Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.     

Growth maintenance of the maize primary root at low water potentials involves increases in cell-wall extension properties, expansin activity, and wall susceptibility to expansins

Previous work on the growth biophysics of maize (Zea mays L.) primary roots suggested that cell walls in the apical 5 mm of the elongation zone increased their yielding ability as an adaptive response to low turgor and water potential (psi w). To test this hypothesis more directly, we measured the acid-induced extension of isolated walls from roots grown at high (-0.03 MPa) or low (-1.6 MPa) psi w using an extensometer. Acid-induced extension was greatly increased in the apical 5 mm and was largely eliminated in the 5- to 10-mm region of roots grown at low psi w. This pattern is consistent with the maintenance of elongation toward the apex and the shortening of the elongation zone in these roots. Wall proteins extracted from the elongation zone possessed expansin activity, which increased substantially in roots grown at low psi w. Western blots likewise indicated higher expansin abundance in the roots at low psi w. Additionally, the susceptibility of walls to expansin action was higher in the apical 5 mm of roots at low psi w than in roots at high psi w. The basal region of the elongation zone (5-10 mm) did not extend in response to expansins, indicating that loss of susceptibility to expansins was associated with growth cessation in this region. Our results indicate that both the increase in expansin activity and the increase in cell-wall susceptibility to expansins play a role in enhancing cell-wall yielding and, therefore, in maintaining elongation in the apical region of maize primary roots at low psi w.     

Root Growth Maintenance at Low Water Potentials (Increased Activity of Xyloglucan Endotransglycosylase and Its Possible Regulation by Abscisic Acid)

Previous work suggested that an increase in cell wall-loosening contributes to the maintenance of maize (Zea mays L.) primary root elongation at low water potentials ([psi]w). It was also shown that root elongation at low [psi]w requires increased levels of abscisic acid (ABA). In this study we investigated the effects of low [psi]w and ABA status on xyloglucan endotransglycosylase (XET) activity in the root elongation zone. XET is believed to contribute to wall-loosening by reversibly cleaving xyloglucan molecules that tether cellulose microfibrils. The activity of XET per unit fresh weight in the apical 10 mm (encompassing the elongation zone) was constant at high [psi]w but increased by more than 2-fold at a [psi]w of -1.6 MPa. Treatment with fluridone to decrease ABA accumulation greatly delayed the increase in activity at low [psi]w. This effect was largely overcome when internal ABA levels were restored by exogenous application. Spatial distribution studies showed that XET activity was increased in the apical 6 mm at low [psi]w whether expressed per unit fresh weight, total soluble protein, or cell wall dry weight, corresponding to the region of continued elongation. Treatment with fluridone progressively inhibited the increase in activity with distance from the apex, correlating with the pattern of inhibition of elongation. Added ABA partly restored activity at all positions. The increase in XET activity at low [psi]w was due to maintenance of the rate of deposition of activity despite decreased deposition of wall material. The loss of activity associated with decreased ABA was due to inhibition of the deposition of activity. The results demonstrate that increased XET activity is associated with maintenance of root elongation at low [psi]w and that this response requires increased ABA.     

Electrophysiological responses of maize roots to low water potentials: relationship to growth and ABA accumulation

The maintenance of root elongation is an important adaptive response to low water potentials (psi(w)), but little is known about its regulation. An important component may be changes in root cell electrophysiology, which both signal and maintain growth maintenance processes. As a first test of this hypothesis, membrane potentials (E(m)) were measured within the cell elongation zone of maize (Zea mays L.) primary roots. Seedlings were grown in oxygenated solution culture, and low psi(w) was imposed by the gradual addition of polyethylene glycol. Cells hyperpolarized approximately 25 mV in response to low psi(w), and after 48 h resting potentials remained significantly hyperpolarized at psi(w) lower than -0.3 MPa compared with roots at high psi(w). Inhibitor experiments showed that the hyperpolarization was dependent on plasma membrane H(+)-ATPase activity. Previous work showed that accumulation of abscisic acid (ABA) is required for the maintenance of maize primary root elongation at low psi(w). To determine if the mechanism of action of ABA involves changes in root electrophysiology, E(m) measurements were made during long-term exposure to low psi(w). Steady-state resting E(m) were measured in regions in which maintenance of cell elongation was dependent on ABA accumulation (2-3 mm from the apex), or in which elongation was inhibited regardless of ABA status (6-8 mm from the apex). E(m) was substantially more negative in ABA-deficient roots specifically in the 2-3 mm region. The results suggest that set-points for ion homeostasis shifted in association with the maintenance of root cell elongation at low psi(w), and that ABA accumulation plays a role in regulating the ion transport processes involved in this response.     

Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production

Previous work showed that primary root elongation in maize (Zea mays L.) seedlings at low water potentials (psi(w)) requires the accumulation of abscisic acid (ABA) (R.E. Sharp, Y. Wu, G.S. Voetberg, I.N. Saab, M.E. LeNoble [1994] J Exp Bot 45: 1743-1751). The objective of the present study was to determine whether the inhibition of elongation in ABA-deficient roots is attributable to ethylene. At a psi(w) of -1.6 MPa, inhibition of root elongation in dark-grown seedlings treated with fluridone to impose ABA deficiency was largely prevented with two inhibitors of ethylene synthesis (aminooxyacetic acid and aminoethoxyvinylglycine) and one inhibitor of ethylene action (silver thiosulfate). The fluridone treatment caused an increase in the rate of ethylene evolution from intact seedlings. This effect was completely prevented with aminooxyacetic acid and also when ABA was supplied at a concentration that restored the ABA content of the root elongation zone and the root elongation rate. Consistent results were obtained when ABA deficiency was imposed using the vp5 mutant. Both fluridone-treated and vp5 roots exhibited additional morphological symptoms of excess ethylene. The results demonstrate that an important role of ABA accumulation in the maintenance of root elongation at low psi(w) is to restrict ethylene production.     

Expansins and coleoptile elongation in wheat

Expansins are now generally accepted to be the key regulators of wall extension during plant growth. The aim of this study was to characterize expansins in wheat coleoptiles and determine their roles in regulating cell growth. Endogenous and reconstituted wall extension activities of wheat coleoptiles were measured. The identification of beta-expansins was confirmed on the basis of expansin activity, immunoblot analysis, and beta-expansin inhibition. Expansin activities of wheat coleoptiles were shown to be sensitive to pH and a number of exogenously applied factors, and their optimum pH range was found to be 4.0 to 4.5, close to that of alpha-expansins. They were induced by dithiothreitol, K(+), and Mg(2+), but inhibited by Zn(2+), Cu(2+), Al(3+), and Ca(2+), similar to those found in cucumber hypocotyls. An expansin antibody raised against TaEXPB23, a vegetative expansin of the beta-expansin family, greatly inhibited acid-induced extension of native wheat coleoptiles and only one protein band was recognized in Western blot experiments, suggesting that beta-expansins are the main members affecting cell wall extension of wheat coleoptiles. The growth of wheat coleoptiles was closely related to the activity and expression of expansins. In conclusion, our results suggest the presence of expansins in wheat coleoptiles, and it is possible that most of them are members of the beta-expansin family, but are not group 1 grass pollen allergens. The growth of wheat coleoptiles is intimately correlated with expansin expression, in particularly that of beta-expansins.     

Translatable RNA Populations Associated with Maintenance of Primary Root Elongation and Inhibition of Mesocotyl Elongation by Abscisic Acid in Maize Seedlings at Low Water Potentials

Previous work indicated that accumulation of abscisic acid (ABA) acts differentially to maintain elongation of the primary root and inhibit elongation of the mesocotyl of maize (Zea mays L.) seedlings at low water potentials ([psi]w). Subsequent results indicated specific locations in the elongation zones where elongation is maintained, inhibited, or unaffected by endogenous ABA at low [psi]w. This information was utilized in this study to identify in vitro translation products of RNA associated with the maintenance or inhibition of elongation in the primary root and mesocotyl, respectively, by endogenous ABA at low [psi]w. The results distinguished products associated specifically with the elongation responses from those nonspecifically associated with ABA accumulation or low [psi]w, as well as normal cell development and maturation. In the primary root, the maintenance of elongation at low [psi]w by ABA was associated with the maintenance of expression of three products that were also expressed during elongation at high [psi]w, the expression of a novel product, and the suppression of two products. In the mesocotyl, the inhibition of elongation by ABA after transplanting to low [psi]w was associated with the induction of a novel translation product. However, the induction of this product, as well as accumulation of ABA and inhibition of elongation, occurred without a decline in tissue water content. The results demonstrate the necessity of examining the association of gene expression with elongation responses to low [psi]w with a high degree of spatial resolution.     

Expansins and cell growth

Expansins are now generally accepted to be key regulators of wall extension during growth. Several alternative roles for expansins have emerged in which the emphasis of their action is on wall breakdown or softening in processes such as fruit ripening, pollination, germination and abscission. Expansins are commonly encoded by substantial gene families and have classically been divided into two subfamilies, referred to as alpha- and beta-expansins. Two further subfamilies have now been identified: gamma-expansins, which were first described in Arabidopsis, and delta-expansins, which were identified in rice and are absent from Arabidopsis. Both are truncated versions of alpha- and beta-expansins, with gamma-expansins representing the amino-terminal half of a mature expansin and delta-expansins the carboxy-terminal half of a beta-expansin. Functional roles for gamma- and delta-expansins have yet to be defined, although recent data indicate a signalling role for gamma-expansins.     

Purification and characterization of four beta-expansins (Zea m 1 isoforms) from maize pollen

Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to well-characterized allergens such as Lol p 1 and Phl p 1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 micro g mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function.     

Increased endogenous abscisic Acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials

Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an alternative to external applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately −1.6 megapascals (roots) or −0.3 megapascals (shoots). Growth occurred in the dark at near-saturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials.     

Grass group I pollen allergens (beta-expansins) lack proteinase activity and do not cause wall loosening via proteolysis.

Group I grass pollen allergens make up a subgroup of the beta-expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain-like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of beta-expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load-bearing components of the plant cell wall. Third, we tested the sensitivity of beta-expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N-ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of beta-expansins do not loosen cell walls via a proteolytic mechanism.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.