Expression characteristics under Zn stress tolerance of ZmHMA3 in maize
In the previous association analysis study, we obtained a candidate gene ZmHMA3 related to the accumulation of heavy metal Cd in leaves (Cao et al. 2019). Due to HMA3 being a Cd/Zn transporter protein, we have focused on studying the function of ZmHMA3 under Cd stress, and by the way, confirmed that overexpression of ZmHMA3 led to a higher Zn accumulation (Liao et al. 2023). To systematically verify the gene function of ZmHMA3 under Zn stress in maize, expression characteristics under Zn stress were firstly taken out. The examination of the expression pattern of the ZmHMA3 gene demonstrated an elevated expression level in both leaves and roots after 48 hours of exposure to Zn stress compared to the control group without Zn stress (Fig. 1A). It suggests a potential involvement of ZmHMA3 in the response to Zn stress.
CRISPR/Cas9-mediated knock-out of ZmHMA3 decreased tolerance in Zn stress
To investigate the functional role of ZmHMA3 in response to Zn stress, ZmHMA3 knock-out (KO) mutants were generated using CRISPR-Cas9 technology, in which ZmHMA3 gene was rare or even not expressed (Fig. 1B). Three guide-RNAs (gRNAs) were designed to target the first two exons of ZmHMA3 (Fig. 2A). In the T0 generation, 54 chimeric and heterozygous mutant maize lines harboring double-site and single-site mutations were identified. Among these, three stable lines without Cas9 were selected as ZmHMA3 homozygous mutants through sanger sequencing analysis and endosperm cutting in the T2 generation. The mutations in these lines resulted in deletions of 11bp, 65bp, and 34bp between the gRNA1 and gRNA2 sequences, leading to frameshift mutations and subsequent alterations in the sequence and conformation of the ZmHMA3 protein (Fig. 2B and 2C). One of these lines, Zmhma3-2, was chosen as the Zmhma3 knock-out (KO) line and was further analyzed in the T3 generation. Subsequently, the relative expression levels and agronomic traits of maize seedlings under varying degrees of Zn stress were compared. Both wild-type (WT) and Zmhma3 plants were subjected to 800 µmol/L ZnSO4·7H2O solutions for specific time periods (0 hour, 24 hours, and 48 hours). The growth phenotypes of WT and Zmhma3 indicated that both experienced wilting after 48 hours of Zn stress treatment, but the extent of wilting was more prominent in Zmhma3 compared to WT plants (Fig. 2D).
ZmHMA3 affects the seedling growth vigor and leaf cell membrane permeability of maize under Zn stress
The agronomic traits of seedlings reflect their growth potential, such as the amount of water content in plants, which often reflects the strength of their life activities. Cell membrane permeability suggests the degree of lipid peroxidation in plant leaf membranes, and the relative conductivity of leaves is positively correlated with the degree of stress on the plant.
We subjected the wild-type and Zmhma3 mutants to Zn stress (800µmol/L ZnSO4·7H2O) treatment, whose groups were named Zn-WT and Zn-Zmhma3. And the control groups with H20 treatment were named CK-WT and CK-Zmhma3. In order to assess the agronomic traits of Zmhma3 and WT plants under Zn stress at different time points, various physiological indices including seedling fresh weight (SFW), seedling dry weight (SDW), seedling water content (SWC), root fresh weight (RFW), root dry weight (RDW), and root water content (RWC) were measured.
After 24 hours of Zn treatment, there was no significant difference in shoots, roots, and total fresh and dry weight between WT and Zmhma3. After 48h, differences began to appear among different treatment groups. Under 48 hours stress, Zmhma3 plants exhibited significantly lower SFW, SDW, SWC, RFW, and RWC compared to WT plants, while RDW showed no significant difference between the two genotypes (Fig. 3A and 3B). Similar trends were observed for total fresh weight (TFW), total dry weight (TDW), and total water content (TWC), which were consistent with RFW, RDW, and RWC (Fig. 3C). Based on the water content, the relative reduction rate of water content for each treatment group at 24 and 48 h can be calculated compared to 0 h. Whether in the aboveground, underground, or entire plant, it can be seen that, after 48h stress, the relative reduction rate of water content in Zn-Zmhma3 was greater than that in Zn-WT, followed by CK-Zmhma3, with CK-WT showing the smallest reduction rate (Fig. 3D). These findings suggest that the knockout of ZmHMA3 resulted in a greater decrease in fresh weight and water content in plants under Zn stress.
Furthermore, the plant height of WT and Zmhma3 showed no significant difference compared to the control during Zn stress for 24 hours. But at 48 hours under Zn stress, the plant height of Zmhma3 was significantly smaller than that of WT, indicating that the knockout of ZmHMA3 enhanced the inhibitory effect of Zn stress on maize growth (Fig. 3E). Additionally, the relative electrical conductivity of Zmhma3 increased after Zn treatment and was significantly higher than that of WT (Fig. 3F). This suggests that Zmhma3 plants experienced more severe damage than WT plants under Zn stress.
ZmHMA3 affects the root growth and architecture of maize under Zn stress
Root is the first organ to contact with heavy metals in soil in plants, which can absorb the heavy metals into root cells, thereby transferring to the above ground of the plant, and then causing the antioxidant activity change. We detected Zn heavy metal content in WT and Zmhma3 at different times of treatments.
With the prolongation of stress time, For WT plants, the root length (Fig. 4A) and surface area (Fig. 4B) were greater than Zmhma3 before Zn stress, but there was no difference in root diameter (Fig. 4C), root volume (Fig. 4D), number of root tips (Fig. 4E), and number of forks (Fig. 4F). After Zn stress, the six root phenotype indicators were greater than those of Zmhma3. With the prolongation of stress time, except for a decrease in root length, all other indicators of CK-WT showed an upward trend. Except for the length and forks that first increase and then decrease, all other indicators of CK-Zmhma3 also showed an upward trend. Zn stress treatment maintained the length, surface area, diameter, and volume of Zn-WT at a level similar to 0h, while the number of root tips and forks still increased. Except for tips, which first decreased and then increased, all other indicators of Zn-Zmhma3 showed a trend of first increasing and then decreasing. These results indicated that knocking out ZmHMA3 exacerbated the inhibition of root growth under Zn stress.
The root antioxidant related enzyme activity was repressed in Zmhma3
Antioxidant enzymes and MDA are closely related to plant stress. CAT, SOD, and POD are effective antioxidant enzymes. And by detecting MDA, the level of membrane lipid peroxidation in plants under adverse conditions can be detected. To determine the resistance to Zn stress, we measured various antioxidant indicators of roots in WT and Zmhma3 under Zn stress.
For antioxidant activity assay, the results showed that CAT content increased after 48h Zn stress in Zn-WT and Zn-Zmhma3, and was significantly higher than that in CK-WT and CK-Zmhma3 (Fig. 5A). The POD content of CK-WT and CK-Zmhma3 tended to stabilize during the treatment, while that of Zn-WT increased, and that of Zn-Zmhma3 decreased. Furthermore, Zn-WT was significantly greater than Zn-Zmhma3 (Fig. 5B). At 0h, there was no significant difference between the treatment groups. At 24h, the SOD content of Zn-Zmhma3 began to significantly decrease and was significantly lower than CK-WT, CK-Zmhma3, and Zn-WT. At 48h, the content of CK-WT, Zn-WT, and Zn-Zmhma3 decreased. The SOD content of CK-Zmhma3 was the highest, followed by CK-WT, then Zn-WT, and that of Zn-Zmhma3 was the lowest (Fig. 5C). The MDA content of Zn-WT significantly increased after treatment, with Zn-Zmhma3 initially increasing and then decreasing, resulting in a significantly higher content of Zn-WT than Zn-Zmhma3 (Fig. 5D). The content of CAT, POD, SOD, and MDA was higher in WT than that in Zmhma3, which suggested that knocking out ZmHMA3 leads to a decrease in plant antioxidant enzyme activity under zinc stress.
The Zn content and transport rate was increased in Zmhma3
Under heavy metal stress, the growth and development of corn are inhibited. As the duration of stress prolongs, the symptoms of victimization first manifest as wilting. The first plant organ that heavy metals come into contact with is the root, which is stored in the root and transferred to the aboveground part of the plant. The results of the study revealed that Zmhma3 plants accumulated higher Zn content compared to WT plants (Fig. 6), particularly in the underground parts of the plant. The Zn content in the underground parts of Zmhma3 plants was significantly higher than that in the aboveground parts after 48 hours of stress (Fig. 6A). Furthermore, the leaf, root, and total Zn content in Zmhma3 plants were higher than those in WT plants, which corresponded to the trend observed in the transport coefficient (Fig. 6A and 6B). In addition, the changes in Zn content in root cells and leaf cells followed a similar trend. After Zn treatment, the Zn content in F2 (soluble part, cell sap, and cytoplasmic matrix) and F3 (cell membrane and organelle) of WT plants was significantly higher than that in Zmhma3 plants, while the Zn content in F1 (cell wall) was significantly lower in WT plants compared to Zmhma3 plants (Fig. 6C). The Zn content in the leaves and roots of Zmhma3 plants was much higher than that in WT plants, but the Zn content within the cells was lower.