The full-length CDS of LbHLH was cloned using primers LbHLH OEAt-S and LbHLH OEAt-A (Table S1) containing NcoI digestion sites for cloning into different vectors. A ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd.) was used to generate p35S::LbHLH via homologous recombination. p35S::LbHLH was introduced into Agrobacterium tumefaciens GV3101 cells, which were used to transform Arabidopsis. After screening for three generations with herbicides (0.1%, v/v), the Col-35S::LbHLH overexpression lines were subjected to physiological measurements.

Three Col-35S::LbHLH overexpression lines were selected for physiological characterization based on LbHLH expression (low, medium, and high). Specifically, positive transgenic plants were first identified using primers LbHLH OEAt-S and LbHLH OEAt-A based on the genomic sequences of the transgenic lines. mRNA was then extracted from different Col-35S::LbHLH lines using a FastPure Plant Total RNA Isolation kit (Vazyme Biotech Co., Ltd.) according to the manufacturer’s instructions. LbHLH expression levels in different Col-35S::LbHLH lines were analyzed by qRT-PCR using primers LbHLH RT-S and LbHLH RT-A (Table S1). Given that no homologs of LbHLH were detected in Arabidopsis, the line with the lowest LbHLH expression level (OE35) was used as a control (relative expression level set to 1) to calculate the expression levels of LbHLH in the Col-35S::LbHLH lines. Three biological replicates were performed for each group. Lines with high (OE40), medium (OE26), and low expression (OE4) levels were retained for analysis.

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