Identification of subcellular localization of ZmSAMDC in tobacco
To study the subcellular localization of ZmSAMDC, the target gene was cloned into the transient expression vector pCAMBIA1302-GFP using Gateway recombination technology. Due to the fusion of ZmSAMDC and GFP, the subcellular localization of ZmSAMDC can be observed under a confocal microscope. Besides, ZmSAMDC can be transformed into tobacco leaves using the green fluorescence characteristics of GFP. The results showed that the ZmSAMDC fusion protein was located only in the nucleus (Fig. 1).
The transgenic maize overexpressing ZmSAMDC
PCR analysis of T3 generation transformed plants using specific primers of the selection marker gene Bar showed that 6 independent transgenic lines were obtained (Fig. 2). WB results showed that compared with the control, the protein expression content of the transgenic lines was significantly increased, and all of them could successfully express 65.53 kDa protein (Fig. 3). Transgenic lines "C3-1", "C3-3", and "C3-6" were selected for analysis of physiological and biochemical indicators and yield traits.
Analysis of PA content in plants overexpressing ZmSAMDC
As a low molecular weight aliphatic nitrogenous base with strong biological activity, PA can bind to the phospholipids of cell membranes under cold stress to prevent intracellular solutes from exuding and improve the cold resistance of plants. The average content of the three PAs of the transgenic line C3 was higher than that of the control (Table 1). The absolute content of Put, Spd, and Spm was 0.04, 0.048, and 0.02 mmol/g higher than that of the control. The relative proportions of Put, Spd, and Spm in the leaves of transgenic plants have also changed. The proportion of Spd in plants overexpressing the ZmSAMDC gene was significantly increased, while the proportion of Put and Spm decreased, and the relative proportion of Put decreased.
Overexpression of ZmSAMDC enhances the cold tolerance of maize
In order to study the role of ZmSAMDC in cold tolerance, we further analyzed the three transgenic maize plants (C3-1, C3-3, and C3-6) overexpressing ZmSAMDC. RT-qPCR analysis of 3 transgenic lines showed that ZmSAMDC was highly expressed in maize (Figure 4A). After 0, 2, 4, and 6 days of cold stress at 4℃, the germination ability of transgenic seeds was higher than that of the control group (Figure 4B). Under normal circumstances, the morphological difference between the transgenic line and the wild-type plant is not statistically significant. After 0, 8 and 12 hours of cold stress at -4 ℃, the damage of the transgenic line at the seedling stage was significantly lower than that of the control group (Figure 4C). The survival rate and relative water content of transgenic lines were significantly higher than those of WT (Figure 4D and 4E). The results indicate that overexpression of ZmSAMDC improves the cold tolerance of transgenic maize.
Overexpression of ZmSAMDC under cold stress significantly increased leaf proline content and malondialdehyde content
Under cold stress, the proline content in plants increases, and varieties with strong cold resistance tend to accumulate more proline. With the increase of the time of low temperature (4 ℃), the Pro content of transgenic plants (C3-1, C3-3, and C3-6) showed an upward trend (Figure 5A). Among them, we found that the most significant change was the 24 h treatment at 4℃. The average proline content of the transgenic lines was 6.1 μg/ml higher than the control group. The results indicate that overexpression of ZmSAMDC changes the protein composition of transgenic maize leaves, resulting in a large accumulation of Pro in plant cells.
Under cold stress, the content of malondialdehyde which could reflect the stress resistance of plants, increased with the increase of the active oxygen content of plant leaves. With the increase of the time of low temperature (4 ℃), the MDA content of transgenic plants showed an upward trend (Figure 5B). These results indicate that the transgenic lines accumulate relatively low ROS under cold stress.
Overexpression of ZmSAMDC under cold stress enhance plant cold resistance by increasing leaf antioxidant enzymes
The metabolic system of the plant will undergo significant changes in a cold environment. The amount of oxygen absorbed by the plant will be reduced, and a large amount of harmful active oxygen will be accumulated, thus causing certain damage to the plant. The level of antioxidant enzyme activity can measure the strength of plant resistance. With the increase of the time of low temperature (4 ℃), the contents of POD, SOD, CAT, and APX of the transgenic plants showed an upward trend (Figure 6A-D). When the transgenic plants were treated at 4 ℃ for 48 h, the average content of POD, SOD, and CAT was higher than the control group. When the transgenic plants were treated at 4 ℃ for 12 h, the average ascorbate peroxidase content of the transgenic line was 5.58 μmol/mg higher than that of the control group. Therefore, the overexpression of ZmSAMDC changes the oxidative stress response of plants, improves the ability of plants to resist oxidation and scavenging cationic free radicals, and promotes the decomposition of H2O2 and the ability to catalyze AsA in plants.
ZmSAMDC positively regulates CBFs and cold-responsive gene expression under cold stress
In order to further clarify the molecular mechanism of ZmSAMDC overexpression lines responding to cold stress, we used qRT-PCR to study the expression patterns of cold-induced CBF family genes and downstream cold-responsive genes. In the transgenic lines and WT plants, the CBF family genes CBF1, CBF2, and CBF3 were induced rapidly and peaked 12h after cold stress. However, the expression levels of these three CBF genes in all transgenic lines were higher than those of WT. RD29A, COR15A, and COR47 are the downstream target genes of CBF, and these COR genes are gradually induced to express under cold stress (Figure 7). These results indicate that overexpression of ZmSAMDC positively regulates the expression of the CBF gene and downstream COR gene, thereby improving the cold tolerance of maize.
Overexpression of ZmSAMDC significantly increased the yield of maize
Field experiments were conducted to observe the agronomic traits of transgenic plants. The results are shown in Table 2. The transgenic materials for plant height, ear length, shaft thickness, and other traits have no significant difference with the control group, indicating that the ZmSAMDC gene may not affect these traits. However, the number of rows and 100-seed weight of ZmSAMDC plants were significantly higher than those of the control group, and the bald tip length was significantly lower than that of the control group. Therefore, the SAMDC gene can effectively increase the yield of maize.