Association mapping for maize stover yield and saccharification efficiency using a multiparent advanced generation intercross (MAGIC) population.

Cellulosic ethanol derived from fast growing C4 grasses could become an alternative to finite fossil fuels. With the potential to generate a major source of lignocellulosic biomass, maize has gained importance as an outstanding model plant for studying the complex cell wall network and also to optimize crop breeding strategies in bioenergy grasses. A genome-wide association study (GWAS) was conducted using a subset of 408 Recombinant Inbred Lines (RILs) from a Multi-Parent Advanced Generation Intercross (MAGIC) Population in order to identify single nucleotide polymorphisms (SNPs) associated with yield and saccharification efficiency of maize stover. We identified 13 SNPs significantly associated with increased stover yield that corresponded to 13 QTL, and 2 SNPs significantly associated with improved saccharification efficiency, that could be clustered into 2 QTL. We have pointed out the most interesting SNPs to be implemented in breeding programs based on results from analyses of averaged and yearly data. Association mapping in this MAGIC population highlight genomic regions directly linked to traits that influence the final use of maize. Markers linked to these QTL could be used in genomic or marker-assisted selection programs to improve biomass quality for ethanol production. This study opens a possible optimisation path for improving the viability of second-generation biofuels.

Exploring tomato Solanum pennellii introgression lines for residual biomass and enzymatic digestibility traits.

BACKGROUND: Residual biomass production for fuel conversion represents a unique opportunity to avoid concerns about compromising food supply by using dedicated feedstock crops. Developing tomato varieties suitable for both food consumption and fuel conversion requires the establishment of new selection methods. RESULTS: A tomato Solanum pennellii introgression population was assessed for fruit yield, biomass phenotypic diversity, and for saccharification potential. Introgression lines 2-5, 2-6, 6-3, 7-2, 10-2 and 12-4 showed the best combination of fruit and residual biomass production. Lignin, cellulose, hemicellulose content and saccharification rate showed a wide variation in the tested lines. Within hemicellulose, xylose value was high in IL 6-3, IL 7-2 and IL 6-2, whereas arabinose showed a low content in IL 10-2, IL 6-3 and IL 2-6. The latter line showed also the highest ethanol potential production. Alkali pre-treatment resulted in the highest values of saccharification in most of lines tested, suggesting that chemical pretreatment is an important factor for improving biomass processability. Interestingly, extreme genotypes for more than one single trait were found, allowing the identification of better genotypes. Cell wall related genes mapping in genomic regions involved into tomato biomass production and digestibility variation highlighted potential candidate genes. Molecular expression profile of few of them provided useful information about challenged pathways. CONCLUSIONS: The screening of S. pennellii introgression population resulted very useful for delving into complex traits such as biomass production and digestibility. The extreme genotypes identified could be fruitfully employed for both genetic studies and breeding.

pH and expansin action on single suspension-cultured tomato (Lycopersicon esculentum) cells.

The aim of this study was to measure key material properties of the cell walls of single suspension-cultured plant cells and relate these to cell-wall biochemistry. To this end, micromanipulation was used to compress single tomato cells between two flat surfaces until they ruptured, and force-deformation data were obtained. In addition to measuring the bursting force, we also determined the elastic (Young's) modulus of the cell walls by matching low strain (< or = 20% deformation) experimental data with a cell compression model, assuming linear elastic cell walls. The walls were most elastic at pH 4.5, the pH optimum for expansin activity, with an elastic modulus of 2.0 +/- 0.1 GPa. Following the addition of exogenous expansins, cell walls became more elastic at all pH values. Western blot analysis of proteins from walls of cultured cells revealed the presence of expansin epitopes, suggesting that the inherent pH dependence of elasticity and other compression phenomena is related to the presence of endogenous expansin proteins and their wall-loosening ability. Although strict application of the linear-elastic model could not be applied to large deformations-for example, up to cell bursting-because of irreversible behaviour, the deviation of the data from the model was generally small enough to allow estimation of the strain in the cell wall at failure. This strain was greater at pH 4.5 and when expansins were added to the suspension. The changes in elasticity are consistent with suggestions about the mode of expansin action. The estimated strains at failure are compatible with data on the failure of Acetobacter-derived cellulose-xyloglucan composites and proposed mechanisms of such failure. Through the measurement of cell-wall material properties using micromanipulation, it may be possible to understand more fully how cell-wall composition, structure and biochemistry lead to cell mechanical behaviour.

From EST sequence to spider silk spinning: identification and molecular characterisation of Nephila senegalensis major ampullate gland peroxidase NsPox.

Spider dragline silk is renowned as one of the toughest materials of its kind. In nature, spider silks are spun out of aqueous solutions under environmental conditions. This is in contrast to production of most synthetic fibres, where hazardous solvents, high temperatures and pressure are used. In order to identify some of the chemical processes involved in spider silk spinning, we have produced a collection of cDNA sequences from specific regions of Nephila senegalensis major ampullate gland. We examined in detail the sequence and expression of a putative Nephila senegalensis peroxidase gene (NsPox) from our EST collection. NsPox encodes a protein with similarity to Drosophila melanogaster and Aedes aegypti peroxidases. Northern analysis and in situ localisation experiments revealed that NsPox is expressed in major and minor ampullate glands of the spider where the main components of the dragline silk are produced. We suggest that NsPox plays a role in dragline silk fibre formation and/or processing.

The molecular basis of plant cell wall extension.

In all terrestrial and aquatic plant species the primary cell wall is a dynamic structure, adjusted to fulfil a diversity of functions. However a universal property is its considerable mechanical and tensile strength, whilst being flexible enough to accommodate turgor and allow for cell elongation. The wall is a composite material consisting of a framework of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides, interlaced with structural proteins and pectic polymers. The assembly and modification of these polymers within the growing cell wall has, until recently, been poorly understood. Advances in cytological and genetic techniques have thrown light on these processes and have led to the discovery of a number of wall-modifying enzymes which, either directly or indirectly, play a role in the molecular basis of cell wall expansion.

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.

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.

Expression of a heterologous expansin in transgenic tomato plants.

Expansins are cell wall proteins thought to play an important role in growth and other events involving cell wall modifications. Whereas the expression patterns of many isoforms have been characterised, the nature of their activity is still poorly understood. Large amounts of active expansins are necessary to undertake biochemical studies and identify their substrates. We report here the successful expression of a recombinant expansin [CsExp1, isolated from cucumber (Cucumis sativus L.) hypocotyls] in transgenic tomato (Lycopersicon esculentum Mill.) plants, under the control of a constitutive promoter. In some transformants, CsExp1 transcript and protein accumulated to high levels, and expansin activity extractable from the cell walls was increased up to about 20-fold the activity measured in wild-type plants. These results confirm the identity of the CsExp1 coding sequence, and will enable large quantities of active expansin to be obtained for further studies.

Probing expansin action using cellulose/hemicellulose composites.

Cellulose-based composite materials containing xyloglucans or mannan-based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical alpha-expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose-only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose-only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1-4) beta-glucan to (1-4) beta-glucan hydrogen bonded contacts can also serve as substrates.

Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding.

The biochemical mechanisms underlying cell wall expansion in plants have long been a matter of conjecture. Previous work in our laboratory identified two proteins (named "expansins") that catalyze the acid-induced extension of isolated cucumber cell walls. Here we examine the mechanism of expansin action with three approaches. First, we report that expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. We conclude that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, we investigated the effects of expansins on stress relaxation of isolated walls. These studies show that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation. In addition, these experiments show that expansins do not cause a progressive weakening of the walls, as might be expected from the action of a hydrolase. Third, we studied the binding of expansins to the cell wall and its components. The binding characteristics are consistent with this being the site of expansin action. We found that expansins bind weakly to crystalline cellulose but that this binding is greatly increased upon coating the cellulose with various hemicelluloses. Xyloglucan, either solubilized or as a coating on cellulose microfibrils, was not very effective as a binding substrate. Expansins were present in growing cell walls in low quantities (approximately 1 part in 5000 on a dry weight basis), suggesting that they function catalytically. We conclude that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting noncovalent bonds within this polymeric network. Our results suggest that a minor structural component of the matrix, other than pectin and xyloglucan, plays an important role in expansin binding to the wall and, presumably, in expansin action.

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls.

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc4-Xyl3-Gal2)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher a pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc4-Xyl3-Gal2), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.