Ectopic expression of a wheat WRKY transcription factor gene TaWRKY71-1 results in hyponastic leaves in Arabidopsis thaliana

Abstract

Leaf type is an important trait that closely associates with crop yield. WRKY transcription factors exert diverse regulatory effects in plants, but their roles in the determination of leaf type have not been reported so far. In this work, we isolated a WRKY transcription factor gene TaWRKY71-1 from a wheat introgression line SR3, which has larger leaves, superior growth capacity and higher yield than its parent common wheat JN177. TaWRKY71-1 specifically expressed in leaves, and produced more mRNA in SR3 than in JN177. TaWRKY71-1 localized in the nucleus and had no transcriptional activation activity. TaWRKY71-1 overexpression in Arabidopsis resulted in hyponastic rosette leaves, and the hyponastic strength was closely correlative with the transcription level of the transgene. The spongy mesophyll cells at abaxial side of leaves were drastically compacted by TaWRKY71-1 overexpression. In TaWRKY71-1 overexpression Arabidopsis, the expression of IAMT1 that encodes a methyltransferase converting free indole-3-acetic acid (IAA) to methyl-IAA ester (MeIAA) to alter auxin homeostatic level was induced, and the induction level was dependent on the abundance of TaWRKY71-1 transcripts. Besides, several TCP genes that had found to be restricted by IAMT1 had lower expression levels as well. Our results suggest that TaWRKY71-1 causes hyponastic leaves through altering auxin homeostatic level by promoting the conversion of IAA to MeIAA.

Figure 1. TaWRKY71-1 are specifically transcribed in wheat leaves with a higher expression level in SR3.

A: The hybridization signals of the probe referring to TaWRKY71-1 in the microarray using wheat seedlings harvested at three-leaf stage. Data present as mean ± standard deviation. B: The sqRT-PCR using two-week old wheat seedlings. A wheat actin gene was used as the internal reference. JN177, a common wheat cultivar; SR3, a wheat introgression cultivar selected from somatic hybrids between JN177 and its close relative grass Thinopyrum ponticum via asymmetric somatic hybridization. 28C, 28 cycles; 38C, 38 cycles.

Figure 2. TaWRKY71-1 encodes a group IIa of WRKY transcription factor.

A: the statistics of single nucleotide polymorphisms of TaWRKY71-1 from SR3 and JN177 and its homologous gene TaWRKY71 from wheat cultivar Chinese Spring. B: The phylogenetic analysis of TaWRKY71-1 and its aligned WRKY transcription factors from NCBI at the amino acid level. C: Sequence alignment of TaWRKY71-1 from SR3 and JN177and its homologous gene TaWRKY71 from Chinese Spring. Underlined peptide sequence is a WRKY domain. Ta, Triticum aestivum; Hv, Hordeum vulgare; Bd, Brachypodium distachyon; Zm, Zea mays; Sb, Sorghum bicolor; Os, Oryza sativa; Vv, Vitis vinifera; At, Arabidopsis thaliana.

Figure 3. TaWRKY71-1 localizes in the nucleus.

Transiently expressed TaWRKY71-1 in-frame fused with GFP in Arabidopsis mesophyll protoplasts (A) and onion epidermis cells (B). Bar in A 10µm and in B 100µm.

Figure 4. TaWRKY71-1 has no transactivation activity.

The full length ORF of TaWRKY71-1 was constructed into pGBKT7, and transformed yeasts were selected on both SD-Trp and SD-Trp-His-Ade media, respectively.

Figure 5. TaWRKY71-1 overexpression results in hyponastic leaves in Arabidopsis.

A: Phenotypic comparison of four-week old Arabidopsis seedlings. B: sqRT-PCR of transgenic TaWRKY71-1 in Arabidopsis, and an Arabidopsis tublin gene was used as the internal reference. Col-0, the ecotype Columbia-0; VC, Vector control; OE1–3, three independent transgenic Arabidopsis lines overexpressing TaWRKY71-1.

Figure 6. TaWRKY71-1 overexpression tightens spongy mesophyll tissue by the paraffin section assay.

A, B: Transverse sections of middle part of the 6^th mature rosette leaves of VC (A) and OE3 (B) lines. C, D: The enlarged view of white pane-labeled section in panels A and B, respectively. VC, Vector control; OE3, an independent transgenic Arabidopsis lines overexpressing TaWRKY71-1. Arrows in A and B show the adaxial direction. mr, main rib; ad, adaxial side; ab, abaxial side; pmc, palisade mesophyll cells; smc, spongy mesophyll cells.

Figure 7. TaWRKY71-1 alters the expression of IAMT1 -mediated leaf curvature genes.

VC, Vector control; OE1–3, Three independent transgenic Arabidopsis lines overexpressing TaWRKY71-1. The leaves of four-week old Arabidopsis seedlings were harvested for real-time PCR. Arabidopsis actin gene ACTIN2 was used as the internal reference. The graphs represent one of the same results from three independent experiments. Data present as mean ± standard deviation. Columns without the same lowercase letter are significantly different using the lowest significant difference test of one-way ANOVA (P<0.05).