Transgenic tobacco plants expressing grass <Emphasis Type="Italic">AstEXPA1</Emphasis> gene show improved performance to several stresses

Expansins are cell wall-loosening proteins and now widely accepted to associate with the plant resistance against various abiotic stresses. In this study, we cloned an expansin gene of AstEXPA1 from Agrostis stolonifera, a heat-resistant creeping bentgrass cultivar, and transformed it into tobacco plants. Physiological index test showed that the transgenic lines were resistant to various abiotic stresses of drought, heat, cold, and salt in comparison to non-transgenic plants. Comprehensive analysis of four physiological response indices showed that the transgenic plants performed much better resistance to drought, following to heat, cold and salt stress, respectively. Meanwhile soluble sugar content displayed more weight to plant resistance by over-expressing AstEXPA1 gene, followed as proline content, REL, and MDA content. The results here would expand our understanding of the expansin roles and drive better insights into plant molecular breeding against stress.     

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.     

Expansins: roles in plant growth and potential applications in crop improvement

Key message

Results from various expansin related studies have demonstrated that expansins present an opportunity to improve various crops in many different aspects ranging from yield and fruit ripening to improved stress tolerance.

Abstract

The recent advances in expansin studies were reviewed. Besides producing the strength that is needed by the plants, cell walls define cell shape, cell size and cell function. Expansins are cell wall proteins which consist of four sub families; α-expansin, β-expansin, expansin-like A and expansin-like B. These proteins mediate cell wall loosening and they are present in all plants and in some microbial organisms and other organisms like snails. Decades after their initial discovery in cucumber, it is now clear that these small proteins have diverse biological roles in plants. Through their ability to enable the local sliding of wall polymers by reducing adhesion between adjacent wall polysaccharides and the part they play in cell wall remodeling after cytokinesis, it is now clear that expansins are required in almost all plant physiological development aspects from germination to fruiting. This is shown by the various reports from different studies using various molecular biology approaches such as gene achieve these many roles through their non-enzymatic wall loosening ability. This paper reviews and summarizes some of the reported functions of expansins and outlines the potential uses of expansins in crop improvement programs.

     

Ectomycorrhizas of two species of <Emphasis Type="Italic">Tuber</Emphasis> (clade Puberulum) in the Mexican subtropical cloud forest

Expansins are non-enzymatic cell wall proteins that mediate plant growth by catalyzing loosening of cell walls without lysing the wall polymers. Advances in the field of bioinformatics have facilitated the prediction of the members of expansin gene family across several model plants. Expansins constitutes into four sub-families; α-expansin, β-expansin, expansin-like A and expansin-like B. Biological functions of expansin gene family include diverse aspects of plant growth and development, shoot and root elongation, leaf morphogenesis, flower and fruit development, embryogenesis, pollen tube growth, stress tolerance, etc. Recent studies have demonstrated the role of expansins in plant-symbiotic interactions. The present review reveals the factors that govern plant-arbuscular mycorrhizal fungi (AMF) and legume-rhizobia symbioses; and the genes that participate in these diverse symbiont interactions. Further, we focus on the expression profiles and the functions of expansins during plant-AMF and legume-rhizobia interactions. The key roles of expansin proteins during AMF invasion, arbuscule formation, rhizobial infection and nodule organogenesis were uncovered during symbioses. This review summarizes discoveries that support the key and versatile roles of various expansin members in the plant-mycorrhizal and legume-rhizobial symbioses.     

Genomic location and expression analysis of expansin gene family reveals the evolutionary and functional significance in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The plant-specific expansin proteins constitute an ancient and major gene family known to have roles in regulating diverse biological processes in plants. Although the functions of many expansin genes have been identified in wheat and other species, little is known about the evolution and genomic locations of the expansin genes in wheat (Triticum aestivum). In this study, a total of 87 expansin genes were identified in the wheat genome, including 52 EXPAs, 42 EXPBs and 4 EXLAs. The EXLB gene was not found in the wheat genome. Phylogenetic tree and comparative analysis revealed amplification of the EXPBs in rice, maize and wheat. The predicted wheat expansins were distributed across 14 of 21 chromosomes with different densities, 3 tightly co-located clusters and 15 paralogous pairs, indicating that tandem duplication and segmental duplication events also played roles in the evolution of expansins in wheat. In addition, the gene structures and conserved protein domains of wheat expansins suggest high levels of conservation within the phylogenetic subgroups. Analysis of a published microarray database showed that most wheat expansin genes exhibit different expression levels in different tissues and developmental stages. To our knowledge, this is the first report of a genome-wide analysis of the wheat expansin gene family, which should provide valuable information for further elucidating the classification and putative functions of the entire gene family.     

Proteins and Metabolites Regulated by Trinexapac-ethyl in Relation to Drought Tolerance in Kentucky Bluegrass

Plants have developed various mechanisms in adaptation to water deficit stress, including growth retardant to reduce water loss. Previous studies reported that plants treated with a growth inhibitor, trinexapac-ethyl (TE), had improved drought tolerance. The objective of this study was to determine alterations in proteins and metabolite accumulation associated with drought tolerance improvement in a perennial grass species, Kentucky bluegrass (Poa pratensis), induced by TE application. Plants were treated with TE [1.95 ml l⁻¹ (v:v); a.i. TE = 0.113%] through foliar spray for 14 days, and then subjected to drought stress by withholding irrigation for 15 days in growth chambers. TE-treated plants exhibited significantly higher relative water content and photosynthetic capacity and lower membrane leakage than nontreated plants under drought stress, suggesting TE-enhanced drought tolerance in Kentucky bluegrass. Physiological improvement in drought tolerance through TE application was associated with the increased accumulation of various proteins and metabolites, including ferritin, catalase, glutathione-S-transferase, Rubisco, heat shock protein 70, and chaperonin 81, as well as fatty acids (palmitic acid, α-linolenic acid, linoleic acid, and octadecanoic acid). Our results suggest that TE may regulate metabolic processes for antioxidant defense, protective protein synthesis, photorespiration, and fatty acid synthesis, and thereby contribute to better drought tolerance in Kentucky bluegrass.     

A genome-wide analysis of the expansin genes in Malus × Domestica

Expansins were first identified as cell wall-loosening proteins; they are involved in regulating cell expansion, fruits softening and many other physiological processes. However, our knowledge about the expansin family members and their evolutionary relationships in fruit trees, such as apple, is limited. In this study, we identified 41 members of the expansin gene family in the genome of apple (Malus × Domestica L. Borkh). Phylogenetic analysis revealed that expansin genes in apple could be divided into four subfamilies according to their gene structures and protein motifs. By phylogenetic analysis of the expansins in five plants (Arabidopsis, rice, poplar, grape and apple), the expansins were divided into 17 subgroups. Our gene duplication analysis revealed that whole-genome and chromosomal-segment duplications contributed to the expansion of Mdexpansins. The microarray and expressed sequence tag (EST) data showed that 34 Mdexpansin genes could be divided into five groups by the EST analysis; they may also play different roles during fruit development. An expression model for MdEXPA16 and MdEXPA20 showed their potential role in developing fruit. Overall, our study provides useful data and novel insights into the functions and regulatory mechanisms of the expansin genes in apple, as well as their evolution and divergence. As the first step towards genome-wide analysis of the expansin genes in apple, our results have established a solid foundation for future studies on the function of the expansin genes in fruit development.     

扩展蛋白与植物抗逆性关系研究进展

扩展蛋白是一种细胞壁蛋白,可调节细胞壁的松弛和伸展。目前研究表明,扩展蛋白几乎参与调节植物生长发育的整个进程。扩展蛋白还与植物的多种抗性反应有关,在植物对干旱、高盐以及病虫害等生物胁迫和非生物胁迫响应方面起着重要的调节作用。干旱胁迫下扩展蛋白基因的表达与植物的抗旱性有一定的关系;植物的耐盐性受到扩展蛋白基因表达的影响;淹水促进植物的伸长生长与扩展蛋白的表达密切相关;扩展蛋白调节细胞壁松弛为植物抗病性研究提供了新的思路。     

Expansin gene loss is a common occurrence during adaptation to an aquatic environment

Expansins comprise a superfamily of plant cell wall loosening proteins that can be divided into four individual families (EXPA, EXPB, EXLA and EXLB). Aside from inferred roles in a variety of plant growth and developmental traits, little is known regarding the function of specific expansin clades, for which there are at least 16 in flowering plants (angiosperms); however, there is evidence to suggest that some expansins have cell‐specific functions, in root hair and pollen tube development, for example. Recently, two duckweed genomes have been sequenced (Spirodela polyrhiza strains 7498 and 9509), revealing significantly reduced superfamily sizes. We hypothesized that there would be a correlation between expansin loss and morphological reductions seen among highly adapted aquatic species. In order to provide an answer to this question, we characterized the expansin superfamilies of the greater duckweed Spirodela, the marine eelgrass Zostera marina and the bladderwort Utricularia gibba. We discovered rampant expansin gene and clade loss among the three, including a complete absence of the EXLB family and EXPA‐VII. The most convincing correlation between morphological reduction and expansin loss was seen for Utricularia and Spirodela, which both lack root hairs and the root hair expansin clade EXPA‐X. Contrary to the pattern observed in other species, four Utricularia expansins failed to branch within any clade, suggesting that they may be the result of neofunctionalization. Last, an expansin clade previously discovered only in eudicots was identified in Spirodela, allowing us to conclude that the last common ancestor of monocots and eudicots contained a minimum of 17 expansins.     

Growth of Transgenic Tobacco Plants with Changed Expression of Genes Encoding Expansins under the Action of Stress Factors

The peculiarities of root growth and stress tolerance of transgenic tobacco plants with constitutive expression of NtEXPA1 and NtEXPA5 genes, as well as plants with reduced expression of NtEXPA4 gene encoding α-expansins of Nicotiana tabacum, were studied during prolonged cultivation under conditions of drought, salinity, and low positive temperatures. Increased expression of expansin genes led to an increase in the growth rate and root length both under normal plant growth conditions and at 12°C and 50 mM NaCl. Increased expression of expansin genes influenced the changes in the fresh and dry mass of a shoot, leading to an increase in their exposure to hypothermia. Transgenic plants with a reduced level of NtEXPA4 expansin gene expression were characterized by a reduction in the fresh and dry weight of a shoot due to drought and low positive temperatures. The totality of the data obtained may indicate the involvement of NtEXPA1, NtEXPA4, and NtEXPA5 tobacco expansin genes in the regulation of growth under hypothermia, drought, and salinity.     

Transgenic creeping bentgrass overexpressing Osa‐miR393a exhibits altered plant development and improved multiple stress tolerance

MicroRNA393 (miR393) has been implicated in plant growth, development and multiple stress responses in annual species such as Arabidopsis and rice. However, the role of miR393 in perennial grasses remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is an environmentally and economically important C3 cool‐season perennial turfgrass. Understanding how miR393 functions in this representative turf species would allow the development of novel strategies in genetically engineering grass species for improved abiotic stress tolerance. We have generated and characterized transgenic creeping bentgrass plants overexpressing rice pri‐miR393a (Osa‐miR393a). We found that Osa‐miR393a transgenics had fewer, but longer tillers, enhanced drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased uptake of potassium and enhanced heat stress tolerance associated with induced expression of small heat‐shock protein in comparison with wild‐type controls. We also identified two targets of miR393, AsAFB2 and AsTIR1, whose expression is repressed in transgenics. Taken together, our results revealed the distinctive roles of miR393/target module in plant development and stress responses between creeping bentgrass and other annual species, suggesting that miR393 would be a promising candidate for generating superior crop cultivars with enhanced multiple stress tolerance, thus contributing to agricultural productivity.     

Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.     

AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis

The calcium-dependent protein kinase (CDPK) family is needed in plant signaling during various physiological pathways. The Arabidopsis AtCPK6 gene belongs to the subclass of stress-inducible CDPKs, which is stimulated by salt and osmotic stress. To elucidate the physiological function of AtCPK6, transgenic Arabidopsis plants under the control of double CaMV 35S promoter were obtained. AtCPK6 over-expressing plants showed enhanced tolerance to salt/drought stresses. The elevated tolerance of the AtCPK6 over-expressing plants was confirmed by the change of proline and malondialdehyde (MDA). Real-time PCR analyses revealed that the expression levels of several stress-regulated genes were altered in AtCPK6 over-expressing plants. However, cpk6 mutant displayed no obvious difference with control. These results are likely to indicate that AtCPK6 is functionally redundant and a positive regulator involved in the tolerance to salt/drought stress in Arabidopsis.     

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

Differential accumulation of proteins in leaves and roots associated with heat tolerance in two Kentucky bluegrass genotypes differing in heat tolerance

To understand the mechanisms of heat stress responses in perennial grasses, differential proteins in leaves and roots of two genotypes of Kentucky bluegrass (Poa pratensis), including heat-tolerant ‘Midnight’ and heat-sensitive ‘Brilliant’, were analyzed with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Plants were exposed to heat stress for 28 days in growth chambers. Under 7–28 days of heat stress, leaf photochemical efficiency declined significantly while electrolyte leakage increased in leaves and roots, and to a lesser extent for heat-tolerant ‘Midnight’ than for heat-sensitive ‘Brilliant’. Compared with leaves, cell membrane damage due to heat stress was more severe in roots. The 2-DE and MS analysis identified 37 heat-responsive proteins in leaves, 28 heat-responsive proteins in roots; 14 proteins in leaves and 9 proteins in roots exhibited differential expression between the two genotypes. The results indicate that proteins involved in metabolism and energy in leaves and those in antioxidant defense in roots are associated with heat tolerance in Kentucky bluegrass. The differential accumulation of these proteins might be the reason for different heat tolerance in two Kentucky bluegrass genotypes in aerial and underground parts.