Maize ZmHSP90 plays a role in acclimation to salt stress

Abstract

Background: Maize is sensitive to salt stress, especially during the germination and seedling stages.

Methods: We conducted germination experiments on 60 maize materials under salt stress, and screened out the most salt-tolerant and salt-sensitive varieties based on germination indicators. Afterwards, transcriptome analysis was performed to screen for key regulators in the plumule and flag leaf at the germination and seedling stages, respectively. Following that, transgenic tobacco was developed to expose the roles and mechanisms of the candidate genes, enabling a deeper investigation of their functions.

Results: Out of the 60 inbred lines of maize, "975-12" exhibits the highest level of salt tolerance, while "GEMS64" displays the lowest. The application of salt stress resulted in a significant increase in the levels of antioxidant enzymes in both "975-12" and "GEMS64". ABA signal transduction and jasmonic acid pathways were the pathways that mainly affected by salt stress. In addition, a significant finding has been made indicating that when exposed to high levels of salt stress, the expression of ZmHSP90 in '975-12' increased while in 'GEMS64' decreased. Furthermore, in tobacco plants overexpressing ZmHSP90, there was an increase in antioxidant enzyme activity associated with salt tolerance. ZmHSP90 enhanced the expression levels of NtSOS1, NtHKT1, and NtNHX1 as compared to those in the salt treatment, causing the maintenance of Na⁺ and K⁺ homeostasis, suggesting that high expression of ZmHSP90 was conducive to regulate Na⁺ transporters to maintain K⁺/Na⁺ balanced in tobacco.

Keywords: Maize; Overexpression; Salt stress; Tobacco; Transcriptome.

Figure 1. Brief sketch of the experimental design.

The experiment is divided into three parts. In part I, 60 maize inbred lines were collected to test the Determination of Germination Index (GRI, GRI = germination rate of salt-treated seeds/germination rate of seeds under control condition). Plant materials with the highest, high, low, and the lowest GRI value were selected to preliminarily evaluate the salt tolerance. Then, salt resistance was further evaluated according the growth status, Na⁺ and K⁺ concentrations, according which the salt salt-tolerant and salt-sensitive maize materials were determined. In part II, the SOD, POD and CAT activity in both salt-tolerant and salt-sensitive maize were determined in germination and seedling periods. Also, transcriptome sequencing was carried out to screen salt-tolerant genes; In part III, the salt tolerance gene was transferred into tobacco and overexpressed to verify the salt tolerance of overexpressed tobacco.

Figure 2. Evaluation of salt tolerance.

(A) Determination of Germination Index (GRI) of 60 maize inbred lines. GRI = germination rate of salt-treated seeds/germination rate of seeds under control condition. The seeds of 60 maize lines were tested under 100 mmol/L salt stress for the preliminary germination test (three repetitions, 150 seeds were evaluated in each repetition). According to the GRI value, “975-12,” “DH3732,” “GML360,” “Jing2146” and “GEMS64” were selected as candidate plant materials. (B to F) The phenotype map of candidate maize lines under different salt stress conditions of “975-12,” “DH3732,” “GML360,” “Jing2146” and “GEMS64”, respectively. The seedlings in each image from left to right were treated with 0 mmol/L, 50 mmol/L, 100 mmol/L and 150 mmol/L NaCl concentration gradients, respectively. Three biological repeats are set for each group. (G) Na⁺/K⁺ ratio of candidate maize seedling leaves. Three biological repeats were set for each group.

Figure 3. Effects of salt stress on maize germination characteristics in candidate maize materials.

(A & B) The germination rate of “975-12” and “GEMS64”. Significantly different groups are indicated by diverse letters in the graphs (p < 0.05), whereas non-significant groups (p > 0.05) share similar letters. Three biological repeats were set for each group.

Figure 4. Effects of salt stress on maize antioxidant enzyme activity.

(A, C and E) The POD, SOD and CAT activity at the germination period, respectively. (B, D and F) The POD, SOD and CAT activity at the seedling stage, respectively. CK, control group (0 mmol/L). Three biological repeats were set for each group. Significantly different groups are indicated by diverse letters in the graphs (p < 0.05), whereas non-significant groups (p > 0.05) share similar letters.

Figure 5. Effects of salt stress on maize transcriptome.

(A) Number of differentially expressed genes (DEGs) at the germination stage. The blue and red bar indicates the number of down-regulated and up-regulated DEGs, respectively. (B) Expression of DEGs in ABA signal transduction and Jasmonic acid pathway. The redder the color is, the higher the expression level is. (C) Expression of ZmHSP90 (candidate gene). (D) Number of differentially expressed genes (DEGs) at the seedling stage. The blue and red bar indicates the number of down-regulated and up-regulated DEGs, respectively. (E) Expression of ZmHSP70 (candidate gene). (F) Interaction expression network of DEGs. The size of the circles indicates the number of connections in the relationship. Three biological repeats were set for each group.

Figure 6. Overexpression of ZmHSP90 in tobacco.

(A) Subcellular localization of ZmHSP90 in Arabidopsis thaliana protoplasts. Protoplasts in green fluorescence (first column) and bright field (second column) were photographed. Merged images are shown in the third column. Protoplasts with DIC are shown in the fourth column. (B) Expression of ZmHSP90 in WT, OE-1 and OE-2. WT, wild type; OE-1, overexpression of tobacco line 1; OE-2, overexpression of tobacco line 2. (D, E and F) represents the POD, SOD and CAT activity, respectively. Values indicated by dissimilar letters are significantly different (p < 0.05). Three biological repeats were set for each group.