A Surprising Diversity of Xyloglucan Endotransglucosylase/Hydrolase in Wheat: New in Sight to the Roles in Drought Tolerance

Abstract

Drought has become a major limiting factor for wheat productivity, and its negative impact on crop growth is anticipated to increase with climate deterioration in arid areas. Xyloglucan endoglycosylases/hydrolases (XTHs) are involved in constructing and remodeling cell wall structures and play an essential role in regulating cell wall extensibility and stress responses. However, there are no systematic studies on the wheat XTH gene family. In this study, 71 wheat XTH genes (TaXTHs) were characterized and classified into three subgroups through phylogenetic analysis. Genomic replication promoted the expansion of TaXTHs. We found a catalytically active motif and a potential N-linked glycosylation domain in all TaXTHs. Further expression analysis revealed that many TaXTHs in the roots and shoots were significantly associated with drought stress. The wheat TaXTH12.5a gene was transferred into Arabidopsis to verify a possible role of TaXTHs in stress response. The transgenic plants possessed higher seed germination rates and longer roots and exhibited improved tolerance to drought. In conclusion, bioinformatics and gene expression pattern analysis indicated that the TaXTH genes played a role in regulating drought response in wheat. The expression of TaXTH12.5a enhanced drought tolerance in Arabidopsis and supported the XTH genes' role in regulating drought stress response in plants.

Keywords: XTH gene family; drought; root plasticity; transgenic; wheat.

Figure 1

Phylogenetic analysis of full-length XTH proteins in wheat and Arabidopsis. The tree was constructed by maximum likelihood (ML) by PhyML 3.0 and bootstrap values based on 1000 replications. The outgroup is highlighted in red, and the branches with different colors correspond to the four groups.

Figure 2

Distribution of XTH genes on T. aestivum chromosomes. The TaXTH genes in red are tandem duplication genes. The number of chromosomes is indicated at the top of each chromosome. The scale on the left is in megabases (Mb).

Figure 3

Characterizations of XTH gene family members in wheat. (A), conserved motif positions. Identification of the motif composition of TaXTH proteins using MEME. Different colored boxes represent different motifs and their positions in the protein sequences. (B), domain location. (C), exon-intron gene structure features.

Figure 4

The expression of the TaXTH gene in the spring wheat line ‘Xinchun11’ was analyzed using real-time quantitative PCR. The relative expression level of TaXTH in wheat was measured after subjecting it to different durations (h) of drought stress with −0.5 MPa D-sorbitol. The 2-day-old germinated wheat seedlings were immersed in −0.5 MPa D-sorbitol and sampled at 0, 24, 48, and 72 h after treatment to assess the expression of TaXTH. Genes in the horizontal direction exhibit similar expression patterns. For additional details, see Supplementary Figure S4.

Figure 5

qRT-PCR analysis of TaXTH genes expressed under drought stress. Samples for expression profiling were collected from wheat at 0, 24, 48, and 72 h post drought stress. Expression profiles were detected by qRT–PCR and normalized to Actin. Note that the relative expression of TaXTHs was on a different scale. Orange dashed lines indicate roots, and green solid lines represent shoots. The name of the gene is shown in the upper left corner of each line graph. Results were analyzed in three biological replicates. Only partial gene expression patterns that were either ‘identical’ or ‘opposite’ were displayed. For additional details, see Supplementary Figure S5.

Figure 6

Calculation of copy number and relative expression level of TaXTH12.5a in four transgenic events. (A) The copy number ratio of TaXTH12.5a and lectin gene (Ta. LOC123165130) in Arabidopsis T0 transgenic plants was determined using digital PCR. When the ratio is equal to 0.5, transgenic Arabidopsis plants obtain one insertion copy, and when the ratio is equal to 1, two insertion copies are obtained. (B) The relative expression of TaXTH12.5a in the roots of T3 homozygous transgenic Arabidopsis was determined by qRT-PCR. The relative level of transcripts was standardized by Actin (NM_001339262). The bar represents the average value of the triplicate. * Significantly different, p < 0.05, using Fisher’s minimum significant difference test.

Figure 7

TaXTH12.5a transgenic plants showed higher germination rates and longer roots and hypocotyls under drought conditions. (A) The 3-day-old seedlings were dehydrated for 5 days. (B) The transgenesis and wild-type seeds were evenly spread on filter paper and subjected to a 5-day drought treatment under the same growth conditions. There were three repeated experiments and we counted their germination rates. (n ≥ 40). Root and hypocotyl lengths of the control and transgenic seedlings under drought conditions. (n ≥ 10). “*” indicates the difference between the transgenic and wild-type plants (p < 0.05).

Figure 8

Arabidopsis TaXTH12.5a transgenic plants exhibit an enhanced drought tolerance phenotype by promoting root development. (A), effect of drought on Arabidopsis seedlings. Two-week-old seedlings were subjected to continuous drought for 5 days. (B), comparison of the primary root length of transgenic and control Arabidopsis under 5-day drought conditions. (C,D), effect of drought on the number of lateral roots and tertiary root tips of the transgenic plants. (n ≥ 40). * indicates the difference between the transgenic and wild-type Arabidopsis (p < 0.05).