https://orcid.org/0000-0003-0693-881X
In eukaryotes, sophisticated translational and posttranslational regulatory processes dramatically expand the functional transcriptome. Alternative splicing (AS) is one such mechanism of differentially processing messenger RNA (mRNA) precursors to generate multiple mature mRNA isoforms from a single gene (reviewed in Lee and Rio, 2015). Because mRNA variants can encode polypeptides with varying sequences and activities, AS increases proteome diversity beyond the set of protein-coding genes present in the genome. Importantly, AS often is employed to orchestrate effective responses to environmental perturbations. In this issue of The Plant Cell, Meina Guo, Yuxin Zhang, Xinqing Jia and colleagues (Guo et al., 2022) show that, during nutritional stress in rice, AS of transcription factors dictates the activation of distinct downstream transcriptional programs. Additionally, the authors present evidence that AS of transcriptional regulators during nutritional stress is evolutionarily conserved in flowering plants, opening avenues for crop improvements.
Phosphorus (Pi) is an essential macronutrient for plant development. Due to its low solubility and motility in soils, Pi availability often represents a limiting factor in plant growth and productivity (Weikard, 2016). Under Pi starvation, monocots, such as rice, typically display inhibition of shoot growth and upright shoot architecture (Carstensen et al., 2018; Figure A). Previously, the authors showed that the transcription factor REGULATOR OF LEAF INCLINATION 1 (RLI1a) controls rice shoot architecture in response to Pi starvation (Ruan et al., 2018). Interestingly, bioinformatic analysis of publicly available transcriptomic datasets revealed two variants for RLI1 mRNA (RLI1a and RLI1b). While both RLI1a and RLI1b encode an MYB DNA-binding domain, only RLI1b harbors a C-terminal coiled-coil (CC) domain. Because CC domains are often associated with protein–protein interaction and dimerization, the authors investigated whether higher-order protein structure dictates the transcriptional activity of RLI1. Biomolecular fluorescence complementation assays confirmed that the RLI1b CC domain is required for protein–protein interaction and dimerization. By combining an array of biochemical and genetic approaches, the authors provide robust evidence that RLI1 isoforms differentially bind to DNA cis-elements in target genes. Furthermore, chromatin immunoprecipitation combined with DNA sequencing showed that, while both RLI1 transcription factors directly target nearly 2,000 genes, RLI1a has an expanded repertoire of over 4,500 genes (Figure B). Together, these data imply that higher-order RLI1 protein structure underlies its DNA-binding affinity and activity.
Phenotypic and transcriptional response to Pi signaling. A, Superficial root foraging and leaf erection in response to Pi deficiency compared to sufficiency. B, RLI1 isoforms selectively regulate transcription in plants. Red color indicates high expression of luciferase reporter gene. Adapted from Guo et al. (2022), Figures 7 and 10, respectively.
So, how do RLI1 isoforms control the response to Pi starvation? Gene expression and immunoblotting analysis showed that transcript and protein levels of RLI1a, but not of RLI1b, are strongly repressed in response to Pi starvation. Likewise, immunoblotting and cell-free degradation assays indicate that Pi-starvation stabilizes RLI1b while promoting RLI1a turnover. To investigate whether RLI1 isoforms have different roles in response to Pi starvation, the authors characterized shoot architecture of RLI1a and RLI1b overexpressing (OE) lines. Phenotypic analysis showed that RLI1a OE plants constitutively display altered lamina joint cell length and looser shoot architecture compared to RLI1b OE and wild-type plants. Surprisingly, however, both RLI1a and RLI1b OE plants accumulate higher levels of Pi and display higher expression of Pi-starvation marker genes compared to wild-type plants. These exciting findings indicate that phenotypic and biochemical responses to Pi starvation can be genetically uncoupled from translational regulatory processes.
By using phylogenetic analysis of MYB-CC protein and structural analysis of the RLI1 clade from representative land plants, corroborated by genetic and biochemical experimentation, Guo et al. (2022) go on to present evidence that AS of RLI1 homologs is functionally conserved in monocots and dicots. Looking forward, it would be interesting to investigate the mechanisms underlying spliceosome recruitment and the impacts of AS on potential interactors of RLI1 transcriptional factors.
