Wheat (Triticum aestivum L.) TaHMW1D Transcript Variants Are Highly Expressed in Response to Heat Stress and in Grains Located in Distal Part of the Spike

Abstract

High-temperature stress during the grain filling stage has a deleterious effect on grain yield and end-use quality. Plants undergo various transcriptional events of protein complexity as defensive responses to various stressors. The "Keumgang" wheat cultivar was subjected to high-temperature stress for 6 and 10 days beginning 9 days after anthesis, then two-dimensional gel electrophoresis (2DE) and peptide analyses were performed. Spots showing decreased contents in stressed plants were shown to have strong similarities with a high-molecular glutenin gene, TraesCS1D02G317301 (TaHMW1D). QRT-PCR results confirmed that TaHMW1D was expressed in its full form and in the form of four different transcript variants. These events always occurred between repetitive regions at specific deletion sites (5'-CAA (Glutamine) GG/TG (Glycine) or (Valine)-3', 5'-GGG (Glycine) CAA (Glutamine) -3') in an exonic region. Heat stress led to a significant increase in the expression of the transcript variants. This was most evident in the distal parts of the spike. Considering the importance of high-molecular weight glutenin subunits of seed storage proteins, stressed plants might choose shorter polypeptides while retaining glutenin function, thus maintaining the expression of glutenin motifs and conserved sites.

Keywords: grain-filling period; high temperature stress; high-molecular glutenin gene; spikelet position in spike; transcript variants; wheat.

Figure 1

Spikelet position and seeds collected from spikes under high-temperature stress. (A) Division of a spike from base to top (spikelet #1–5). (B) Seeds taken from the five different sections of the spike. (C) Seeds from mature plants collected from the five different spike sections. Con, nontreated; DAT6-T, 6 days of treatment; DAT6-C, 6 days of nontreated control; DAT10-T, 10 days of treatment; DAT10-C, 10 days of nontreated control.

Figure 2

High-molecular weight glutenin subunit (HMW-GS) profiles of spikelets from different sections of the spike (#1–5, Figure 1A) of heat-treated plants. Glutenins were extracted from mature seeds harvested from high-temperature stress-treated wheat and nontreated control wheat. Ax, Dx, Bx, By, and Dy indicate HMW-GSs. M, Molecular size marker; Con, nontreated control; DAT6-T, 6 days of treatment; DAT10-T, 10 days of treatment.

Figure 3

Identification of six glutenin spots with significantly decreased expression levels under high-temperature stress during grain filling. PI ranges are displayed at the top. Protein sizes are displayed to the left. Con, nontreated control; DAT6-T, 6 days of treatment; DAT10-T, 10 days of treatment. Three biological replicates were used (Rep. 1–3). The numbers near the circles indicate spot numbers.

Figure 4

Complete Keumgang TaHMW1D gene sequence, including the (A) promoter, (B) 5′ UTR, (C) CDS, and (D) 3′ UTR. Bold and underlined, transcription factor binding sites; green, signal peptide region; pink, N-terminal region; gray, repetitive region; blue, C-terminal region.

Figure 5

Identification of transcript variants. (A) Agarose gel (1%) showing the 2242-bp TaHMW1D gene product from control plants (left). The PCR products of high-temperature stress-treated wheat show smaller-sized (1000–2242 bp) transcript variants (right). (B) Sizes of the TaHMW1D transcript variants.

Figure 6

Conserved motif sequence alignments of TaHMW1D and its transcript variants in Keumgang. Motifs with identical sequences are given the same number. Signal peptide (1, 2), N-terminal region (3–8), C-terminal region (9–11). The region between motifs 8 and 9 is a repetitive sequence region in which deletion events occur.

Figure 7

Protein sequence alignment of TaHMW1D and its transcript variants (TaHMW1D-1, 2, 3, 4) in Keumgang. Green, signal peptide; pink, N-terminal peptide; black, repetitive region; blue, C-terminal peptide. The conserved cysteine is marked in yellow.

Figure 8

Features of TaHMW1D and its transcript variants TaHMW1D-1, 2, 3, and 4. qRT-PCRs amplifying either the conserved or repetitive regions are indicated. Specific deletion sites (CAAGG/TG GGGCAA) are indicated. Transcript regions that are deleted out during high-temperature stress are marked with empty boxes.

Figure 9

qRT-PCR of spikelets taken from different locations along the spike (position #3 or 5), targeting the conserved (A) or repetitive (B) region of TaHMW1D. * p < 0.05; *** p < 0.001.