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Seeds exhibit primary dormancy to prevent germination under unfavourable conditions. Previous studies have shown that the gibberellin signalling intermediate RGA-LIKE2 (RGL2) forms a transcription factor complex with DNA-BINDING ONE ZINC FINGER6 (DOF6) in regulating seed dormancy in Arabidopsis. Using an RNA-sequencing approach, we identified MAJOR LATEX PROTEIN-LIKE PROTEIN329 (MLP329) as a downstream target of DOF6. MLP329 was found to be a positive regulator of primary seed dormancy, because freshly harvested unstratified mlp329 mutant seeds showed early germination, while unstratified transgenic seeds overexpressing MLP329 showed poor germination. MLP329 expression level was reduced in wild-type seeds upon dry storage and cold stratification. MLP329 expression level was enhanced by DOF6; however, DOF6-dependent MLP329 expression was suppressed in the presence of RGL2. MLP329 expression was enhanced in seeds treated with ABA and auxin IAA. Moreover, the mlp329 mutant seeds exhibited enhanced expression of the GA biosynthetic gene GA1 and suppression of the ABA biosynthetic gene ZEP compared to the overexpression lines. The observed suppression of DOF6-dependent MLP329 expression by RGL2 reveals a possible negative feedback mechanism to modulate seed dormancy. MLP329 also probably enhances the endogenous ABA/GA ratio to positively regulate primary seed dormancy., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: LRRop-1, induced by DOF6 transcription factor, negatively regulates abiotic stress responses during Arabidopsis seed germination. The lrrop-1 mutant has reduced ABA signaling, which is part of the underlying stress-remediation mechanism. The large family of leucine-rich repeat (LRR) proteins plays a role in plant immune responses. Most LRR proteins have multiple functional domains, but a subfamily is known to possess only the LRR domain. The roles of these LRR-only proteins in Arabidopsis remain largely uncharacterized. In the present study, we have identified 44 LRR-only proteins in Arabidopsis and phylogenetically classified them into nine sub-groups. We characterized the function of LRRop-1, belonging to sub-group V. LRRop-1 encodes a predominantly ER-localized LRR domain-containing protein that is highly expressed in seeds and rosette leaves. Promoter motif analysis revealed an enrichment in binding sites for several GA-responsive and stress-responsive transcription factors. The lrrop-1 mutant seeds showed enhanced seed germination on medium containing abscisic acid (ABA), paclobutrazol and NaCl compared to the wild type (WT), demonstrating higher abiotic stress tolerance. Also, the lrrop-1 mutant seeds have lower levels of endogenous ABA, but higher levels of gibberellic acid (GA) and jasmonic acid-Ile (JA-Ile) compared to the WT. Furthermore, lrrop-1 mutant seeds imbibed with ABA exhibited reduced expression of ABA-responsive genes compared to similarly treated WT seeds, suggesting suppressed ABA signaling events in the mutant. Furthermore, chromatin immunoprecipitation (ChIP) data showed that DNA BINDING1 ZINC FINGER6 (DOF6), a negative regulator of seed germination, could directly bind to the LRRop-1 promoter and up-regulate its expression. Thus, our results show that LRRop-1 regulates ABA-mediated abiotic stress responses during Arabidopsis seed germination.

The DELLA protein RGA-LIKE2 (RGL2) is a key transcriptional repressor of gibberellic acid (GA) signaling that regulates seed germination. We identified GATA12, a gene encoding a GATA-type zinc finger transcription factor, as one of the downstream targets of RGL2 in Arabidopsis thaliana. Our data show that freshly harvested (unstratified) seeds of GATA12 antisense suppression lines have reduced dormancy compared with the wild-type, while ectopic expression lines show enhanced seed dormancy. We show that GATA12 expression is negatively regulated by GA, and its transcript levels decline dramatically under dormancy-breaking conditions such as dry storage and cold stratification of seeds. GATA12 promoter has several GAMYB- and DOF-associated motifs that are known to be GA- and RGL2-responsive, respectively. Chromatin immunoprecipitation assay showed that a protein complex containing RGL2 can bind to GATA12 promoter and thereby regulate its expression. RGL2 lacks a DNA binding domain and requires a transcription factor to induce GATA12 expression. Our data show that this RGL2-containing protein complex includes DNA BINDING1 ZINC FINGER6 (DOF6), which is a known negative regulator of germination in freshly harvested seeds. We further show that this novel RGL2-DOF6 complex is required for activating GATA12 expression, thus revealing a molecular mechanism to enforce primary seed dormancy., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Background: Seed germination is of immense significance for agriculture and has been studied for centuries. Yet, our understanding of the molecular mechanisms underlying regulation of dormancy and germination is still in its infancy. Gibberellins are the key phytohormones that promote germination, and the DELLA protein RGL2 is the main signalling intermediate involved in this response. Germination is completely inhibited if functional RGL2 is overexpressed and/or stabilized; however, the molecular mechanisms of RGL2 function are still largely unknown. We therefore attempted to shed light onto some of the genetic events downstream of RGL2., Results: Gene ontology of the transcriptome differentially regulated by RGL2, as well as extensive cross-comparison with other available microarray data indicates that RGL2-mediated inhibition of germination causes seeds to enter a state of dormancy. RGL2 also appears to differentially regulate a number of transcription factors, many of which are known to be involved in light- or phytohormone-mediated aspects of germination. A promoter analysis of differentially expressed genes identified an enrichment of several motifs that can be bound by specific transcription factors, for example GAMYB, ARF1, or Dof-type zinc fingers. We show that Dof-binding motifs indeed play a role in RGL2-mediated transcription. Using Chromatin Immunoprecipitation (ChIP), we show that RGL2 directly downregulates at least one cell wall modifying enzyme, which is predicted to constrain cell growth thereby leading to inhibition of seed germination., Conclusions: Our results reveal that RGL2 controls various aspects of germination. Through the repression of cell wall modifying enzymes, cell growth is directly constrained to inhibit germination. Furthermore, RGL2 likely interacts with various types of proteins to regulate transcription, and differentially regulates several transcription factors. Collectively, our data indicate that gibberellins, acting via RGL2, control several aspects of seed germination.