Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat

Expansin protein is a component of the cell wall generally accepted to be the key regulator of cell wall extension during plant growth. Plant hormones regulate expansin gene expression as well as plant growth during drought stress. However, the relationship between expansin and plant hormone is far from clear. Here, we studied the involvement of expansin in plant cell growth mediated by the hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) under osmotic stress which was induced by polyethylene glycol (PEG)-6000. Wheat coleoptiles from a drought-resistant cultivar HF9703 and a drought-sensitive cultivar 921842 were used to evaluate cell growth and expansin activity. Osmotic stress induced the accumulation of ABA. ABA induced expansin activity mainly by enhancing expansin expression, since ABA induced cell wall basification via decreasing plasma membrane H⁺-ATPase activity, which was unfavorable for expansin activity. Although ABA induced expansin activity and cell wall extension, treatment with exogenous ABA and/or fluridone (FLU, an ABA inhibitor) suggested that ABA was involved in the coleoptile growth inhibition during osmotic stress. IAA application to detached coleoptiles also enhanced coleoptile growth and increased expansin activity, but unlike ABA, IAA-induced expansin activity was mainly due to the decrease of cell wall pH by increasing plasma membrane H⁺-ATPase activity. Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

Cloning,characterization, and expression of the <Emphasis Type="Italic">BjEXPA1</Emphasis> gene and its promoter region from <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

To explore the regulation of expansin expression under heavy metal stresses, a novel expansin gene, BjEXPA1, and its upstream region were obtained from Indian mustard (Brassica juncea L.). BjEXPA1 encodes a cell wall protein of the α-subgroup of the expansin family. The expression of BjEXPA1 is regulated by exogenous phytohormones, NaCl, and heavy metals. Sequence analysis of its upstream region revealed the presence of several putative cis-acting elements, including phytohormone response, abiotic stress response, and tissue-specific elements. GUS activity under the control of the BjEXPA1 promoter allowed observation of BjEXPA1 spatial and temporal expression patterns. The reporter construct indicated that BjEXPA1 is induced by exogenous gibberellin and auxin, and inhibited by abscisic acid, CdCl₂ and PEG6000. Transgenic tobacco seedlings overexpressing BjEXPA1 showed an altered phenotype and enhanced sensitivity to cadmium. Higher H₂O₂ accumulation and extensive plasma membrane damage were detected in the leaves of transgenic seedlings when exposed to Cd, compared with wild-type plants. Our findings support the conclusion that overexpression of BjEXPA1 in tobacco renders plants susceptible to Cd stress, probably due to disruption of the growth process and/or the breakdown of cell walls.     

Expansin-regulated cell elongation is involved in the drought tolerance in wheat

Water stress restrains plant growth. Expansin is a cell wall protein that is generally accepted to be the key regulator of cell wall extension during plant growth. In this study, we used two different wheat cultivars to study the involvement of expansin in drought tolerance. Wheat coleoptile was used as the material in experiment. Our results indicated that water stress induced an increase in acidic pH-dependant cell wall extension, which is related to expansin activity; however, water stress inhibited coleoptile elongation growth. The increased expansin activity was mainly due to increased expression of expansin protein that was upregulated by water stress, but water stress also resulted in a decrease in cell wall acidity, a negative factor for cell wall extension. Decreased plasma membrane H⁺-ATPase activity was involved in the alkalinization of the cell wall under water stress. The activity of expansin in HF9703 (a drought-tolerant wheat cultivar) was always higher than that in 921842 (a drought-sensitive wheat cultivar) under both normal and water stress conditions, which may be correlated with the higher expansin protein expression and plasma membrane H⁺-ATPase activity observed in HF9703 versus 921842. However, water stress did not change the susceptibility of the wheat cell wall to expansin, and no difference in this susceptibility was observed between the drought-tolerant and drought-sensitive wheat cultivars. These results suggest the involvement of expansin in cell elongation and the drought resistance of wheat.     

AtEXP2 Is Involved in Seed Germination and Abiotic Stress Response in Arabidopsis

Expansins are cell wall proteins that promote cell wall loosening by inducing pH-dependent cell wall extension and stress relaxation. Expansins are required in a series of physiological developmental processes in higher plants such as seed germination. Here we identified an Arabidopsis expansin gene AtEXPA2 that is exclusively expressed in germinating seeds and the mutant shows delayed germination, suggesting that AtEXP2 is involved in controlling seed germination. Exogenous GA application increased the expression level of AtEXP2 during seed germination, while ABA application had no effect on AtEXP2 expression. Furthermore, the analysis of DELLA mutants show that RGL1, RGL2, RGA, GAI are all involved in repressing AtEXP2 expression, and RGL1 plays the most dominant role in controlling AtEXP2 expression. In stress response, exp2 mutant shows higher sensitivity than wild type in seed germination, while overexpression lines of AtEXP2 are less sensitive to salt stress and osmotic stress, exhibiting enhanced tolerance to stress treatment. Collectively, our results suggest that AtEXP2 is involved in the GA-mediated seed germination and confers salt stress and osmotic stress tolerance in Arabidopsis.     

Antagonistic relationship between AtRALF1and brassinosteroid regulates cellexpansion-related genes

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.     

Overexpression of β‐expansin gene GmEXPB2 improves phosphorus efficiency in soybean

Soybean (Glycine max) is an important oil crop in agricultural production, but low phosphorus (P) availability limits soybean growth and production. Expansin is a family of plant cell wall proteins and involved in a variety of physiological processes, including cell division and enlargement, root growth and leaf development. To test the potential effects of expansins on crop production, we have developed soybean transgenic plants overexpressing a soybean β‐expansin gene GmEXPB2, which was significantly induced by phosphate (Pi) starvation. The results indicated that constitutive overexpression of GmEXPB2 promoted leaf expansion, sequentially stimulated root growth and consequently resulted in improved P efficiency in the transgenic plants under P‐limited conditions in hydroponics. In particular, when tested in calcareous (CS) and acid soils (AS), the two GmEXPB2 transgenic soybean lines showed above 25 and 40% increases in plant dry weight and P content, respectively to wild‐type plants in low‐P CS, but not in AS. To our knowledge, this is the first report in which improvement of P efficiency could be achieved through constitutive overexpression of an endogenous EXPB gene in soybean. These findings suggest that genetic modification of root and leaf traits might be a suitable strategy for improving crop production in low‐P soils.     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

Impaired growth in transgenic plants over-expressing an expansin isoform

Expansins are cell wall proteins characterised by their ability to stimulate wall loosening during cell expansion. The expression of some expansin isoforms is clearly correlated with growth and the external application of expansins can stimulate cell expansion in vivo in several systems. We report here the expression of a heterologous expansin coding sequence in transgenic tomato plants (Lycopersicon esculentum Mill.) under the control of a constitutive promoter. In some transgenic lines with high levels of expansin activity extractable from cell walls, we observed alterations of growth: mature plants were stunted, with shorter leaves and internodes, and dark-grown seedlings had shorter and wider hypocotyls than their wild-type counterparts. Examination of hypocotyl sections revealed similar differences at the cellular level: cortical and epidermal cells were shorter and wider than those from wild-type seedlings. The observed stimulation of radial expansion did not compensate for the decreased elongation, and overall growth was reduced in the transgenics. As this observation can seem paradoxical given the known effect of expansins on isolated cell walls, we examined the mechanical behaviour of transgenic tissue. We measured a decrease in hypocotyl elongation in response to acidic pH in the transformants. This result may account for the alterations in cell expansion, and could itself be explained by a reduced susceptibility of transgenic cell walls to expansin action.     

Expansins in the bryophyte Physcomitrella patens

Expansins are cell wall proteins which play a key function in basic processes of plant growth and differentiation. It has been proposed that expansins are likely to be present in all land plants and, to date, they have been reported in angiosperms, gymnosperms and pteridophytes. In this paper, we provide the first report and analysis of genes encoding expansin-like proteins in the bryophyte, Physcomitrella patens. Our analysis indicates that both - and -expansins are present as gene families in this plant and expression analysis indicates that these genes are subject to a complex regulation by both hormonal and environmental factors. In particular, the expression of many expansin genes in P. patens is upregulated by stress conditions, suggesting that they play a role in the specific cellular differentiation displayed by P. patens in response to such stress. Finally, we provide the first report on the generation and analysis of a series of knockout mutants for individual expansin genes. Abbreviations: IAA, indole-acetic acid; BAP, 6-benzylaminopurine; ABA, abscisic acid; npt, neomycin phospotransferase; KO, knockout     

Plant Responses to Drought Stress and Exogenous ABA Application are Modulated Differently by Mycorrhization in Tomato and an ABA-deficient Mutant (<Emphasis Type="Italic">Sitiens</Emphasis>)

The aims of the present study are to find out whether the effects of arbuscular mycorrhizal (AM) symbiosis on plant resistance to water deficit are mediated by the endogenous abscisic acid (ABA) content of the host plant and whether the exogenous ABA application modifies such effects. The ABA-deficient tomato mutant sitiens and its near-isogenic wild-type parental line were used. Plant development, physiology, and expression of plant genes expected to be modulated by AM symbiosis, drought, and ABA were studied. Results showed that only wild-type tomato plants responded positively to mycorrhizal inoculation, while AM symbiosis was not observed to have any effect on plant development in sitiens plants grown under well-watered conditions. The application of ABA to sitiens plants enhanced plant growth both under well-watered and drought stress conditions. In respect to sitiens plants subjected to drought stress, the addition of ABA had a cumulative effect in relation to that of inoculation with G. intraradices. Most of the genes analyzed in this study showed different regulation patterns in wild-type and sitiens plants, suggesting that their gene expression is modulated by the plant ABA phenotype. In the same way, the colonization of roots with the AM fungus G. intraradices differently regulated the expression of these genes in wild-type and in sitiens plants, which could explain the distinctive effect of the symbiosis on each plant ABA phenotype. This also suggests that the effects of the AM symbiosis on plant responses and resistance to water deficit are mediated by the plant ABA phenotype.