SUMMARYAdvanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, limited effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Here we reveal, in the plant model Arabidopsis thaliana, that the PIN7 gene, which encodes a polarly localized transporter for the phytohormone auxin, produces two evolutionarily conserved transcripts. These isoforms PIN7a and PIN7b, differing in a 4 amino acid motif, are present at nearly equal levels in most cells, except some early developing tissues where the expression of PIN7b is moderately prevalent. Both proteins also transport auxin with similar capacity and directionality. However, only PIN7a but not PIN7b cDNA rescues the phenotypes associated with the pin7 knock-out mutation, consistent with their differences in the subcellular trafficking and dynamics at the plasma membrane. Further phenotypic analyses suggested a joint, mutually opposing activity of both isoforms as being required for correct seedling apical hook formation and auxin-mediated tropic responses. These results establish alternative splicing of the PIN family as an evolutionary conserved, functionally relevant mechanism, taking part in the auxin-mediated plant development.

Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators.Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b.PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses.Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.

Rapid progress in plant molecular biology in recent years has uncovered the main players in hormonal pathways and characterized transcriptomic networks associated with hormonal response. However, the role of RNA processing, in particular alternative splicing (AS), remains largely unexplored. Here, using example genes involved in cytokinin signaling, brassinosteroid synthesis and auxin transport, we present a set of reporters devised to visualize their AS events in vivo. These reporters show a differential tissue-specific expression of certain transcripts and reveal that expression of some of the them can be changed by the application of the exogenous hormone. Finally, based on the characterized AS event of the PIN7 auxin efflux carrier, we designed a system that allows a rapid genetic screening for the factors upstream of this AS event. Our innovative toolset can be therefore highly useful for exploring novel regulatory nodes of hormonal pathways and potentially helpful for plant researchers focusing on developmental aspects of AS.