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.