The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy

Polarized one- and two-dimensional infrared spectra were obtained from the epidermis of onion (Allium cepa) under hydrated and mechanically stressed conditions. By Fourier-transform infrared microspectroscopy, the orientation of macromolecules in single cell walls was determined. Cellulose and pectin exhibited little orientation in native epidermal cell walls, but when a mechanical stress was placed on the tissue these molecules showed distinct reorientation as the cells were elongated. When the stress was removed the tissue recovered slightly, but a relatively large plastic deformation remained. The plastic deformation was confirmed in microscopic images by retention of some elongation of cells within the tissue and by residual molecular orientation in the infrared spectra of the cell wall. Two-dimensional infrared spectroscopy was used to determine the nature of the interaction between the polysaccharide networks during deformation. The results provide evidence that cellulose and xyloglucan associate while pectin creates an independent network that exhibits different reorientation rates in the wet onion cell walls. The pectin chains respond faster to oscillation than the more rigid cellulose.     

Hypergravity increases the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity in azuki bean epicotyls.

Elongation growth of dark-grown azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls was suppressed by hypergravity at 30 x g and above. Acceleration at 300 x g significantly decreased the mechanical extensibility of cell walls. The amounts of cell wall polysaccharides (pectin, hemicellulose-II and cellulose) per unit length of epicotyls increased under the hypergravity condition. Hypergravity also increased the amounts and the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction, while decreasing the activity of xyloglucan-degrading enzymes extracted from epicotyl cell walls. These results suggest that hypergravity increases the amounts and the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity. Modification of xyloglucan metabolism as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of azuki bean epicotyls.     

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots

Arabidopsis root hair formation is determined by the patterning genes CAPRICE ( CPC ), GLABRA3 ( GL3 ), WEREWOLF ( WER ) and GLABRA2 ( GL2 ), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy–principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase ( CESA1 – 10 ) and 4 of 33 xyloglucan endotransglucosylase ( XTH1 – 33 ) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.     

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.     

Stimulation of elongation growth and xyloglucan breakdown in Arabidopsis hypocotyls under microgravity conditions in space

Seedlings of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia and an ethylene-resistant mutant etr1-1) were cultivated for 68.5, 91.5 and 136 h on board during the Space Shuttle STS-95 mission, and changes in the elongation growth and the cell wall properties of hypocotyls were analyzed. Elongation growth of dark-grown hypocotyls of both Columbia and etr1-1 was stimulated under microgravity conditions in space. There were no clear differences in the degree of growth stimulation between Columbia and etr1-1, indicating that the ethylene level was not abnormally high in the cultural environment of this space experiment. Microgravity also increased the mechanical extensibility of cell walls in both cultivars, and such an increase was attributed to the increase in the apparent irreversible extensibility. The levels of cell wall polysaccharides per unit length of hypocotyls decreased in space. Microgravity also reduced the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction. Also, the activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls increased under microgravity conditions. These results suggest that microgravity reduces the molecular mass of xyloglucans by increasing xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell wall polysaccharides seem to be involved in an increase in the cell wall extensibility, leading to growth stimulation of Arabidopsis hypocotyls in space.     

Genetic dissection of plant cell-wall biosynthesis

The plant cell wall is a complex structure consisting of a variety of polymers including cellulose, xyloglucan, xylan and polygalacturonan. Biochemical and genetic analysis has made it possible to clone genes encoding cellulose synthases (CesA). A comparison of the predicted protein sequences in the Arabidopsis genome indicates that 30 divergent genes with similarity to CesAs exist. It is possible that these cellulose synthase-like (Csl) proteins do not contribute to cellulose synthesis, but rather to the synthesis of other wall polymers. A major challenge is, therefore, to assign biological function to these genes. In an effort to address this issue we have systematically identified T-DNA or transposon insertions in 17 Arabidopsis Csls. Phenotypic characterization of "knock-out" mutants includes the determination of spectroscopic profile differences in mutant cell walls from wild-type plants by Fourier-transform IR microscopy. A more precise characterization includes cell wall fractionation followed by neutral sugar composition analysis by anionic exchange chromatography.     

Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations

Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P  ≤ 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24–67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.     

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.     

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.     

Quantitative trait loci analysis of nitrogen use efficiency in Arabidopsis

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 x Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL x QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.     

Natural variation for carbohydrate content in Arabidopsis. Interaction with complex traits dissected by quantitative genetics

Besides being a metabolic fuel, carbohydrates play important roles in plant growth and development, in stress responses, and as signal molecules. We exploited natural variation in Arabidopsis (Arabidopsis thaliana) to decipher the genetic architecture determining carbohydrate content. A quantitative trait locus (QTL) approach in the Bay-0 x Shahdara progeny grown in two contrasting nitrogen environments led to the identification of 39 QTLs for starch, glucose, fructose, and sucrose contents representing at least 14 distinct polymorphic loci. A major QTL for fructose content (FR3.4) and a QTL for starch content (ST3.4) were confirmed in heterogeneous inbred families. Several genes associated with carbon (C) metabolism colocalize with the identified QTL. QTLs for senescence-related traits, and for flowering time, water status, and nitrogen-related traits, previously detected with the same genetic material, colocalize with C-related QTLs. These colocalizations reflect the complex interactions of C metabolism with other physiological processes. QTL fine-mapping and cloning could thus lead soon to the identification of genes potentially involved in the control of different connected physiological processes.     

Effect of hypergravity stimulus on XTH gene expression in Arabidopsis thaliana.

There are several reports indicating that hypergravity and microgravity influence the mechanical properties of cell walls in shoots, resulting in changes in the growth rate. The mechanical properties of cell walls in dicots are mainly determined by the physicochemical properties of xyloglucan, a matrix polysaccharide. An increase in the molecular mass of xyloglucan correlated with a decrease in cell wall extensibility. Hypergravity is known to increase the molecular mass of xyloglucan. The cell wall enzyme, xyloglucan endotransglucosylase/hydrolase (XTH) is involved in xyloglucan metabolism. Using Arabidopsis, it was examined whether or not the expression of XTH genes in the floral stem and rosette leaf is influenced by hypergravity. RT-PCR analysis revealed that the expression of XTH genes changes in response to hypergravity of 300 g.     

QTL architecture of resistance and tolerance traits in Arabidopsis thaliana in natural environments

Quantitative-genetic approaches have offered significant insights into phenotypic evolution. However, quantitative-genetic analyses fail to provide information about the evolutionary relevance of specific loci. One complex and ecologically relevant trait for plants is their resistance to herbivory because natural enemies can impose significant damage. To illustrate the insights of combined molecular and ecological research, we present the results of a field study mapping quantitative trait loci (QTL) for resistance and tolerance to natural rabbit herbivory in the genetic model, Arabidopsis thaliana. Replicates of the Ler x Col recombinant inbred lines were planted into field sites simulating natural autumn and spring seasonal germination cohorts. Shortly after flowering, herbivores removed the main flowering inflorescence (apical meristem). We found several main-effect QTL for resistance within each seasonal cohort and significant QTL-season interactions, demonstrating that the loci underlying resistance to a single herbivore differ across seasonal environments. The presence of QTL x environment also shows that variation at specific loci is only available to selection in some environments. Despite significant among-line variance components, no QTL for tolerance were detected. The combined results of the quantitative-genetic and QTL analyses demonstrate that many loci of small effect underlie tolerance to damage by rabbits, and counter the hypothesis of locus-specific tradeoffs between resistance and tolerance. The results also provide insights as to the locus-specific nature of evolutionary constraints, i.e. some loci influence flowering time and resistance in both seasonal cohorts. Our results show how linking molecular-genetic tools with field studies in ecologically relevant settings can clarify the role of specific loci in the evolution of quantitative traits.     

Genomic approaches to analyzing natural variation in Arabidopsis thaliana

The genomics tools available for studying Arabidopsis thaliana are a great resource for researchers trying to characterize and understand the genetic basis of natural variation. Abundant polymorphic markers aid quantitative trait locus (QTL) mapping, the fully sequenced genome provides rapid identification of candidate loci, and extensive knockout collections allow those candidate loci to be tested. Combining QTL mapping of classic phenotypic traits with biochemical or expression analysis is providing mechanistic insight into the traits of interest. Conversely, natural variation studies are now being done on genomic traits such as methylation or chiasma frequency.     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.