A reevaluation of the role of the ASIL trihelix transcription factors as repressors of the seed maturation program

Abstract

Developmental transitions are typically tightly controlled at the transcriptional level. Two of these transitions involve the induction of the embryo maturation program midway through seed development and its repression during the vegetative phase of plant growth. Very little is known about the factors responsible for this regulation during early embryogenesis, and only a couple of transcription factors have been characterized as repressors during the postgerminative phase. Arabidopsis 6b-INTERACTING PROTEIN-LIKE1 (ASIL1), a trihelix transcription factor, has been proposed to repress maturation both embryonically and postembryonically. Preliminary data also suggested that its closest paralog, ASIL2, might play a role as well. We used a transcriptomic approach, coupled with phenotypical observations, to test the hypothesis that ASIL1 and ASIL2 redundantly turn off maturation during both phases of growth. Our results indicate that, contrary to what was previously published, neither of the ASIL genes plays a role in the regulation of maturation, at any point during plant development. Analyses of gene ontology (GO)-enriched terms and published transcriptomic datasets suggest that these genes might be involved in responses during the vegetative phase to certain biotic and abiotic stresses.

Keywords: Arabidopsis; embryo; embryonic maturation program; trihelix factor.

FIGURE 1

Characterization of the asil alleles. (a) Levels of expression of the LAFL genes in the embryo proper at different developmental stages. (b) Expression of a seed storage protein gene in the embryo proper at different developmental stages. Data from Hofmann et al. (2019). Stages: pg: preglobular, gl: globular, eh: early heart, lh: late heart, et: early torpedo, lt: late torpedo, bc: bent cotyledon. (c) Schematic of the ASIL1 and ASIL2 proteins with the location of the insertions in the mutant alleles and sequence of the C‐terminal portion of the ASIL2 and asil2‐1 alleles. (d) Expression of ASIL1 and ASIL2 in wild type and mutant 5 DAP seeds. (e) Expression of ASIL1 and ASIL2 in wild type and mutant 14 days after germination (DAG) seedlings. (d) and (e) represent data from the RNAseq experiments (three biological replicates). (f) Expression of ASIL2 in wild type and mutant 5 DAP seeds and 14 DAG seedlings, measured by RT‐qPCR (three biological replicates). Analysis of variance (ANOVA) with Tukey's post hoc test *p < .05, **p < .01. Error bars: in a,b,f: ±SEM; in d,e: ±SD

FIGURE 2

Embryonic phenotypes of asil mutants and overexpressors. (a) Percentages of embryos at different stages in 5 DAP siliques for the different genotypes (n = 120–160 embryos per genotype). (b) Percentage of embryos at different stages that are green for the different genotypes (n = 60–250 embryos per stage per genotype). (b,c) Percentage of embryos at different stages that express At2S3p:GFP for Col and asil1‐1 asil2‐1 (n = 35–88 embryos per stage per genotype). For (b) and (c), there were no significant differences between wild type and mutants (Fisher's exact test). (d) Expression of ASIL1 and ASIL2 in overexpressor lines, measured by RT‐qPCR (biological triplicates). Error bars: ±SEM. *p < .05 (t test). For ASIL2 in 35S:ASIL2, p = .08 (t test). (e) Percentages of embryos at different stages in 6 DAP siliques for overexressor lines (n = 62–132 embryos per genotype, two plants per genotype)

FIGURE 3

Gene expression in asil mutant and overexpressing siliques measured by RT‐qPCR. (a–c) Expression of LAFL genes in mutant siliques at (a) 3 DAP, (b) 5 DAP, (c) 6 DAP. (d–f) Expression of genes encoding maturation products in mutant siliques at (d) 5 DAP, (e) 6 DAP, and (f) 10 DAP. (g,h) Expression of genes encoding maturation products in siliques of overexpressors at (g) 7 DAP, (h) 10 DAP. *p < .05, **p < .01 (analysis of variance [ANOVA] followed by Tukey's post‐hoc test). Error bars: ±SEM of three biological replicates

FIGURE 4

Characterization of asil mutant seedlings. (a–d) Expression of selected maturation genes in 14 days after germination (DAG) seedlings (a) asil1‐1 versus Col, (b) all asil mutants versus their corresponding wild types, (c,d) val1‐2 val2‐1 versus Col. *p < .05, **p < .01 (analysis of variance [ANOVA] followed by Tukey's post hoc test). Error bars: ±SEM of three biological replicates. (e–l) 14 DAG seedlings (the last 2 days exposed to 50 μM ABA) stained with Fat Red. Magnification: (e–k) 0.75×, (l) 1.25×

FIGURE 5

Comparisons and overlaps of differentially expressed genes (DEGs) in 14 days after germination (DAG) seedlings. Comparisons of sets of DEGs derived from the RNAseq experiment, overlaps of these sets between single and double asil mutants. (a,b) asil2 alleles: asil2‐1 versus asil2‐2, (a) up versus up and down versus down, (b) up versus down and vice versa, (c) asil1‐1 versus asil2‐1 versus asil1‐1 asil2‐1. (d) asil1‐1 versus asil2‐2 versus asil1‐1 asil2‐2. Overlap lists generated with BioVenn

FIGURE 6

Overlaps of differentially expressed genes (DEGs) in our experiment versus Gao's. Comparison of our RNAseq data (asil1‐1) versus microarray data from Gao et al. (2009)