Distinct growth patterns in seedling and tillering wheat plants suggests a developmentally restricted role of HYD2 in salt-stress response

Abstract

Mutants lacking functional HYD2 homoeologs showed improved seedling growth, but comparable or increased susceptibility to salt stress in tillering plants, suggesting a developmentally restricted role of HYD2 in salt response. Salinity stress threatens global food security by reducing the yield of staple crops such as wheat (Triticum ssp.). Understanding how wheat responds to salinity stress is crucial for developing climate resilient varieties. In this study, we examined the interplay between carotenoid metabolism and the response to salt (NaCl) stress, a specific form of salinity stress, in tetraploid wheat plants with mutations in carotenoid β-hydroxylase 1 (HYD1) and HYD2. Our investigation encompassed both the vulnerable seedling stage and the more developed tillering stage of wheat plant growth. Mutant combinations lacking functional HYD2 homoeologs, including hyd-A2 hyd-B2, hyd-A1 hyd-A2 hyd-B2, hyd-B1 hyd-A2 hyd-B2, and hyd-A1 hyd-B1 hyd-A2 hyd-B2, had longer first true leaves and slightly enhanced root growth during germination under salt stress compared to the segregate wild-type (control) plants. Interestingly, these mutant seedlings also showed decreased levels of neoxanthin and violaxanthin (xanthophylls derived from β-carotene) and an increase in β-carotene in roots. However, tillering hyd mutant and segregate wild-type plants generally did not differ in their height, tiller count, and biomass production under acute or prolonged salt stress, except for decreases in these parameters observed in the hyd-A1 hyd-B1 hyd-A2 hyd-B2 mutant that indicate its heightened susceptibility to salt stress. Taken together, these findings suggest a significant, yet developmentally restricted role of HYD2 homoeologs in salt-stress response in tetraploid wheat. They also show that hyd-A2 hyd-B2 mutant plants, previously demonstrated for possessing enriched nutritional (β-carotene) content, maintain an unimpaired ability to withstand salt stress.

Keywords: Carotenoid; Carotenoid β-hydroxylase (HYD); Hydroxylation; Salinity; Salt-stress response; Wheat; Xanthophyll; β-Carotene.

Fig. 1

Growth of hyd mutant and segregate wild-type seedlings subjected to control (0 mM NaCl) and salt-stress treatments. Representative seedlings for each genotype are shown for the 0 mM NaCl treatment (a) and the salt-stress treatment (100 mM NaCl) (b). Effects of salt stress on first-true-leaf length (c), root count (d), root length (e), and root convex area (f) are shown. Error bars represent ± standard deviation (n = 20). Statistically significant differences (p < 0.05) between groups within a treatment are denoted with different letters

Fig. 2

Relative expression of candidate carotenoid cleavage genes in tetraploid wheat tissues. Transcript quantification was carried out using the relative standard curve method (Applied Biosystems 2008), with gene expression normalized using the geometric mean of two reference genes, Ta2291 and Ta54227. Values shown are the mean ± SD of 3–4 biological replicates. CCD, carotenoid cleavage dioxygenase; NCED, nine-cis-epoxycarotenoid dioxygenase. Expression of CCD7, CCD8, and NCED3 were below the limit of reliable detection by real-time qPCR (nd) in first true leaves and roots of 4-day-old seedlings

Fig. 3

Growth of tillering hyd mutant and segregate wild-type plants subjected to control (0 mM NaCl) and salt-stress treatments. Salt stress was applied at 250 mM NaCl, followed by recovery in a soil-based system. Phenotypic values after harvest for shoot dry mass (a), shoot water fraction (b), leaf count (c), and tiller count (d) are shown. Representative plants for each genotype are shown in (e) (note that the genotypes are ordered differently from panels a–d). Error bars represent ± standard deviation (n = 9). Statistically significant differences (p < 0.05) between groups within a treatment are denoted with different letters

Fig. 4

Growth of tillering hyd mutant and segregate wild-type plants subjected to a prolonged salt stress at 50 mM NaCl in a hydroponic system. Tiller counts were determined over the course of growth (a). Statistically significant differences (p < 0.05) in tiller count among the genotypes at each time point are indicated with different letters. Phenotypic values at harvest for shoot height (b), root length (c), and shoot fresh mass (d) are shown. Shoot dry mass (e) and root dry mass (f) after drying are also shown. Error bars represent ± standard deviation (n = 14 for segregate wild type; n = 10 for hyd-A2 hyd-B2 and hyd-A1 hyd-B1 hyd-A2 hyd-B2). Statistically significant differences (p < 0.05) between groups are denoted with different letters