responses of Contrasting Maize (Zea mays L.) Hybrids to Repeated Drought

Maize is the most important crop worldwide in terms of production and yield, but every year a considerable amount of yield is lost due to drought. The foreseen increase in the number of drought spells due to climate change raises the question whether the ability to recover quickly after a water pulse may be a relevant trait for overall drought resistance. We here address the following hypotheses: (i) different maize hybrids exhibit distinct physiological adaptive responses to drought stress and (ii) these responses affect the ability to recover from the stress. (iii) The relative biomass production of maize hybrids, which show severe drought symptoms but are able to recover quickly after a water pulse, is comparable to those hybrids, which invest more energy into tolerance mechanisms. The physiological responses of eight maize hybrids to repeated drought were elucidated employing physiological parameters such as electrolyte leakage, osmolality, relative water content, growth rate and gas-exchange measurements. Only one hybrid was able to maintain biomass production under drought conditions. Amongst the others, two hybrids with similar growth inhibition but contrasting physiological responses were identified by a PCA analysis. Both strategies, i.e. stabilization of leaf water content via resistance mechanisms versus high recovery potential were equally effective in maintaining aboveground biomass production in the scenario of a long drought intermitted by a water-pulse. However, each strategy might be advantageous under different drought stress scenarios. Overall, the recovery potential is underestimated in drought resistance under natural conditions, which includes periodic cycles of drought and rewatering, and should be considered in screening trials.

     

Chlorophyll Synthesis from Protochlorophyll(ide) in Chill-Stressed Maize (Zea mays L.)

Illumination of aetiolated maize at temperatures lower than20 °C results in negligible accumulation of chlorophyll.Illumination of leaf tissue, previously incubated in 10 molm^–3 ALA in darkness, shows only a slight conversion ofprotochlorophyll(ide) to chlorophyll a and b at temperaturesless than 20 °C. A refined procedure for measuring photosynthesisby photo-acoustic spectroscopy in leaves that differ in chlorophyllcontent is presented. Studies of photosynthesis in aetiolatedseedlings illuminated at different temperatures by photo-acousticspectroscopy suggests that impairment of the chlorophyll pathwayis paralleled by an aberrant development of the thylakoid membrane. Key words: Protochlorophyll(ide), temperature, photo-acoustic spectroscopy, membrane biogenesis     

Protein Changes in Response to Pyrene Stress in Maize （Zea mays L.） Leaves

Phytoremedlation is a relatively new approach to remove polycyclic aromatic hydrocarbons （PAHs） from the environment. When plants are grown under pyrene treatment, they respond by synthesizing a set of protective proteins. To learn more about protein changes in response to pyrene treatment, we extracted total proteins from the leaves of maize （Zea mays L.） 1 week after pyrene treatment. The proteins extracted were separated with twodimensional gel electrophoresis. In total, approximately 54 protein spots were found by comparing gels from treated and control groups. According to the Isoelectric point, molecular weight, and abundance of these protein spots, 20 pyrene-lnduced proteins were found to have changed abundance. Of these, 15 protein spots were Increased and five protein spots were newly appeared in pyrene-treated plant leaves. Six model upregulated protein spots of different molecular weights were excised from the gels and subjected to trypsin digestion followed by peptide separation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptlde masses were used to search the matrix-science database for protein Identification. Two of the proteins were Identified on the basis of the homology of their peptide profiles with existing protein sequences as pyruvate orthophosphate diklnase and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunlt. These proteins are Involved in the regulation of carbohydrate and energy metabolism. The present study gives new Insights into the pyrene stress response In maize leaves and demonstrates the power of the proteomlc approach in phytoremedlation of PAHs.     

Proline Accumulation in Maize (Zea mays L.) Primary Roots at Low Water Potentials (I. Requirement for Increased Levels of Abscisic Acid)

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.     

Proliferation of Maize (Zea mays L.) Roots in Response to Localized Supply of Nitrate

Maize (Zea mays L.) plants with two primary nodal root axeswere grown for 8 d in flowing nutrient culture with each axisindependently supplied with . Dry matter accumulation by roots was similar whether 1.0 mol m^–3 was supplied to on( or both axes. When was supplied to only one axis, however, accumulationof dry matter within the root system was significantly greaterin the axis supplied with . The increased dry matter accumulation by the +N-treated axis was attributableentirely to increased density and growth of lateral branchesand not to a difference in growth of the primary axis. Proliferation of lateral branches for the + N axis was associatedwith the capacity for in situ reduction and utilization of aportion of the absorbed , especially in the apical region where lateral primordia are initiated. Althoughreduced nitrogen was translocated to the –N axis, concentrationsin the –N axis remained significantly lower than in the+N axis. The concentratio of reduced nitrogen, as well as invitro reductase activity, was greater in apical than in more basal regions of the +N axis. The enhancedproliferation of lateral branches in the + N axis was accompaniedby an increase in total respiration rate of the axis. Part ofthe increased respiration was attributable to increased massof roots. The specific respiration rate (umol CO₂ exolved perhour per gram root dry weight) was also greater for the +N thanfor the –N axis. If respiration rate is taken as representativeof sink demand, stimulation of initiation and growth of lateralsby in situ utilization of a localized exogenous supply of establishes an increased sink demand through enhancedmetabolic activity and the increased partitioning of assimilatesto the + N axis responds to the difference in sink demand between+N and –N axes. Key words: NO₃^- reduction, NO₃^- uptake nitrogen partitioning, root respiration, sink demand     

Growth and Graviresponsiveness of Primary Roots of Zea mays Seedlings Deficient in Abscisic Acid and Gibberellic Acid

Moore, R. and Dickey, K. 1985. Growth and graviresponsivenessof primary roots of Zea mays seedlings deficient in abscisicacid and gibberellic acid.—J. exp. Bot. 36: 1793–1798. The objective of this research was to determine if gibberellicacid (GA) and/or abscisic acid (ABA) are necessary for graviresponsivenessby primary roots of Zea mays. To accomplish this objective wemeasured the growth and graviresponsiveness of primary rootsof seedlings in which the synthesis of ABA and GA was inhibitedcollectively and individually by genetic and chemical means.Roots of seedlings treated with Fluridone (an inhibitor of ABAbiosynthesis) and Ancymidol (an inhibitor of GA biosynthesis)were characterized by slower growth rates but not significantlydifferent gravicurvatures as compared to untreated controls.Gravicurvatures of primary roots of d-5 mutants (having undetectablelevels of GA) and vp-9 mutants (having undetectable levels ofABA) were not significantly different from those of wild-typeseedlings. Roots of seedlings in which the biosynthesis of ABAand GA was collectively inhibited were characterized by gravicurvaturesnot significantly different from those of controls. These results(1) indicate that drastic reductions in the amount of ABA andGA in Z. mays seedlings do not significantly alter root graviresponsiveness,(2) suggest that neither ABA nor GA is necessary for root gravicurvature,and (3) indicate that root gravicurvature is not necessarilyproportional to root elongation. Key words: Abscisic acid, Ancymidol, Fluridone, gibberellic acid, root gravitropism, Zea mays     

Mapping QTLs for Component Traits Influencing Drought Stress Tolerance of Maize (Zea mays L) in India

The present study was aimed at mapping of Quantitative Trait Loci (QTL) for various traits influencing the performance of maize genotypes under drought stress conditions in India. A set of 210 Recombinant Inbred Lines (RILs) developed at CIMMYT (Mexico) was analyzed in drought trials undertaken at Karimnagar (2002-03) and Hyderabad (2003-04). Analyses of the RIL datasets using Composite Interval Mapping (CIM) models led to the detection of 52 QTLs, including 22 QTLs under the control conditions and 30 QTLs under drought stress conditions at Karimnagar, and 14 QTLs influencing various characters under drought stress conditions at Hyderabad. A significant digenic epistatic QTL effect, other than the main effect QTLs, was detected for kernel number per ear under drought stress conditions. A comparison of the QTL information obtained from independent analyses of the Karimnagar and Hyderabad datasets revealed colocalization of QTLs on chromosomes 1, 2, 8 and 10 in the RILs influencing specific characters under drought stress conditions. Comparison of the QTL information with that reported from previous analyses of the same set of RILs at Mexico, Kenya and Zimbabwe revealed some ‘consensus QTLs’, which could be of significance in molecular marker-assisted breeding for drought tolerance in maize, besides functional genomics.     

Chloroplast Reactions of Photosynthetic Mutants in Zea mays

Three seedling lethal mutants of Zea mays with impaired photosynthesis are described. These recessive mutants were selected on the basis of high chlorophyll fluorescence. They have normal chlorophyll pigmentation but are unable to fix CO₂ fully. Evidence is presented from fluorescence characteristics of isolated chloroplasts that both photosystem I and II mutants were isolated. Using conventional measures of photosynthetic electron transport, we suggest that the photosystem I mutant has limited ability to reduce NADP. The other two mutants are clearly blocked in photosystem II, one possibly lacking the primary electron acceptor.     

Accumulation of Group 3 Late Embryogenesis Abundant Proteins in Zea mays Embryos : Roles of Abscisic Acid and the Viviparous-1 Gene Product

Several different types of proteins that are modulated by abscisic acid (ABA) accumulate in developing embryos of maize (Zea mays L.). Some of these proteins are specific to the developing seed, such as the storage globulin, GLB1, whereas others are involved in general responses to water deficit. Here we describe a maize protein family of this second type, a Group 3 late embryogenesis abundant (MLG3). Like other proteins of this class, MLG3 polypeptides are ABA-responsive. They are found in maturing seeds and in dehydrating plant tissues. Antigenically related proteins are found in other cereals. To distinguish the regulation of developmentally programmed ABA responses from those that are environmentally induced, we compared the ontological pattern and accumulation requirements of MLG3 polypeptides with those we previously described for GLB1. GLB1 accumulation begins early in the maturation phase and specifically requires high levels of ABA and the participation of the Viviparous-1 (Vp1) gene product. Vp1 is required for other ABA-modulated events in maize seed development as well. In experiments using vp1 mutants and mutants deficient in ABA synthesis (vp5 mutation), we show that MLG3 accumulation also is dependent upon ABA, but it shows striking differences from GLB1. MLG3 accumulates much later in embryogenesis, coincident with the onset of dehydration. In contrast to GLB1, MLG3 proteins can be induced by de novo ABA synthesis in response to culturing in high osmoticum. Unlike GLB1, MLG3 has no specific requirement for the Vp1 gene product.     

Physiological and Biochemical Mechanisms Involved in the Response to Abscisic Acid in Maize Coleoptiles

The role of abscisic acid (ABA) in controlling growth and developmenthas been studied in maize (Zea mays L.) coleoptile segments.Application of ABA reduces the elongation rate by about 50%and affects ion fluxes. In particular, proton extrusion is decreasedwhile potassium efflux is greatly enhanced. Apparently, ABAdoes not: seem to influence calcium influx from the apoplastinto the cytosol, but more likely it influences its efflux.Alteration of cytosolic calcium concentration may also be obtainedby increasing its release from internal stores. This possibilitymight be sustained by the increased hydrolysis of phosphatidylinositolupon ABA application. Change in the balance of ion fluxes shouldresult from regulation of transport mechanisms at the membranelevel and should produce changes in the transmembrane electricalpotential. The H⁺- ATPase and the ATP-dependent calcium transportactivities are both influenced by the treatment with ABA, –55%and –40%, respectively. Under these conditions [Ca²⁺]_cytand pH_cyt can be modified and, as a consequence of their regulation,they may play an important role in mediating the physiologicaland biochemical effects of ABA, acting as second intracellularmessengers. ¹Research supported by National Research Council of Italy, SpecialProject RAISA, Sub-Project N. 2, Paper n. 2782.     

Synthesis and Deposition of Callose in Anthers and Ovules of Meiotic Mutants of Maize (Zea mays)

The pattern of callose deposition was studied in anthers and ovules of three meiotic mutants of Zea mays L. The synthesis of the callose wall in sporogenous cells was related to their transfer to meiotic division.     

Cytosolic Ascorbate Peroxidase in Seedlings and Leaves of Maize (Zea mays)

Ascorbate peroxidase (APX) was purified to homogeneity frommaize (Zea mays L. cv.) coleoptiles. APX was a monomer witha molecular mass of 28 kDa, as determined by gel nitration andSDS-polyacrylamide gel electrophoresis. It contained one protohememoiety per molecule, with the oxidized form giving a Soret peakat 403 nm with small peaks at 502 and 638 nm, and the reducedform giving peaks at 435 and 556 nm. The enzyme was not inactivatedby depletion of ascorbate. Cell fractionation and immunohistochemicalstudies using polyclonal antibodies raised against maize APXrevealed that the enzyme was not located in the chloroplastsof green leaves. It was abundant in the cytoplasm but not inthe vacuoles of cells in the coleoptile, mesocotyl and youngleaves of seedlings. In mature green leaves, small amounts ofthe enzyme were distributed in vascular systems, in particularin the companion cells. The N-terminal amino acid sequence ofmaize APX exhibited high homology to pea cytosolic APX, spinachAPX and Arabidopsis APX, but not to APX from tea chloroplasts. (Received February 15, 1993; Accepted May 6, 1993)     

Continuity of the Chloroplast Membrane Systems in Zea mays L

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.