A transcription factor TaMYB5 modulates leaf rolling in wheat

Abstract

Leaf rolling is an important agronomic trait in wheat (Triticum aestivum L.). Moderate leaf rolling keeps leaves upright and maintains the relatively normal photosynthesis of plants under drought stress. However, the molecular mechanism of wheat leaf rolling remains unclear. Here, we identified a candidate gene TaMYB5-3A that regulates leaf rolling by using a genome-wide association study (GWAS) in a panel of 323 wheat accessions. Phenotype analysis indicated that the leaves of tamyb5 mutants were flatter than that of the wild type under drought condition. A nucleotide variation in the TaMYB5-3A coding region resulted in a substitution of Thr to Lys, which corresponds to two alleles SNP-3A-1 and SNP-3A-2. The leaf rolling index (LRI) of the SNP-3A-1 genotype was significantly lower than that of the SNP-3A-2 genotype. In addition, TaMYB5-3A alleles were associated with canopy temperature (CT) in multiple environments. The CT of the SNP-3A-1 genotype was lower than that of the SNP-3A-2 genotype. Gene expression analysis showed that TaMYB5-3A was mainly expressed in leaves and down-regulated by PEG and ABA treatment. TaMYB5 induces TaNRL1 gene expression through the direct binding to the AC cis-acting element of the promoter of the target gene, which was validated by EMSA (electrophoretic mobility shift assay). Our results revealed a crucial molecular mechanism in wheat leaf rolling and provided the theoretical basis and a gene resource for crop breeding.

Keywords: GWAS; NRL1; TaMYB5; canopy temperature; leaf rolling; wheat.

FIGURE 1

A natural variation in TaMYB5 was associated with wheat leaf rolling. (A) A regional Manhattan diagram of the TaMYB5-3A genomic region (477,138,673 bp–482,177,523 bp). One hundred twenty-five SNPs were used in the association analysis. The SNP in the TaMYB5 coding region is represented by a red dot. The P-value is shown on a −log10 scale. (B) Linkage disequilibrium (LD) heat map of the chromosome region. These genes within the block region are shown with arrows. The position of TaMYB5-3A (TraesCS3A01G257800) is shown with a red arrow. Red triangle box highlight the strong LD of a haplotype block.

FIGURE 2

The phenotypes of tamyb5 mutants and WT (wild-type AK58). (A) Phenotypes were observed under 18 days of drought treatment at the post-anthesis stage. The leaves of tamyb5 mutants were flatter than that of the WT. There is no significant phenotypic difference in leaf rolling between tamyb5 mutants and WT before drought stress. (B) Leaf rolling index (LRI) of WT and tamyb5. Data are presented as mean ± SE (n = 9). ***P < 0.001.

FIGURE 3

Nucleotide polymorphisms and molecular marker development of TaMYB5-3A. (A) Schematic diagram of the TaMYB5-3A structure, including two exons and one intron. One SNP was detected in the coding region of TaMYB5-3A. (B) The TaMYB5-3A-dCAPS marker was developed based on the SNP (C/A) at 544 bp. Red rectangle and dot represent the introduction of the Sal I restriction site by base mismatched (A to T), and red letters represent the different bases at 544 bp of the TaMYB5-3A coding region. (C) PCR products were digested by Sal I. The 153 bp fragment amplified from accessions with SNP-3A-1 could be digested by Sal I into 132 bp and 21 bp, while the 153 bp fragment amplified from accessions with SNP-3A-2 could not be digested. M, 100 bp DNA ladder. SNP-3A-1 (C) was the majority in the wheat population, and this sequence was used in subsequent experiments if not specified.

FIGURE 4

Comparisons of LRI (leaf rolling index), and CT (canopy temperature) in the two TaMYB5-3A genotypes SNP-3A-1 and SNP-3A-2. (A) LRI comparisons of two TaMYB5-3A genotypes in six environments. LRI data were collected in 2020 and 2021; 20, 2020; 21, 2021; DS, drought stress; WW, well-watered; 1 or 2 after 20DS or 20WW refers to two replicates set for each treatment, respectively. (B) CT comparisons of two TaMYB5-3A genotypes in nine environments. CT data were collected in 2017. Plant growth stages: J, jointing stage; G, grain-filling stage. Planting locations: CP, Changping, SY, Shunyi. Treatments: DS, drought stress; WW, well-watered; HS, heat stress. The significance of data differences is tested by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. Error bar, ± SE.

FIGURE 5

Frequency and distribution of two TaMYB5-3A genotypes, SNP-3A-1 and SNP-3A-2. Distribution of two TaMYB5-3A genotypes from 157 landraces (A) and 348 modern cultivars (B) in 10 Chinese wheat production zones. I, Northern Winter Wheat Zone; II, Yellow and Huai River Valleys Facultative Wheat Zone; III, Middle and Low Yangtze Valleys Autumn-Sown Spring Wheat Zone; IV, Southwestern Autumn-Sown Spring Wheat Zone; V, Southern Autumn-Sown Spring Wheat Zone; VI, Northeastern Spring Wheat Zone; VII, Northern Spring Wheat Zone; VIII, Northwestern Spring Wheat Zone; IX, Qinghai-Tibetan Plateau Spring-Winter Wheat Zone; X, Xinjiang Winter-Spring Wheat Zone.

FIGURE 6

Expression patterns of TaMYB5 in wheat. (A) Expression patterns of TaMYB5-3A in different plant tissues at seedling and jointing stage. L, leaf; S, stem; PN, penultimate node; RB, root base; R1, R2, R3, and R4 indicate the root section from the ground to 30, 30–60, 60–90, and 90–120 cm depth, respectively. (B) Relative expression of TaMYB5-3A following H₂O (Control), PEG, and ABA treatment. Two-week-old seedlings of Hanxuan 10 were treated with PEG-6000 and 50 μM ABA. Leaves were collected for expression experiments. All data are the means ± SE of three independent experiments.

FIGURE 7

Subcellular localization and transcriptional activity analysis of TaMYB5. (A) 35S-TaMYB5-GFP and the nuclear localization marker (mCherry) were transiently co-transformed into tobacco leaf cells. Scale bar = 20 μm. (B) Transcriptional activity of TaMYB5. The LUC/REN ratio of TaMYB5. Data are means of three independent experiments. *P < 0.05, ***P < 0.001. Error bar, ± SE.

FIGURE 8

TaMYB5 binds to the wheat TaNRL1 promoter region. (A) TaMYB5 activated expression in yeast of the LacZ reporter gene driven by the AC box of TaNRL1 promoter. proNRL1 are base pairs from 1,150 bp to 2,000 bp in the promoter of TaNRL1. LacZ and pB42AD were used as negative controls. TaMYB5-1 and TaMYB5-2 correspond to SNP-3A-1 and SNP-3A-2 amino acid sequences, respectively. (B) EMSA (electrophoretic mobility shift assay) of the TaMYB5-GST protein and the AC box probe. The lower bands show the free probes and the upper bands show that the TaMYB5-GST protein binds to the biotin-labeled AC box probe. (C) Dual-luciferase assay of transformed tobacco leaves to detect the interaction between TaMYB5 and the proNRL1. Schematic diagrams of the effector and reporter constructs are shown. Data are means (± SE) of three biological replicates.

FIGURE 9

A proposed model for the TaMYB5 gene in the regulation of adaxial rolling of leaf. Under well-watered, TaNRL1 was expressed normally and inhibited leaf rolling and leaves showed a flat state. Under drought stress, the expression of TaNRL1 decreased along with the decrease of TaMYB5, which reduced its ability to inhibit leaf rolling. The promoting role is represented by →, and ⊣ shows the inhibiting role.