Cell wall proteome in the maize primary root elongation zone. I. Extraction and identification of water-soluble and lightly ionically bound proteins

Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4-beta-D-glucanase and alpha-L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.     

The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH

Growth of elongating primary roots of maize (Zea mays) seedlings was approximately 50% inhibited after 48 h in aerated nutrient solution under water deficit induced by polyethylene glycol 6000 at -0.5 MPa water potential. Proton flux along the root elongation zone was assayed by high resolution analyses of images of acid diffusion around roots contacted for 5 min with pH indicator gel. Profiles of root segmental elongation correlated qualitatively and quantitatively (r(2) = 0.74) with proton flux along the surface of the elongation zone from water-deficit and control treatments. Proton flux and segmental elongation in roots under water deficit were remarkably well maintained in the region 0 to 3 mm behind the root tip and were inhibited from 3 to 10 mm behind the tip. Associated changes in apoplastic pH inside epidermal cell walls were measured in three defined regions along the root elongation zone by confocal laser scanning microscopy using a ratiometric method. Finally, external acidification of roots was shown to specifically induce a partial reversal of growth inhibition by water deficit in the central region of the elongation zone. These new findings, plus evidence in the literature concerning increases induced by acid pH in wall-extensibility parameters, lead us to propose that the apparently adaptive maintenance of growth 0 to 3 mm behind the tip in maize primary roots under water deficit and the associated inhibition of growth further behind the tip are related to spatially variable changes in proton pumping into expanding cell walls.     

Water Deficit Rapidly Stimulates the Activity of a Protein Kinase in the Elongation Zone of the Maize Primary Root

The mechanisms by which plants detect water deficit and transduce that signal into adaptive responses is unknown. In maize (Zea mays L.) seedlings, primary roots adapt to low water potentials such that substantial rates of elongation continue when shoot growth is completely inhibited. In this study, in-gel protein kinase assays were used to determine whether protein kinases in the elongation zone of the primary root undergo activation or inactivation in response to water deficit. Multiple differences were detected in the phosphoprotein content of root tips of water-stressed compared with well-watered seedlings. Protein kinase assays identified water-deficit-activated protein kinases, including a 45-kD, Ca2+-independent serine/threonine protein kinase. Water-deficit activation of this kinase occurred within 30 min after transplanting seedlings to conditions of low water potential and was localized to the elongation zone, was independent of ABA accumulation, and was unaffected by cycloheximide-mediated inhibition of protein translation. These results provide evidence that the 45-kD protein kinase acts at an early step in the response of maize primary roots to water deficit and is possibly involved in regulating the adaptation of root growth to low water potential.     

Genetic variability of oxalate oxidase?activity and elongation in water-stressed primary roots of diverse maize and rice lines

A previous study of maize primary roots under water stress showed pronounced increases in oxalate oxidase activity and apoplastic hydrogen peroxide in the apical region of the growth zone where cell elongation is maintained. We examined whether increased oxalate oxidase activity in water-stressed roots is conserved across diverse lines of maize and rice. The maize lines exhibited varied patterns of activity, with some lines lacking activity in the apical region. Moreover, none of the rice lines showed activity in the apical region. Also, although the genotypic response of root elongation to water stress was variable in both maize and rice, this was not correlated with the pattern of oxalate oxidase activity. Implications of these findings for root growth regulation under water stress are discussed.     

Effects of water deficit stress on the developmental growth of excised tomato roots cultured in vitro

Summary Excised tomato roots (Lycopersicon esculentum Mill. cv Bonny Best) were cultured in the presence of mannitol to determine the effects of varying degrees of mild water deficit on their developmental growth. It was found that over the 7-d culture period, the cultured roots could regulate their own developmental responses to the water deficit such that elongation of the primary root axis was favored over that of the lateral roots. Higher degrees of water deficit proportionately decreased lateral root number and density, but lateral root primordia (visualized by clearing roots in chromium trioxide) continued to be formed in water-stressed roots. Measurements of water and osmotic (solute) potentials of the root tips showed that the cultured roots osmoregulated and did not suffer a loss in turgor pressure as a result of the mannitol treatments. However, reciprocal transfer experiments showed that root cultures were unable to resume growth after removal from water deficit conditions, thus indicating a probable requirement for the shoot for complete recovery.     

Effects of low water potential on cortical cell length in growing regions of maize roots

Roots growing under low water potential commonly exhibit a marked decrease in growth rate and in diameter. Using median longitudinal sections of fixed maize (Zea mays L. cv WF9 × Mo 17) seedling roots, we investigated the cellular basis for these effects. Cortical cells in the shortened elongation zone of water stressed roots were longer than cortical cells in the comparable location of well-watered roots. Nearly twofold differences in cell length were seen in the region 2 to 4 millimeters behind the root apex. The shortened growth zone, however, leads to a final mean cortical cell length approximately 30% shorter in the stressed roots. These differences were present regardless of the age of the control roots. These data, and the slower growth rate seen in water-stressed roots, suggest that the water deficit causes a significant reduction in the rate of cell supply to the cortical cell files.     

Maize lateral root developmental plasticity induced by mild water stress. II: Genotype-specific spatio-temporal effects on determinate development

Maize lateral roots exhibit determinate growth, whereby the meristem is genetically programmed to stop producing new cells. To explore whether lateral root determinacy is modified under water deficits, we studied two maize genotypes (B73 and FR697) with divergent responses of lateral root growth to mild water stress using an experimental system that provided near-stable water potential environments throughout lateral root development. First-order laterals of the primary root system of FR697 exhibited delayed determinacy when grown at a water potential of −0.28 MPa, resulting in longer and wider roots than in well-watered (WW) controls. In B73, in contrast, neither the length nor width of lateral roots was affected by water deficit. In water-stressed FR697, root elongation continued at or above the maximum rate in WW roots for 3 days longer, and was still 45% of maximum when WW roots approached their determinate length. Maintenance of root elongation was associated with sustained rates of cell production. In addition, kinematic analyses showed that reductions in tissue expansion rates with aging were delayed in the longitudinal, radial and tangential planes throughout the root growth zone. Thus, this study reveals large genotypic differences in the interaction of water stress with developmental determinacy of maize lateral roots.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Differential expression profiles of growth-related genes in the elongation zone of maize primary roots

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. Rates of root segmental elongation change from accelerating to decelerating as cell development proceeds from newly formed to fully elongated status. One of the primary variables regulating these changes in elongation rates is the extensibility of the elongating cell walls. To help decipher the complex molecular mechanisms involved in spatially variable root growth, we performed a gene identification study along primary root tips of maize (Zea mays) seedlings using suppression subtractive hybridization (SSH) and candidate gene approaches. Using SSH we isolated 150 non-redundant cDNA clones representing root growth-related genes (RGGs) that were preferentially expressed in the elongation zone. Differential expression patterns were revealed by Northern blot analysis for 41 of the identified genes and several candidate genes. Many of the genes have not been previously reported to be involved in root growth processes in maize. Genes were classified into groups based on the predicted function of the encoded proteins: cell wall metabolism, cytoskeleton, general metabolism, signaling and unknown. In-situ hybridization performed for two selected genes, confirmed the spatial distribution of expression shown by Northern blots and revealed subtle differences in tissue localization. Interestingly, spatial profiles of expression for some cell wall related genes appeared to correlate with the profile of accelerating root elongation and changed appropriately under growth-inhibitory water deficit.     

Differential expression of cell wall related genes in the elongation zone of rice roots under water deficit

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. It has been known that rice seminal root elongation could be enhanced by water stress. In the present study, 17 cell-wall related genes were identified by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Five genes encoded cell-wall loosening enzymes and six genes were involved in the lignin biosynthesis. The six other genes were related to the metabolism of polysaccharide and protein matrices in cell wall. Northern blot analysis confirmed that they were differentially expressed in the elongation zone of rice seminal roots under water stress, and none of them was root-specific. The results indicated that the activity of cell-wall loosening enzymes was enhanced in the early stage (within 16 h), and some cell wall matrices were synthesized rapidly in the middle stages (from 16 to 48 h), while lignin biosynthesis was enhanced in the middle and late stages of water stress (from 48 to 72 h). Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 437–443. The text was submitted by the authors in English.     

Cell wall proteome of wheat roots under flooding stress using gel-based and LC MS/MS-based proteomics approaches

Cell wall proteins (CWPs) are important both for maintenance of cell structure and for responses to abiotic and biotic stresses. In this study, a destructive CWP purification procedure was adopted using wheat seedling roots and the purity of the CWP extract was confirmed by minimizing the activity of glucose-6-phosphate dehydrogenase, a cytoplasmic marker enzyme. To determine differentially expressed CWPs under flooding stress, gel-based proteomic and LC-MS/MS-based proteomic techniques were applied. Eighteen proteins were found to be significantly regulated in response to flood by gel-based proteomics and 15 proteins by LC MS/MS-based proteomics. Among the flooding down-regulated proteins, most were related to the glycolysis pathway and cell wall structure and modification. However, the most highly up-regulated proteins in response to flooding belong to the category of defense and disease response proteins. Among these differentially expressed proteins, only methionine synthase, β-1,3-glucanases, and β-glucosidase were consistently identified by both techniques. The down-regulation of these three proteins suggested that wheat seedlings respond to flooding stress by restricting cell growth to avoid energy consumption; by coordinating methionine assimilation and cell wall hydrolysis, CWPs played critical roles in flooding responsiveness.     

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.     

植物根系和叶片生长对水分亏缺的原初反应

细胞扩张生长是植物受水分亏缺影响最敏感的生理过程之一。主要在对细胞水分导性、细胞壁特性和延伸组织中溶质传输结果分析的基础上 ,从细胞、组织和器官水平上对细胞扩展生长进行了探讨。根系和叶片细胞主要通过以下 2个过程来补偿水分胁迫的作用 :调节扩展生长需要的细胞临界膨压 ;溶质在延伸组织中的运移。此外 ,还探讨了植物根系和叶片生长对水分亏缺的生理适应机制     

Hydrotropism in roots: sensing of a gradient in water potential by the root cap

Roots of the agravitropic pea (Pisum sativum L.) mutant, ageotropum, responded to a gradient in water potential as small as 0.5 MPa by growing toward the higher water potential. This positive response occurred when a sorbitol-containing agar block was unilaterally applied to the root cap but not when applied to the elongation region. Unilateral application of higher concentrations of sorbitol to the elongation region caused root curvature toward the sorbitol source, presumably because of growth reduction on the water-stressed side. The control blocks of plain agar applied to either the root cap or the elongation region did not cause significant curvature of the roots. These results demonstrate that hydrotropism in roots occurs following perception of a gradient in water potential by the root cap.     

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots (I. Spatial Distribution of Longitudinal,Radial, and Tangential Expansion Rates)

As a system to study the regulation of growth anisotropy, we studied thinning of the primary root of maize (Zea mays L.) occurring developmentally or induced by water stress. Seedlings were transplanted into vermiculite at a water potential of approximately -0.03 MPa (well-watered) or -1.6 MPa (water-stressed). The diameter of roots in both treatments decreased with time after transplanting; the water-stressed roots became substantially thinner than well-watered roots at steady state, showing that root thinning is a genuine response to water stress. To analyze the thinning responses we quantified cell numbers and the spatial profiles of longitudinal, radial, and tangential expansion rates separately for the cortex and stele. The results showed that there was no zone of isotropic expansion and the degree of anisotropy varied greatly with position and treatment. Thinning over time in well-watered roots was caused by rates of radial and tangential expansion being too low to maintain the shape of the root. In response to low water potential, cell number in both tissues was unchanged radially but increased tangentially, which shows that thinning was caused wholly by reduced cell expansion. Water stress substantially decreased rates of tangential and radial expansion in both the stele and cortex, but only in the apical 5 mm of the root; basal to this, rates were similar in well-watered and water-stressed roots. By contrast, as in previous studies, longitudinal expansion was identical between the treatments in the apical 3 mm but in water-stressed roots was inhibited at more basal locations. The results show that expansion in longitudinal and radial directions can be regulated independently.     

Reactive oxygen species localization in roots of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedlings grown under phosphate deficiency

Arabidopsis plants responding to phosphorus (P) deficiency increase lateral root formation and reduce primary root elongation. In addition the number and length of root hairs increases in response to P deficiency. Here we studied the patterns of radical oxygen species (ROS) in the roots of Arabidopsis seedlings cultured on media supplemented with high or low P concentration. We found that P availability affected ROS distribution in the apical part of roots. If plants were grown on high P medium, ROS were located in the root elongation zone and quiescent centre. At low P ROS were absent in the elongation zone, however, their synthesis was detected in the primary root meristem. The proximal part of roots was characterized by ROS production in the lateral root primordia and in elongation zones of young lateral roots irrespective of P concentration in the medium. On the other hand, plants grown at high or low P differed in the pattern of ROS distribution in older lateral roots. At high P, the elongation zone was the primary site of ROS production. At low P, ROS were not detected in the elongation zone. However, they were present in the proximal part of the lateral root meristem. These results suggest that P deficiency affects ROS distribution in distal parts of Arabidopsis roots. Under P-sufficiency ROS maximum was observed in the elongation zone, under low P, ROS were not synthesized in this segment of the root, however, they were detected in the apical root meristem.     

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.