Natural variation and domestication selection of ZmSULTR3;4 is associated with maize lateral root length in response to salt stress

Abstract

Soil salinity is a major constraint that restricts crop productivity worldwide. Lateral roots (LRs) are important for water and nutrient acquisition, therefore understanding the genetic basis of natural variation in lateral root length (LRL) is of great agronomic relevance to improve salt tolerance in cultivated germplasms. Here, using a genome-wide association study, we showed that the genetic variation in ZmSULTR3;4, which encodes a plasma membrane-localized sulfate transporter, is associated with natural variation in maize LRL under salt stress. The transcript of ZmSULTR3;4 was found preferentially in the epidermal and vascular tissues of root and increased by salt stress, supporting its essential role in the LR formation under salt stress. Further candidate gene association analysis showed that DNA polymorphisms in the promoter region differentiate the expression of ZmSULTR3;4 among maize inbred lines that may contribute to the natural variation of LRL under salt stress. Nucleotide diversity and neutrality tests revealed that ZmSULTR3;4 has undergone selection during maize domestication and improvement. Overall, our results revealed a regulatory role of ZmSULTR3;4 in salt regulated LR growth and uncovered favorable alleles of ZmSULTR3;4, providing an important selection target for breeding salt-tolerant maize cultivar.

Keywords: ZmSULTR3;4; domestication selection; lateral root length; maize (Zea mays); natural variation; salt stress.

Figure 1

GWAS performed on the lateral root length (LRL) of maize under 100 mM NaCl condition. (A) Manhattan plot for the GWAS. The red dotted line represents the significance threshold (P = 1.96 × 10⁻⁵). Two SNPs located in ZmSULTR3;4 were highlighted in red. (B) Local manhattan plot of the ZmSULTR3;4 genomic region on chromosome 9. The 0.1-Mb genomic region on both sides of the most significant SNP was displayed. The most significant SNP was highlighted with a black diamond, while others were shown by dots and colored according to their LD (r ²) with the most significant SNP.

Figure 2

Phylogenetic and expression patterns of ZmSULTR genes in different types of tissue and stress response. (A) The phylogenetic tree of putative SULTR transporters in Arabidopsis, rice and maize. The neighbor-joining tree was constructed by MEGA-X with 1000 bootstrap values and the Poisson model. The scale denoteed the branch lengths. The gene identifiers and annotation were illustrated as black dots for maize, diamonds for rice, and triangles for Arabidopsis, respectively. (B) A heatmap showing the transcript levels of 8 ZmSULTRs in fifteen different tissues at various developmental stages. Normalized gene expression values were indicated in different colors. (C) Expression patterns of ZmSULTR genes in the leaves of 7-d-old hydroponically grown maize plants in response to salt, drought, heat, and cold stresses. The bar color represents the Z score of the FPKM of each gene under five treatments. (D) Expression patterns of ZmSULTR genes in the roots of 7-d-old hydroponically grown maize plants under salt stress conditions. The bar color for the Z score of the FPKM of each gene is shown on the right.

Figure 3

Tissue-specific expression and subcellular localization analysis of ZmSULTR3;4. (A) The tissue-specific expression of ZmSULTR3:4 detected via in situ RNA hybridization in the roots of 7-d-old hydroponically grown maize seeldings. The tissue-specific expression of ZmSULTR3;4 was detected using DIG-labeled RNA sense probes (left) and antisense probes (right). Ep, epidermis; en, endodermis; pe, pericycle; xy, xylem; pa, parenchyma; co, cortex. Bar = 100 μm. (B) ZmSULTR3:4 is localized exclusively in the plasma membrane (PM). The Ubi : ZmSULTR3:4-GFP expression cassette was transfected into maize B73 protoplasts. The transformed protoplasts were stained with FM4-64 stain for 1 min to marker the PM. Bar = 5 μm.

Figure 4

Genetic variation in ZmSULTR3:4 is associated with the lateral root length (LRL) of maize seedlings under salt stress conditions. (A) Association analysis between the genetic variation of ZmSULTR3:4 and the LRL of maize seedlings under salt stress. Black dots represent SNPs, and triangles denote InDels. The position of the start codon (ATG) is defined as ‘‘+1’’. The 5’- and 3’-UTRs and exons of ZmSULTR3:4 are shown as open and filled boxes, respectively. The black lines represent gene promoters and introns. The p value is shown on a -log₁₀ scale. The nine significant polymorphisms in the promoter are connected to the pairwise LD diagram with black vertical lines, illustrating that the nine variants are in strong LD (r² >0.8). (B) Significant markers of ZmSULTR3:4 associated with LRL in different haplotypes. (C) The distribution of i LRL under salt stress conditions. n is the number of inbred lines belonging to each haplotype. Statistical significance was determined using a two-sided t test. "**”denotes statistical significance at the p < 0.01 level.

Figure 5

LUC enzyme activity driven by 0.8-kb and 1.3-kb promoter fragments of ZmSULTR3:4 ^A404 and ZmSULTR3:4 ^A207 under normal and salt stress conditions. (A) Vector diagram used to detect the effect of genetic variation of the promoter region on ZmSULTR3:4 expression. A404-800, A207-800, A404-1300, and A207-1300 constructs harbor the promoter fragments of different ZmSULTR3:4 alleles, including 800 bp from A404, 800 bp from A207, 1300 bp from A404, and 1300 bp from A207. (B) Transient expression assays of different promoter fragments from two. 35S:Renilla luciferase was used as a positive control for transfection efficiency. Statistical significance was determined by a two-sided t test: *p < 0.05, **p < 0.01. (C) Relative expression of ZmSULTR3:4 in roots of 14-d-old hydroponically grown A404 and A207 inbred lines under normal and 100 mM NaCl conditions.

Figure 6

Analysis of nucleotide diversity (π) and allele frequency of ZmSULTR3;4 in teosinte, landrace, and inbred lines. (A) Evaluation of nucleotide polymorphisms and neutrality tests of ZmSULTR3;4. Hd represents haplotype diversity; Dens. denotes the number of single nucleotide polymorphisms (SNPs) per 1000 bp; C represents sequence conservation; D and F represent Fu and Li’s D* and F*, respectively. Asterisks indicate statistical significance at the level of *p < 0.05, and **p < 0.01. (B) Nucleotide diversity (π) of teosinte, landrace, and inbred lines. The sliding window method was used to calculate π with a window size of 100 bp and a step size of 25 bp. A schematic illustrating the genomic region of ZmSULTR3;4, encompassing the upstream promoter region, introns (black lines), exons (filled boxes), and the 5’- and 3’-UTRs (open boxes). (C) Detection of allele frequency of SNP-705 in teosinte, landrace, and inbred lines. Number in the brackets indicated the count of accessions carrying the corresponding allele within respective population.