The LbSAD2 ORF was cloned into the vector pCAMBIA3301 under the control of the CaMV 35S promoter to generate p35S:LbSAD2, using the primers LbSAD2 F0 and LbSAD2 R0 (Supplementary Table 1), according to the instructions of the In-Fusion HD Cloning Kit (Clontech Laboratories, Inc.). The p35S:LbSAD2 vector was introduced into the A. tumefaciens strain GV3101, which was then transformed into Arabidopsis Col-0 and CS65878 by the Agrobacterium-mediated floral dip method (Clough and Bent, 2010). After screening with herbicide for three consecutive generations, homozygous Col-35S:LbSAD2 and CS65878-35S:LbSAD2 lines were retained for qPCR.

Plants heterologously expressing LbSAD2 were identified by PCR using the primers SAD2-S and pCAMBIA3301-A after extraction of genomic DNA. Total RNA of several strains of Col-35S:LbSAD2 and CS65878-35S:LbSAD2 was then extracted using the FastPure Plant Total RNA Isolation kit (Vazyme, China) according to the manufacturer’s instructions. qPCR was conducted to evaluate the expression level of LbSAD2 in Col-35S:LbSAD2 and CS65878-35S:LbSAD2 using LbSAD2 RTS and LbSAD2 RTA primers. Amplification of the ACTIN2 gene of Arabidopsis was used as an internal control (primers ACTIN2 sense and ACTIN2 anti). Three replications were carried out for each transgenic line. Three lines each with high, medium, and low LbSAD2 expression levels, respectively, of Col-35S:LbSAD2 and CS65878-35S:LbSAD2 were used for further experiments.

In the calculation of the expression levels of LbSAD2 in the Col-35S:LbSAD2 plants, the line OE26 with the lowest expression level was used as the control (relative level is 1) to calculate the relative expression level of different overexpression strains (Leng et al., 2021). The same method was used to calculate the expression level of LbSAD2 in CS65878-35S:LbSAD2 lines using CL6 as the control.

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