Genome-wide characterization of ZmCRY genes: unveiling stress response mechanisms and the role of ZmCRYPHR2 in salinity tolerance

Abstract

Background: Blue light serves as a crucial environmental signal regulating plant growth and development. The cryptochrome (CRY) family represents a key class of blue light receptors involved in these processes, as well as plant growth, development, and defense. However, the functions of CRYs in maize remain largely unexplored.

Results: In this study, nine ZmCRY genes were identified and found to be unevenly distributed across five chromosomes. Gene structure and conserved motif analyses revealed that ZmCRYs within the same phylogenetic groups are highly conserved. Synteny analysis indicated a close evolutionary relationship between ZmCRYs and their homologs in Oryza sativa. Promoter analysis identified diverse cis-regulatory elements linked to light response, stress tolerance, and hormone signaling. RT-qPCR analysis showed that ZmCRYs respond to various abiotic and biotic stresses, including high salinity, drought, nitrogen deficiency, Fusarium verticillioides, and Puccinia polysora. Functional studies demonstrated that ZmCRYPHR2, localized in chloroplasts and the cytoplasmic membrane, plays a role in scavenging reactive oxygen and regulating maize salt tolerance. Haplotype 2 of ZmCRYPHR2 was identified as the preferred haplotype in a panel of 269 inbred lines.

Conclusions: These findings provide a comprehensive genomic and functional characterization of the ZmCRY gene family, with ZmCRYPHR2 identified as a pivotal regulator of salt tolerance, offering valuable genetic insights for the development of stress-resilient maize breeding.

Keywords: ZmCRYPHR2; Cryptochrome; Haplotype; Maizes; Salt tolerance; Stress.

Fig. 1

Chromosomal distribution, phylogeny, conserved motifs, gene structure, and protein domain of ZmCRYs. A, The distribution of ZmCRYs across maize chromosomes. B, Phylogeny, gene structure, conserved motifs, and functional domain diagram of ZmCRYs

Fig. 2

Inter-species phylogeny and collinearity of ZmCRYs. A, Inter-species phylogenetic tree showing CRYs in Arabidopsis thaliana, Glycine max, Solanum tuberosum, Oryza sativa, and Zea mays. B, Collinearity analysis of CRYs in Zea mays, Oryza sativa, and Arabidopsis thaliana with collinear genes denoted by red lines and other collinear blocks by gray lines. C, Intraspecies collinearity analysis of ZmCRYs

Fig. 3

Tissue-specific and blue light-responsive expression patterns of ZmCRYs. A, Heatmaps illustrating the expression of representative ZmCRYs across various tissues by RT-qPCR. n = 3 biologically independent samples with three plants per biological replicate. B, Expression patterns of ZmCRYs under blue light or white light treatment. Light intensities: B (blue light), 31 µmol m⁻² s⁻¹; W (white light), 34 µmol m⁻² s⁻¹. Statistical significance was determined using a multiple unpaired t-test (* P < 0.05, ** P < 0.01, n = 3 biologically independent samples with three plants per biological replicate)

Fig. 4

ZmCRYs’ responses to biotic and abiotic stresses. A, The expression analysis of ZmCRYs in the B73 plants with high salinity. 0 h, normal conditions; 24 h, indicate hours of a 300 mM NaCl treatment. B, The expression analysis of ZmCRYs in the B73 plants with drought. 0 h, normal conditions; 16 h, indicate hours of a 15% polyethylene glycol (PEG) treatment. C, The expression analysis of ZmCRYs in the B73 plants with nitrogen deficiency. 0d, normal conditions; 5d, indicate hours of a 0.04 mmol NO³⁻ treatment. D, The expression analysis of ZmCRYs in the B73 plants after inoculation by F. verticillioides. 0 h, normal conditions; 48 h, indicate hours of F. verticillioides inoculation. E, The expression analysis of ZmCRYs in the B104 plants after inoculation by P. polysora. 0 h, normal conditions; 48 h, indicate hours of P. polysora inoculation. Data are means ± s.e.m. N = 3 biologically independent samples with three plants per biological replicate (* P < 0.05, ** P < 0.01), statistical significance was determined using a multiple unpaired t-test

Fig. 5

ZmCRYPHR2 negatively regulates maize’s resistance to high salinity stress. A Subcellular localization of ZmCRYPHR2 proteins. B–C Phenotypic differences between B104/OE-1/OE-2 and B73/ZmCRYPHR2 under control and 300 mM NaCl stress. Bar = 6 cm. D Measurement of morphological indicators and SPAD indices in B104/OE-1/OE-2 and B73/ZmCRYPHR2. Values represent mean ± SE from three biological replicates. Statistically significant differences were identified between pairs of measurements using a multiple unpaired t-test (*P < 0.05, ** P < 0.01). N = 3 biologically independent samples with nine plants per biological replicate

Fig. 6

Analysis of physiological processes under high salinity stress. A Relative expression levels of ROS-related genes. B–E Assessment of plant physiological indices. F NBT and DAB staining of leaves under stress conditions. Bar = 2 cm. Statistically significant differences were identified between pairs of measurements using a multiple unpaired t-test (* P < 0.05, ** P < 0.01). Data are means ± s.e.m. N = 3 biologically independent samples with three plants per biological replicate

Fig. 7

Identification of ZmCRYPHR2 haplotypes, phenotypic differences, and frequency changes. A Haplotypes of ZmCRYPHR2 were identified from two SNPs. B–E Comparison of salt stress tolerance based on PHR, SFR, RFR and RLR under high salinity stress. F–M Distribution of ZmCRYPHR2 haplotypes across nine maize inbred line groups: BSSS, Indent, PA, LRC, Mix, Lan, PB, SPT. Statistically significant differences were identified between pairs of measurements using a t-test (*p < 0.05)