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.     

Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases.

Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.     

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.     

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.     

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.     

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.     

Cell wall composition of leaves of an inherently fast- and an inherently slow-growing grass species

Differences in the relative growth rules of the inherently slow-growing Deschampsia flexuosa L. and the inherently fast-growing Holcus lanatus L. were reflected in cell wall synthesis in the elongation zone of the leaves. Leaf elongation rates depended on the size of the plant and ranged from 6 to 14 mm d^?1 in Deschampsia and from 12 to 42 mm d^?1 in Holcus. Anatomical data showed that the epidermis and vascular tissue are the important tissues controlling leaf extension. The cell wall polysaccharides of fully expanded leaves of the two species were identical in sugar composition. Enzymatic hydrolysis of polymeric sugars in the cell walls of the sheath and the lamina gave glucose (85%), arabinose (3.5%), fucose (0.5%), xylose (5.0%), mannose (0.5%), galaclose (0.8%) and galacturonic acid (3–4%). This composition applied throughout the blade and the sheath and did not change with ageing. Polysaccharides in the meristems of the two species showed identical sugar compositions with 51–55% glucose, 13–15% galactoronic acid and 13–14% arabinose as the main components. The extension zone was marked by a gradual increase of driselase-digestable polymers (per mm tissue) and a concurrent shift in sugar composition. The massive increase of glucose in the cell wall polymers of the elongation zone is probably caused by cellulose synthesis. The rate of synthesis of cell wall polysaccharides in Holcus was twice as high as that in Deschampsia. The slower-growing Deschampsia has more ferulic acid esterified with cell walls, which might contribute to the slowing of leaf growth. Lignin is not significantly deposited until growth has essentially ceased and is not responsible for the difference in growth rate.     

Biochemical properties and localization of the beta-expansin OsEXPB3 in rice (Oryza sativa L.)

Alpha-expansins are bound to the cell wall of plants and can be solubilized with an extraction buffer containing 1 M NaCl. Localization of alpha-expansins in the cell wall was confirmed by immunogold labeling and electron microscopy. The subcellular localization of vegetative beta-expansins has not yet been studied. Using antibodies specific for OsEXPB3, a vegetative beta-expansin of rice (Oryza sativa L.), we found that OsEXPB3 is tightly bound to the cell wall and, unlike alpha-expansins, cannot be solubilized with extraction buffer containing 1 M NaCl. OsEXPB3 protein could only be extracted with buffer containing SDS. The subcellular localization of the OsEXPB3 protein was confirmed by immunogold labeling and electron microscopy. Gold particles were mainly distributed over the primary cell walls. Immunohistochemistry showed that OsEXPB3 is present in all regions of the coleoptile and root tissues tested.     

Expansins: ever-expanding numbers and functions

Expansins were first identified as cell-wall-loosening proteins that, at least in part, mediate pH-dependent extension of the plant cell wall and growth of the cell. More recently, it has been realized that expansins belong to two protein families, the alpha-and beta-expansins, and that they appear to be involved in regulating, besides cell expansion, a variety of plant processes, including morphogenesis, softening of fruits, and growth of the pollen tube of grasses through the stigma and the style. The Arabidopsis genome contains 26 alpha-expansin genes and the rice genome at least 26. There are more beta-expansin genes in monocots than in dicots, at least 14 in rice and five in Arabidopsis. Expansin genes are differentially regulated by environmental and hormonal signals, and hormonal regulatory elements have been found in their promoter regions. An analysis of exon/intron structure led to the hypothesis that alpha-and beta-expansins evolved from a common ancestral gene.     

Leaf area establishment of a maize (Zea Mays L.) field crop under potassium deficiency

The effect of K deficiency on leaf area index (LAI) establishment of a maize field crop (Zea Mays L.) was studied. The experimental work was carried out in 2000 and 2001 on a long-term K fertilization trial. Three K fertilization regimes (K0, K1 and K4) have been applied since 1995, thus leading to contrasted levels of available K in soils (14, 23 and 44 µg exchangeable K per g of dry soil for the three fertilization regimes, respectively). The rate of leaf appearance, the leaf elongation rate (LER), the leaf elongation duration (LED), their final length and width and the number of senescent leaves were investigated. K concentrations in shoot tissue water were lower in K0 plants, whereas concentrations of Ca and Mg were higher. The LAI was reduced in the K0 treatment, mainly because of a slower rate of leaf appearance and a reduced final size of individual leaves. The reduced final length of individual leaves was almost entirely accounted for by a reduced LER during the quasi linear elongation phase. The LED was only slightly affected. A rough parallelism was observed between the relative reduction of leaf length and the relative reduction of plant water content during leaf elongation. Conversely, there was no evidence that leaf elongation was limited by carbohydrate availability in leaf growing zones. This suggests that K deficiency reduced LER probably because of altered plant-water relationships. On the whole, these results strengthen the idea that leaf growth is a key variable for analyzing, and later on modeling, crop growth under K deficiency.     

Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl

A creep extensiometer technique was used to provide direct evidence that short (20 min) and long-term (3d) exposures of roots to growth inhibitory levels of salinity (100mol m^-3 NaCl) induce reductions in the irreversible extension capacity of cell walls in the leaf elongation zone of intact maize seedlings (Zea mays L.). The long-term inhibition of cell wall extension capacity was reversed within 20 min of salt withdrawal from the root medium. Inhibited elongation of leaf epidermal tissues was also reversed after salt removal. The salt-induced changes in wall extension capacity were detected using in vivo and in vitro assays (shortly after localized freeze/thaw treatment of the basal elongation zone). The rapid reversal of the inhibition of wall extensibility and leaf growth after salt removal from root medium of long-term salinized plants, suggested that neither deficiencies in growth essential mineral nutrients nor toxic effects of NaCl on plasmamembrane viability were directly involved in the inhibition of leaf growth. There was consistent agreement between the scale, direction and timing of salinity-induced changes in leaf elongation growth and wall extension capacity. Rapid metabolically regulated changes in the physical properties of growing cell walls, caused by osmotic (or other) effects, appear to be a factor regulating maize leaf growth responses to root salinization.     

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.