Authors: Paola PunzoAlessandra RuggieroMarco PossentiGiorgio PerrellaRoberta NurcatoAntonello CostaGiorgio MorelliStefania GrilloGiorgia Batelli

Institutions:

  • National Research Council of Italy, Institute of Biosciences and Bioresources, 80055 Portici, Naples, Italy
  • Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics, 00178 Rome, Italy
  • Italian National Agency for New Technologies, Energy and Sustainable Economic Development, 75026 Rotondella, Matera, Italy

Publication: Plant Physiology

Date: June 2020

Full paper: http://www.plantphysiol.org/content/183/2/793

Abstract:

RNA splicing is a fundamental mechanism contributing to the definition of the cellular protein population in any given environmental condition. DNA-DAMAGE REPAIR/TOLERATION PROTEIN111 (DRT111)/SPLICING FACTOR FOR PHYTOCHROME SIGNALING is a splicing factor previously shown to interact with phytochrome B and characterized for its role in splicing of pre-mRNAs involved in photomorphogenesis. Here, we show that DRT111 interacts with Arabidopsis (Arabidopsis thaliana) Splicing Factor1, involved in 3′ splicing site recognition. Double- and triple-mutant analysis shows that DRT111 controls splicing of ABI3 and acts upstream of the splicing factor SUPPRESSOR OF ABI3–ABI5. DRT111 is highly expressed in seeds and stomata of Arabidopsis and is induced by long-term treatments of polyethylene glycol and abscisic acid (ABA). DRT111 knock-out mutants are defective in ABA-induced stomatal closure and are hypersensitive to ABA during seed germination. Conversely, DRT111 overexpressing plants show ABA-hyposensitive seed germination. RNA-sequencing experiments show that in dry seeds, DRT111 controls expression and splicing of genes involved in osmotic-stress and ABA responses, light signaling, and mRNA splicing, including targets of ABSCISIC ACID INSENSITIVE3 (ABI3) and PHYTOCHROME INTERACTING FACTORs (PIFs). Consistently, expression of the germination inhibitor SOMNUS, induced by ABI3 and PIF1, is upregulated in imbibed seeds of drt1112 mutants. Together, these results indicate that DRT111 controls sensitivity to ABA during seed development, germination, and stomatal movements, and integrates ABA- and light-regulated pathways to control seed germination.

The phytohormone abscisic acid (ABA) regulates physiological and developmental processes, including stress responses, seed development, and germination. Perhaps the most well-defined mechanism mediated by ABA is induction of stomatal closure. In plants subjected to hyperosmotic stress, ABA is synthesized predominantly in leaf vascular tissues and guard cells. Here, ABA activates a signaling pathway that coordinately modulates activity of membrane-located transporters, leading to efflux of solutes. The consequent reduction of guard cell turgor causes stomatal closure, thus reducing evapotranspiration under abiotic stress conditions (Qin and Zeevaart, 1999Schroeder et al., 2001Nambara and Marion-Poll, 2005Bauer et al., 2013Kuromori et al., 2018).

In seeds, ABA induces maturation, dormancy, and plays a key role during germination. Transcription factors such as LEAFY COTYLEDON1 (LEC1) and LEC2, FUSCA3, and ABSCISIC ACID INSENSITIVE3 (ABI3) are involved in reserve accumulation and inhibition of premature germination (Santos-Mendoza et al., 2008Mönke et al., 2012Yan and Chen, 2017). At early stages of seed maturation, LEC1LEC2, and FUSCA3 are expressed to prevent germination of the developing embryo, whereas ABI3 expression is maintained at high levels until final maturation stages (Perruc et al., 2007). In this phase, ABI3 and LEC1 regulate expression of genes involved in storage reserve accumulation and acquisition of desiccation tolerance, such as late embryogenesis abundant proteins (Parcy et al., 1994).

In addition, ABA prevents germination by inhibiting water uptake and endosperm rupture (Finch-Savage and Leubner-Metzger, 2006). When favorable conditions are restored, ABA levels decrease, with a concomitant increase of gibberellic acid (GA) to allow embryos to expand and break the seed-covering layers (Manz et al., 2005). The endogenous levels of ABA and GA are regulated by different signaling pathways, and recent studies have highlighted the crosstalk between light and hormonal pathways in the regulation of germination (Kim et al., 2008Lau and Deng, 2010de Wit et al., 2016). Phytochrome A (phyA) and phyB are photoreceptors that perceive Far Red (FR) and Red (R) light, respectively. During early stages of germination, phyB signaling involves a family of basic helix–loop–helix transcription factors, namely PHYTOCHROME INTERACTING FACTORs (PIFs). After R or white-light illumination, phyB translocates to the nucleus in its active Pfr conformation, where it binds and inhibits PIF1, also known as PIF3-LIKE5 (PIL5), promoting light-induced germination (Lee et al., 2012). In the dark or in low R/FR light, when phyB is in the inactive, Pr cytosolic form, PIF1 is stabilized and represses germination. PIF1 promotes ABA biosynthesis and signaling, and represses GA signaling, inducing expression of genes such as ABI3ABI5REPRESSOR OF GA13, and DOF AFFECTING GERMINATION1 (Oh et al., 2009). Interestingly, ABI3 protein also interacts with PIF1 to activate the expression of direct targets, such as SOMNUS (SOM), a key regulator of light-dependent seed germination acting on ABA and GA biosynthetic genes (Kim et al., 2008Park et al., 2011).

In seeds initiating germination, ABI3 expression is repressed. Perruc et al. (2007) reported that the chromatin-remodeling factor PICKLE negatively regulates ABI3 by promoting silencing of its chromatin during seed germination. ABI3 activity is also controlled by alternative splicing (AS) of the corresponding precursor mRNA (pre-mRNA), with different splice forms predominating at different seed developmental stages. This process is regulated by splicing factor SUPPRESSOR OF ABI3-5 (SUA) through the splicing of a cryptic intron in ABI3 mRNA (Sugliani et al., 2010).

AS occurs when the spliceosome differentially recognizes the splice sites. The selection of alternative 5′ or 3′ splice sites leads to an inclusion of different parts of an exon, whereas failure to recognize splicing sites causes intron retention (IR) in the mature mRNA. These alternative splice forms can produce proteins with altered domains and function (Nilsen and Graveley, 2010Staiger and Brown, 2013Fu and Ares, 2014Laloum et al., 2018). In plants, this mechanism is highly induced in response to external stimuli. Recent studies report an emerging link between splicing and ABA signaling (Zhu et al., 2017Laloum et al., 2018). For example, the transcript encoding type 2C phosphatase HYPERSENSITIVE TO ABA1 (HAB1), a negative regulator of ABA signaling, undergoes AS. In the presence of ABA, the last intron is retained, leading to a truncated protein. The two encoded proteins, HAB11 and HAB12, play opposite roles by competing for interaction with OPEN STOMATA1 during germination, which then results in switching of the ABA signaling on and off (Wang et al., 2015). Likewise, SR45, a member of the Ser/Arg-rich proteins (an important class of essential splicing factors that influence splice site selection), regulates Glc signaling through downregulation of the ABA pathway during seedling development (Carvalho et al., 2010). In addition, several splicing regulators were reported to influence ABA sensitivity, such as SAD1ABH1SKB1, and Sf1 (Hugouvieux et al., 2001Xiong et al., 2001Zhang et al., 2011Jang et al., 2014).

In this study, we show that the splicing factor DNA-DAMAGE REPAIR/TOLERATION PROTEIN111 (DRT111), previously characterized to play a role in the control of pre-mRNA splicing in light-regulated developmental processes (Xin et al., 2017), is involved in ABA response mechanisms. Manipulation of DRT111 expression results in a modified sensitivity to ABA regarding stomatal movements and seed germination. Accordingly, DRT111 is highly expressed in stomata and seeds, and upregulated upon long-term exposure to ABA. Moreover, ABI3 alternative transcript quantification as well as analysis of double and triple mutants shows that DRT111 controls splicing of ABI3 upstream of SUA. Transcriptome analysis in drt111 dry seeds revealed extensive alteration in gene expression and splicing of genes involved in light- and ABA-dependent control of germination. Consistently, we show that expression of the germination inhibitor SOM is induced in drt111. Taken together, our results suggest that DRT111 functions in the integration of ABA and light quality stimuli for seed germination under appropriate conditions