Periodic Reporting for period 1 - InScope (Integrated analysis of regulatory networks modulating seed coat permeability in natural accessions)
Reporting period: 2019-11-01 to 2021-10-31
Seed coat permeability affects key traits that impact seed quality, such as dormancy, longevity, and germination tolerance to abiotic stress. Seed coats are formed of several overlying cell layers that accumulate large amounts of metabolites such as polyphenols, lignin, lignan, polysaccharides, cutin and suberin, whose properties impart physical and chemical resistance to the cells. A number of mutants related to seed coat properties have been isolated and they often have altered seed coat permeability. The regulatory mechanisms that modulate seed coat permeability are, however, not well understood. As a preliminary study for this project, a method for quantitative analysis of seed coat permeability was established and used in a genome-wide association study (GWAS) with Arabidopsis accessions. Type-A ARABIDOPSIS RESPONSE REGULATOR 16 (ARR16) was identified as a novel causal gene of seed permeability that encodes a component of the signal transduction pathway for the plant hormone cytokinins (CKs).
The overall objective of this project was to elucidate the molecular mechanisms underlying seed coat permeability variation between naturally occurring Arabidopsis accessions. In particular, this project aimed to address three key questions: (i) what is the causal cis-sequence controlling ARR16 expression and its effect on seed coat permeability? (ii) which seed coat metabolites and/or structures are regulated by ARR16? and (iii) how is the transcriptional network of the CKs signaling pathway structured during seed coat development? The project consisted of three experimental, and one management, work packages (WPs).
The overall objective of this project was to elucidate the molecular mechanisms underlying seed coat permeability variation between naturally occurring Arabidopsis accessions. In particular, this project aimed to address three key questions: (i) what is the causal cis-sequence controlling ARR16 expression and its effect on seed coat permeability? (ii) which seed coat metabolites and/or structures are regulated by ARR16? and (iii) how is the transcriptional network of the CKs signaling pathway structured during seed coat development? The project consisted of three experimental, and one management, work packages (WPs).
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.
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.
The involvement of CKs in the regulation of seed coat development is a completely novel function for this hormone. In agreement with this new role, the InScope project revealed that ARR16 modulates the accumulation of a subset of lipid polyesters, suberin phenolics. While several transcription factors, biosynthetic enzymes and transporters are known to be involved in suberin accumulation in seeds, the implication of a CKs signal transduction factor is a completely new finding. Analysis of CKs signaling is challenging due to functional redundancy between the numerous CKs response regulators, which show overlapping expression patterns. Nevertheless, single arr16 mutants have clear seed phenotypes, suggesting that ARR16 plays a non-redundant role in CKs signaling in developing seeds. Additionally, CKs metabolism and signaling are known to be involved in the regulation of seed size, and InScope analyses found that arr16 seeds were larger than those of WT. Moreover, the permeability of the seed coat is often linked to seed longevity/storability and arr16 mutant seeds had a longer lifespan than WT. Results from InScope are expected to contribute to the elucidation of the molecular mechanisms that control seed size and longevity, which are important agronomic traits. In conclusion, InScope has identified a novel function for a plant hormone in a fundamental development process, seed differentiation, and this affects key seed traits that have wide-ranging impact on plant fitness as well as agricultural production.