Analysis of Cell Wall-Related Genes in Organs of Medicago sativa L. under Different Abiotic Stresses

Abstract

Abiotic constraints are a source of concern in agriculture, because they can have a strong impact on plant growth and development, thereby affecting crop yield. The response of plants to abiotic constraints varies depending on the type of stress, on the species and on the organs. Although many studies have addressed different aspects of the plant response to abiotic stresses, only a handful has focused on the role of the cell wall. A targeted approach has been used here to study the expression of cell wall-related genes in different organs of alfalfa plants subjected for four days to three different abiotic stress treatments, namely salt, cold and heat stress. Genes involved in different steps of cell wall formation (cellulose biosynthesis, monolignol biosynthesis and polymerization) have been analyzed in different organs of Medicago sativa L. Prior to this analysis, an in silico classification of dirigent/dirigent-like proteins and class III peroxidases has been performed in Medicago truncatula and M. sativa. The final goal of this study is to infer and compare the expression patterns of cell wall-related genes in response to different abiotic stressors in the organs of an important legume crop.

Keywords: abiotic stresses; cell wall; cellulose synthases; dirigent proteins; gene expression; peroxidases.

Figure 1

Phylogenetic relationships of dirigent and dirigent-like protein from Medicago truncatula, Arabidopsis thaliana, Picea sitchensis, Oryza sativa, Hordeum vulgare and Triticum aestivum. The scale bar indicates an evolutionary distance of 1 amino acid substitution per position. The different subfamilies are indicated with different branch colours (according to [28]). Only bootstrap values >0.90 are indicated (small gray circle) for visual clarity. At: A. thaliana, P: P. sitchensis, Ta: T. aestivum, Hv: H. vulgare, Os: O. sativa. The accession numbers used to build the tree are indicated in the Materials and Methods section.

Figure 2

Organ-specific expression profiles of 16 M. truncatula genes coding for dirigent and dirigent-like proteins (retrieved from the M. truncatula eFP browser [33]). The heat map is drawn on the values retrieved from the eFP browser (the values ± standard deviations are shown in Supplementary Table S2). Pixel colour intensity is proportional to the actual expression values. The length of the branches, represented by the numbers, refers to the Pearson correlation coefficients among tissues and genes. Nodule mature (4w) refers to nodules samples from plants aged four weeks. Petiolules refer to the stalk present at the base of each of the three leaflets composing the trifoliate leaf of M. truncatula.

Figure 3

Phylogenetic relationships of class III peroxidases from M. truncatula and M. sativa. The peroxidases belonging to the same class are indicated with the same name colour (class I: blue, class II: fuchsia, class III: pink, class IV: red, class V: green, class VI: orange, class VII: turquoise, class VIII: violet). The peroxidase from Marcanthia polymorpha (MpPrx04, GenBank Accession BJ842248) was used to root the tree. The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position. Only bootstrap values >0.90 are indicated (small gray circle) for visual clarity. M. truncatula gene accessions are indicated in Supplementary Table S3. M. sativa gene accessions are indicated in the Materials and Methods section.

Figure 4

Organ-specific expression profiles of M. truncatula class III peroxidases (retrieved from the M. truncatula eFP database [33]). The values ± standard deviation retrieved from the eFP browser are shown in Supplementary Table S4. Pixel colour intensity is proportional to the actual expression values. The length of the branches, represented by the numbers, refers to the Pearson correlation coefficient among tissues and genes. Nodule mature (4w) refers to nodules samples from plants aged 4 weeks. Petiolules refer to the stalk present at the base of each of the three leaflets composing the trifoliate leaf of M. truncatula.

Figure 5

Heat map representation of the data reported in Table S5 and Table S6 showing the hierarchical clustering of cell wall-related genes in response to abiotic stresses at different time points in alfalfa roots. The numbers indicate the Pearson gene correlation coefficient. Pixel colour intensity is proportional to the actual expression values.

Figure 6

Heat map representation of the data reported in Supplementary Tables S7 and S8 showing the hierarchical clustering of cell wall-related genes in response to abiotic stresses at different time points in alfalfa leaves. The numbers indicate the Pearson gene correlation coefficient. Pixel colour intensity is proportional to the actual expression values.

Figure 7

Heat map representation of the data reported in Supplementary Table S9 showing the hierarchical clustering of cell wall-related genes in response to abiotic stresses at different time points in alfalfa stems. The numbers indicate the Pearson gene correlation coefficient. Pixel colour intensity is proportional to the actual expression values.

Figure 8

Graphs showing the change in expression of primary and secondary CesAs in roots (A,C) and leaves (B,D) of alfalfa plants subjected to different abiotic stresses. The data correspond to the values reported in Supplementary Tables S5 and S7. The control condition and the different stress treatments are boxed. The standard error of the mean is not represented in the graphs.