Cloning and Characterization of TaSAP7-A, a Member of the Stress-Associated Protein Family in Common Wheat

Abstract

Stress association proteins (SAPs) are A20/AN1 zinc-finger domain proteins, which play important roles in plant adaptation to abiotic stress and plant development. The functions of SAPs in some plants were reported, but little is known about it in wheat (Triticum aestivum L.). In this study, we characterized a novel 2AN1-type stress association protein gene TaSAP7-A, which was mapped to chromosome 5A in wheat. Subcellular localization indicated that TaSAP7-A was distributed in the nucleus and cytoplasm. Unlike previously known A20/AN1-type SAP genes, TaSAP7-A was negatively regulated to abiotic stress tolerance. Overexpressing TaSAP7-A Arabidopsis lines were hypersensitive to ABA, osmotic and salt stress at germination stage and post-germination stage. Overexpression of TaSAP7-A Arabidopsis plants accelerated the detached leaves' chlorophyll degradation. Association analysis of TaSAP7-A haplotypes and agronomic traits showed that Hap-5A-2 was significantly associated with higher chlorophyll content at jointing stage and grain-filling stage. These results jointly revealed that TaSAP7-A is related to the chlorophyll content in the leaves of Arabidopsis and wheat. Both in vivo and in vitro experiments demonstrated that TaSAP7-A interacted with TaS10B, which was the component of regulatory subunit in 26S proteasome. In general, TaSAP7-A was a regulator of chlorophyll content, and favorable haplotypes should be helpful for improving plant chlorophyll content and grain yield of wheat.

Keywords: TaS10B; TaSAP7-A; abiotic stress; chlorophyll content; wheat.

FIGURE 1

Clone and chromosomal location of TaSAP7-A. (A) TaSAP7-A isolated from Hanxuan 10. Lane 1 is the PCR product of TaSAP7-A. (B) TaSAP7-A was located on chromosome 5A using nulli-tetrasomic lines of Chinese Spring. M is Marker III.

FIGURE 2

Wheat TaSAP7-A belongs to the stress association proteins (SAPs) protein family. (A) Alignment of SAPs from different plant species; Ta from Triticum aestivum, XP_015611004 from Oryza sativa, XP_002462330 from Sorghum bicolor, NP_189461 from Arabidopsis thaliana, and XP_014627640 from Glycine max. Numbers on the right indicate amino acid position. Amino acid residues with 100% similarity are shown in black background; amino acid residues with no less than 75% similarity are shown in gray background. The conserved AN1 domains are marked above the alignment with lines. (B) Phylogenetic tree of SAP proteins. The neighbor-joining tree was built with 1,000 bootstrap replicates. TaSAP7-A are marked with red dots.

FIGURE 3

Subcellular localization of TaSAP7-A in wheat protoplasts (A) and tobacco leaf cells (B). The vector control (35S::GFP) and fusion protein construct 35S::TaSAP7-A::GFP were introduced into wheat protoplast and tobacco leaf cells, respectively. For wheat protoplast transformation, the nucleus marker D53-mCherry was co-transformed into the protoplasts and GFP was detected at 16 h with a laser scanning confocal microscope. For tobacco, GFP was detected at 3 days. Then, 4,6-diamino-2-phenyl indole (DAPI) was used to stain cell nuclei. Scale bars: 10 μm for wheat protoplasts, 20 μm for tobacco leaf cells.

FIGURE 4

Expression patterns of TaSAP7-A. (A) Tissue expression of TaSAP7-A in wheat seedlings detected by qRT-PCR. (B–E) Expression patterns of TaSAP7-A in wheat seedlings exposed to 50 μM abscisic acid (ABA), under salt stress (250 mM NaCl), cold stress (4°C) and osmotic stress (16.1% PEG-6000), respectively. The histogram represents mean ± SE of three biological replicates.

FIGURE 5

Overexpression of TaSAP7-A in Arabidopsis enhances sensitivity to ABA, high salinity and osmotic stress. Phenotypes of TaSAP7-A overexpressing Arabidopsis on MS solid medium (A), MS containing 0.5 μM ABA (B), MS containing 100 mM NaCl (C) and MS containing 200 mM mannitol (D). (E) The expression level of TaSAP7-A in two transgenic lines (L1 and L2). (F) Comparison of seedlings with cotyledon of transgenic lines treated with 0.5 μM ABA, 100 mM NaCl and 200 mM mannitol. WT, wild type; VC, plants transformed with the empty pCAMBIA1300 vector; L1 and L2, transgenic lines. The histogram represents mean ± SE of three biological replicates. ^∗∗P < 0.01.

FIGURE 6

Overexpression TaSAP7-A accelerated chlorophyll degradation in detached leaves of Arabidopsis. (A) Phenotypes of overexpressing TaSAP7-A Arabidopsis detached leaves under darkness for 3 days. (B) Comparison of chlorophyll content in detached leaves of transgenic, WT and VC Arabidopsis plants under darkness for 3 days. The histogram represents mean ± SE of three biological replicates. ^∗∗P < 0.01.

FIGURE 7

Marker development of TaSAP7-A. Partial single nucleotide polymorphisms in three TaSAP7-A haplotypes identified among 32 wheat accessions. PCR products of dCAPS (left) and CAPS (right) markers were restrictively digested by Swal I and EcoR I, respectively. M is Marker III.

FIGURE 8

Phenotypic comparisons of three TaSAP7-A haplotypes in 10 environments. (A) Plant height. (B) 1,000 grain weight. (C) Chlorophyll content at jointing stage. (D) Chlorophyll content at grain filling stage. *P < 0.05, **P < 0.01. E1, 2014-SY-WW; E2, 2014-SY-DS; E3, 2014-SY-WW+HS; E4, 2014-SY-DS+HS; E5, 2015-CP-WW; E6, 2015-CP-DS; E7, 2015-SY-WW; E8, 2015-SY-DS; E9, 2015-SY-WW+HS; E10, 2015-SY-DS+HS. CP, Changping; SY, Shunyi; WW, well-watered; DS, drought-stressed; HS, heat-stressed. Error bars denote 1 SE.

FIGURE 9

Transcriptional activation activity assay and TaSAP7-A interacts with TaS10B in yeast system. (A) According to the amino acid position of the conserved domain, transcriptional activation activity of TaSAP7-A of the full-length and two truncations. (B) The transformants were placed on SD/−2 (−Trp, −Leu) medium to examine growth. Protein-protein interactions were assessed on SD/−4 (−Ade, −His, −Trp, −Leu) medium and further confirmed by monitoring α-galactosidase activity.

FIGURE 10

TaSAP7-A interacts with TaS10B in plant system. (A) LCI assays showed that TaSAP7-A interacted with TaS10B in tobacco leaves which were infiltrated with Agrobacterium tumefaciens strain GV3101 containing the indicated constructs. The signals were collected at 48 h after infiltration. (B) BiFC assays made clear that interaction location of TaSAP7-A and TaS10B exist in the cytoplasm of tobacco. Strain GV3101 carrying the indicated constructs were infiltrated into tobacco leaves. Fluorescence was detected 3 days after transformation. Images are in dark field (1, 4, 7), bright field (2, 5, 8), and combined (3, 6, 9). Scale bar, 50 μm.