Generation of transgenic plants
Generation of the constructs used for moderate overexpression and genetic complementation experiments was previously described (Anderson et al. 2021). The ProUBI10:GET3B-STREP construct contains the promoter for the UBIQUITIN 10 gene (Norris et al., 1993), genomic sequence for the coding region of GET3B , sequence encoding a C-terminal 1X STREP tag, and the terminator of the NOS (nopaline synthase ) gene. For theProUBI10:GET3B(D124N)-STREP construct, the D124N mutation was introduced into the ProUBI10:GET3B-STREP construct by site-directed mutagenesis. The Arabidopsis plants transformed for the genetic complementation experiments were homozygous for the alb4 and get3b alleles, and heterozygous for the srp54 allele. Plants transformed for moderate overexpression studies were homozygous for either the alb4 or stic2-4 allele. The plant transformation procedure was previously described (Anderson et al. 2021), and transformants were selected in the T1 generation based on gentamycin resistance. Transformants containing mutant alleles as well as the introduced transgene were identified by PCR genotyping using primers listed in Supplemental Table S2. Genetic complementation was assessed in the T2 generation based on the phenotype of plants that were homozygous for alb4 , get3b and srp54 and carried the introduced transgene. The effects of moderate overexpression inalb4 or stic2 homozygous backgrounds were assessed in either the T2 or T3 generations.