SAMBA controls cell division rate during maize development

Abstract

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

Figure 1

samba mutants obtained through CRISPR/Cas9 gene editing. A, Structural representation of the maize SAMBA gene showing the target sites of the two gRNAs. B, The gRNA sequences (blue), PAM (orange), and mutation sites (red) of samba alleles. C, The amino acid sequences of SAMBA WT and putative samba mutant isoforms from translation re-initiation (TRI). SAMBA homology region 1 (SHR1) and SHR2, as defined by Eloy et al. (2012) are marked in green and the missense AAs are indicated in red. SAMBA-qF and SAMBA-qR indicate the position of the forward and reverse primers, respectively, used for SAMBA RT-qPCR. Asterisk represents a stop codon.

Figure 2

Overview of the phenotypes of the WT and samba mutant plants. A, WT and samba mutant plants grown in the greenhouse 70 d after sowing. The internodes (B) and ligule morphology (C) of WT plant and samba mutants from leaf 6 to leaf 12. Scale bars = 20 cm.

Figure 3

Quantification of the phenotypes of phenotypes of the WT and samba mutant plants. Quantification of internode (A), leaf sheath (B) and blade (C) length and blade width (D) of samba mutants in comparison to the WT. Error bars represent standard error and significant differences were determined using Student’s t test: *P < 0.05, **P < 0.01 (n ≥ 3).

Figure 4

Complementation of the samba mutant plants by introducing SAMBA-GS5. Plant phenotype of the WT, SAMBA-GS5, samba complemented plants and samba-1 (A), or samba-3 (B). Bars = 50 cm.

Figure 5

Co-IP results of C-terminally YFP-tagged WT SAMBA and samba alleles with C-terminally RFP-tagged APC3. Co-IP (A) and input of the extracted whole proteins (B).