The binding pocket properties were fundamental to functional diversification of the GDSL-type esterases/lipases gene family in cotton

Abstract

Cotton is one of the most important crops in the world. GDSL-type esterases/lipases (GELPs) are widely present in all kingdoms and play an essential role in regulating plant growth, development, and responses to abiotic and biotic stresses. However, the molecular mechanisms underlying this functional diversity remain unclear. Here, based on the identification of the GELP gene family, we applied genetic evolution and molecular simulation techniques to explore molecular mechanisms in cotton species. A total of 1502 GELP genes were identified in 10 cotton species. Segmental duplication and differences in evolutionary rates are the leading causes of the increase in the number and diversity of GELP genes during evolution for ecological adaptation. Structural analysis revealed that the GELP family has high structural diversity. Moreover, molecular simulation studies have demonstrated significant differences in the properties of the binding pockets among cotton GELPs. In the process of adapting to the environment, GELPs not only have segmental duplication but also have different evolutionary rates, resulting in gene diversity. This diversity leads to significant differences in the 3D structure and binding pocket properties and, finally, to functional diversity. These findings provide a reference for further functional analyses of plant GELPs.

Keywords: GDSL-type esterases/lipases; Gossypium species; binding pocket; functional diversity; molecular mechanism; molecular simulation.

Figure 1

The phylogeny of the 10 cotton species and the number of GELP genes used in this study. The phylogeny of the 10 cotton species (A genome, D genome, and At/Dt) was constructed based on data from previous studies (Grover et al., 2019; Chen, ZJ et al., 2020b; Huang, G et al., 2021). - represents the absence of gene at this branch.

Figure 2

Evolutionary tree of the GELP family in the 10 cotton species (A genome, D genome, and At/Dt) and in Arabidopsis thaliana. A total of 1502 GELPs could be classified into four clades (I–IV) indicated with four branch colours. Every clade had 3-5 subclades (such as, A, B, C, D and E).

Figure 3

The occurrence pattern of shared orthologous groups among 10 cotton species and Arabidopsis thaliana.

Figure 4

The tertiary structure of representative GDSL lipases belonging to subclades in the evolutionary tree. Green, co-occurrence motifs of four clades; Violet motifs specific to each clade. The catalytic triad Ser, Asp, and His are shown as sticks. The names of the tertiary structure of representative GDSL lipases consist of two parts: the subclade of the evolutionary tree and the gene name.

Figure 5

The binding pocket structure of GELPs. SAS: Creates a surface coloured by the solvent accessibility of the receptor residues from blue for ‘exposed’ to green for ‘buried’. The names of the binding pocket structures of representative GDSL lipases consist of two parts: the subclade of the evolutionary tree and the gene name.

Figure 6

Expression patterns of GELP genes. Heatmap of the differential expression of GELP genes involved in the growth and development of ovule and fibre (A); eight tissues (root, stem, leaf, torus, petal, stamen, pistil and calycle) (B); the growth and development of seed, cotyledon, and root (C). Heatmap of differential expression of GELP genes in response to stress, including cotton aphid attack (0 h, 6 h, 12 h, 24 h, 48 h, and 72 h) (D), four abiotic stresses (cold, hot, salt and PEG) (E), and atoxigenic and toxigenic strains of Aspergillus flavus (F). The colour bar indicates log₂ expression levels.

Figure 7

Identification of salt tolerance of GbaGELP142D overexpressing Arabidopsis thaliana. (A) qRT-PCR analysis of GbaGELP142D expressing in the wild-types and transgenic Arabidopsis thaliana plants. Different letters on the bar graph show a significant difference (P < 0.05). (B) Germination percentage of the wild-types and transgenic GbaGELP142D Arabidopsis thaliana with different NaCl treatments. (C) The phenotype of the seedling of the overexpression lines of the GbaGELP142D gene (L19, L20 and L27) and WT under salt stress. (D) The phenotype of wild-type plants and transgenic GbaGELP142D plants after NaCl treatments.

Figure 8

Determination of biological indicators of salt tolerance of GbaGELP142D overexpressing Arabidopsis thaliana. (A) plant height of wild-type plants and transgenic GbaGELP142D plants after NaCl treatments. (B) root length of wild-type plants and transgenic GbaGELP142D plants after NaCl treatments. (C) aboveground fresh weight of wild-type plants and transgenic GbaGELP142D plants after NaCl treatments. (D) leaf chlorophyll content of wild-type plants and transgenic GbaGELP142D plants after NaCl treatments. Different letters on the bar graph show a significant difference (P<0.05).