With the aim of determining the spatio-temporal expression of ARR16 in seed tissues of permeable and non-permeable accessions, GFP reporter lines (pARR16-GFP-ARR16) were generated using the GoldenBraid cloning system. A specific GFP signal was observed in early meristemoid cells in the epidermis of young cotyledons in T2 plants of reporter lines in agreement with a previous study of ARR16 localization (Vatén et al. (2018) Developmental Cell 47: 53–66), indicating that the GFP reporter lines generated here were functional. However, no clear GFP signal was observed in developing seed tissues. RT-qPCR analysis detected ARR16 transcript expression in the mid- to late stages of seed formation, although the level of expression was relatively low, as generally observed for transcription factors. Furthermore, the observation of fluorescent markers such as GFP is technically challenging for genes expressed at low-levels in seeds due to various endogenous autofluorescent molecules. GUS reporter lines (pARR16-GUS) generated at the end of the project will provide a useful alternative to specify where ARR16 is expressed in developing seed tissues.
Based on naturally-occurring variation within the ARR16 gene, eight haplotypes were detected that could be associated with permeable or impermeable phenotypes. RT-qPCR analysis demonstrated that ARR16 transcripts were more abundant in developing seeds of representative accessions having permeable haplotypes than in those having impermeable haplotypes, indicating that these haplotypes were causal for the level of ARR16 transcripts and thereby regulate seed coat permeability in natural accessions.
Among the components of the seed coat known to affect permeability, suberin, a lipid and phenolic cell wall heteropolymer, is mainly accumulated in the hilum region of the seed coat and acts as a water-repellant. It was found that arr16 mutant seeds showed more suberin-related autofluorescence (AF) than those of wild type (WT) under UV light. Additionally, GWAS based on hilum AF measured by image analysis was performed and resulting SNPs associated with hilum AF level were compared with those associated with seed coat permeability. Interestingly, the 10 significantly associated SNPs located in the promoter of ARR16 were common to both traits. This suggests that ARR16 regulates the variation of permeability in natural accessions, at least partially, by affecting suberin deposition in the hilum. Analysis of seeds for lipid polyester monomer composition revealed that certain dicarboxylic acids (DCAs) and phenolics were accumulated to significantly higher levels in arr16 compared to WT.
Transcriptome analysis to determine potential downstream targets of ARR16 was initiated. Initially, laser-assisted microdissection coupled with RNA-Seq using Illumina HiSeq was planned, however, as the spatio-temporal expression of ARR16 in developing seeds was not known, RNA was extracted from tissues following a simple manual dissection to separate developing embryo and seed coat/endosperm tissues RNA-Seq analysis is in progress. The association of ARR16 with modified suberin accumulation in seeds, was investigated further by RT-qPCR analysis of suberin biosynthesis gene expression in developing seeds. The differential gene expression patterns observed between arr16 and WT were coherent with ARR16 playing a role in the transcriptional modulation of suberin production. Moreover, these were correlated with higher levels of certain lipid polyester monomers, notably dicarboxylic acids and suberin phenolics, in the arr16 mutant. The RNA-Seq data will yield further key information about ARR16 targets, and is expected to provide a more comprehensive model of the ARR16 transcriptional network during seed development, in particular in relation to the expression of suberin biosynthesis-related genes.
