Overexpression of the ZmSAMDC Enhances Cold Tolerance in Transgenic Maize (Zea Mays L.)

Maize (Zea mays L.) is a food crop sensitive to low temperatures. Low temperature, as one of the abiotic stress hazards, seriously affects the yield of corn. However, the genetic basis of low-temperature adaptation in maize is still poorly understood. In this study, maize S-adenosylmethionine decarboxylase (SAMDC) was localized on the nucleus. We introduced the SAMDC gene into the excellent maize inbred line variety GSH9901 and used Agrobacterium-mediated transformation to produce cold-tolerant transgenic maize lines. After a 3-year single-location eld trial, the contents of polyamine (PA), proline, malondialdehyde, an antioxidant enzyme, and APX in the leaves of transgenic maize plants overexpressing SAMDC were signicantly increased, and the introduction of the SAMDC gene was signicantly increased the expression of CBFs and cold-related genes.The agronomic traits of overexpression maize changed and the yield traits were signicantly improved, but no signicant changes were found in plant height, ear length, and shaft thickness.Thus, engineering the SAMDC enzyme is an effective strategy to improve the cold tolerance and value of maize.


Introduction
Maize (Zea mays L.) is an annual herbaceous plant and the second-largest food crop in the world (Jiao et al. 2021). Chilling damage has become a major adverse factor for the growth and development of corn, seriously affecting its growth and yield . Therefore, the differences in physiological and biochemical indicators and agronomic traits of plants were studied using biotechnological methods such as genetic modi cation under cold stress, providing an important theoretical basis for cultivating new cold-tolerant maize varieties. In this study, to cultivate cold-tolerant maize lines, Agrobacterium was used to transform callus, and the SAMDC gene was overexpressed into the excellent maize inbred line GSH9901. Under cold stress, the SAMDC gene was overexpressed to achieve the PA content and proteolytic content in the leaves. The signi cant increase in acid content, malondialdehyde content, antioxidant enzyme content, and yield proves that the SAMDC gene can improve the cold resistance of maize effectively.

Materials And Methods
Plant material and cold treatment Wild-type (WT) and transgenic maize plants (C3) were grown in soil in a growth chamber at 25

Sub-Cellular localization assay of ZmSAMDC
To study the subcellular localization properties of ZmSAMDC, we modi ed the pCAMBIA1302 vector to construct a fusion expression vector p1302-Ubi-ZmSAMDC-GFP for ZmSAMDC and the green uorescent protein reporter gene GFP. The maize Ubiquitin promoter was used in this vector to regulate gene expression. A control vector p1302-Ubi-GFP was also constructed. Agrobacterium carrying both vectors was immersed in tobacco leaf cells. The infected tobacco leaf cells within 24 hours were observed with an LSM710 microscope.

Transformation and molecular characterization
In order to obtain SAMDC-overexpressed transgenic maize, primer pairs containing BstEII (5'-ACTCTTGACCATGGTAGATCTTCCCTCCATCTCCAGCATTG-3') and BglII T3 generation plants were identi ed using PCR by selecting bar genes with the primer pair 5′-TCAAATCTCGGTGACGGGC-3 ′ and 5 ′-ATGAGCCCAGAACGACGCC-3′ (552 bp). ZmSAMDC protein expression was tested with western blot analysis in T3 generation plants. The protein from young leaves was fractionated by dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) using a Mighty Small II electrophoresis system (Hoefer Scienti c Instruments, San Francisco, CA, USA) ). The slab gel was composed of a 12% (w/v) separation gel and a 5% (w/v) concentration gel. The wet transfer technique used the wet transfer technique to separate the leaf protein and then blotted onto the PVDF membrane. The primary antibody was added to the protein. Besides, the protein was incubated with the secondary antibody. Finally, the color with DAB horseradish peroxidase coloring solution was developed to stain the protein in the dark until obvious bands appeared. Then the sample was washed with PBST to stop the reaction, drain, and saved with pictures.

Physiological and biochemical index detection
The leaves of the three transgenic lines and WT were sampled at the three-leaf stage. High-performance liquid chromatography (HPLC) was used to determine the PA content of plant leaves (Xu et al. 2016). The acid ninhydrin method was used to analyze the proline content of maize leaves (E et al. 2015). The thiobarbituric acid method was used to analyze the malondialdehyde content of corn leaves (Yun et al.

2013
). The dry weight and fresh weight analysis techniques were applied to determine the relative water content of plant leaves (Wopereis et al. 1996). The guaiacol method was used to analyze the peroxidase content of corn leaves ). The nitrogen blue tetrazolium method was used to analyze the superoxide dismutase content of maize leaves (Samuilov et al. 2021). The UV absorption method was used to analyze the catalase content of corn leaves (Svetlana et al. 2011). An absorbance photometer at 290nm (absorption coe cient 2.8mM-1cm-1) was used to determine APX activity (Hameed et al. 2012). The experiment was repeated three times, and the average refers to the calculation of various physiological indicators.
Expression analysis of cold-responsive genes regulated by ZmSAMDC RT-qPCR was used to detect cold signal pathway genes CBF1/2/3, RD29A, COR15A, and COR47. WT plants were used as controls, and ACTIN2 was used as an internal reference gene. The 2-ΔΔCt method calculated the relative expression changes between samples (Livak and Schmittgen, 2001). Each experiment was repeated 3 times. The data represents the mean ± standard deviation. The single-factor Duncan, a multiple comparison test, was used to determine the signi cance of the experimental mean when P<0.05.

Identi cation of Agronomic Characters of Transgenic Plants
Three transgenic plants and WT were planted in the transgenic base of Jilin Agricultural University. A total of 20 plants were randomly selected during the growth period to analyze their agronomic traits after they matured.

Statistical analysis
The statistical data analyses were performed using the IBM SPSS Statistics 19 (the general term for a series of software products and related services for statistical analysis operations, data mining, predictive analysis, and decision support tasks launched by IBM). The signi cant differences were determined using paired Student's t-test, and data were presented as means ± standard deviations. P < 0.05 were considered to be statistically signi cant.

Identi cation 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 uorescence 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 speci c 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 signi cantly 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 signi cantly 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 signi cant. After 0, 8 and 12 hours of cold stress at -4 ℃, the damage of the transgenic line at the seedling stage was signi cantly lower than that of the control group ( Figure 4C). The survival rate and relative water content of transgenic lines were signi cantly 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 signi cantly 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 signi cant 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 re ect 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 signi cant 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 signi cantly 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 signi cant 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 signi cantly higher than those of the control group, and the bald tip length was signi cantly lower than that of the control group. Therefore, the SAMDC gene can effectively increase the yield of maize. Agronomic traits are an important index for breeding excellent varieties of maize. In this study, the baldness, number of rows, and 100-seed weight of the overexpression plants were improved, indicating that the ZmSAMDC gene could effectively hinder the decreased yield caused by low temperature. Bais et al. (2002) found that PA was widely involved in life activities and growth and development processes in plants, including fruit development and maturation, senescence, and stress response. Zhu et al. used the antisense RNA of the SAMDC gene to decrease the transcription level of SAMDC rapidly. With decreased Spd and Spm content, plants showed growth inhibition, internode shortening, stem branching, and leaf reduction (Zhu et al. 2020).

Discussion
The most important nding of this study is that overexpression of the SAMDC gene increases the PA content, proline content, malondialdehyde content, and antioxidant enzyme content in the leaves, thereby increasing the yield of maize under cold stress. Therefore, engineering the SAMDC enzyme is an effective strategy to improve the chilling tolerance and value of maize. Tables   Table 1 Determination Figure 1 Sub-cellular localization analysis of the ZmSAMDC gene bar=50 μm   Overexpression of ZmSAMDC enhances the cold tolerance of maize.a Analysis of ZmSAMDC expression in positive transgenic maize. Using ACTIN2 as an internal reference gene, the mRNA levels in transgenic lines C3 and WT were detected by RT-qPCR.The frozen phenotype (b and c), survival rate (d) and relative water content (e) of transgenic lines C3-1, C3-3 and C3-6 and WT. Corn seeds germinate after cold acclimation (2-6 days at 4°C), and are treated at -4°C for 8 hours and 12 hours after the three-leaf period.