Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Summary Advanced transcriptome sequencing has uncovered 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 plant model Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionary-conserved transcripts PIN7a and PIN7b. PIN7a and PIN7b, differing in a 4-amino acid motif, exhibit almost identical expression pattern and subcellular localization. We reveal that they closely associate and mutually influence their 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, unveiling an additional regulatory level of 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.Peer reviewe

CLE Peptides can Negatively Regulate Protoxylem Vessel Formation via Cytokinin Signaling

Cell–cell communication is critical for tissue and organ development. In plants, secretory CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides function as intercellular signaling molecules in various aspects of tissue development including vascular development. However, little is known about intracellular signaling pathways functioning in vascular development downstream of the CLE ligands. We show that CLE peptides including CLE10, which is preferentially expressed in the root vascular system, inhibit protoxylem vessel formation in Arabidopsis roots. GeneChip analysis displayed that CLE10 peptides repressed specifically the expression of two type-A Arabidopsis Response Regulators (ARRs), ARR5 and ARR6, whose products act as negative regulators of cytokinin signaling. The arr5 arr6 roots exhibited defective protoxylem vessel formation. These results indicate that CLE10 inhibits protoxylem vessel formation by suppressing the expression of type-A ARR genes including ARR5 and ARR6. This was supported by the finding that CLE10 did not suppress protoxylem vessel formation in a background of arr10 arr12, a double mutant of type-B ARR genes. Thus, our results revealed cross-talk between CLE signaling and cytokinin signaling in protoxylem vessel formation in roots. Taken together with the indication that cytokinin signaling functions downstream of the CLV3/WUS signaling pathway in the shoot apical meristem, the cross-talk between CLE and cytokinin signaling pathways may be a common feature in plant development

In situ hybridization for mRNA detection in Arabidopsis tissue sections

Plant biology is currently confronted with an overflow of expression profile data provided by high-throughput microarray transcription analyses. However, the tissue and cellular resolution of these techniques is limited. Thus, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here we present an in situ mRNA hybridization method that is routinely used in the analysis of gene expression patterns. The protocol is optimized for mRNA localizations in sectioned tissue of Arabidopsis seedlings including embryos, roots, hypocotyls, young primary leaves and flowers. The detailed protocol, recommended controls and troubleshooting are presented along with examples of application. The total time for the process is 10 days

Cell-surface receptors enable perception of extracellular cytokinins

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses. The main site of cytokinin perception in plant cells is thought to be the endoplasmic reticulum where most cytokinin receptors localise. Here via the use of bioactive probes that cannot enter plant cells and super-resolution microscopy, Antoniadi et al. show that cytokinin can also be perceived at the plasma membrane

In situ hybridization method for localization of mRNA molecules in medicago tissue sections

Here we describe an in situ hybridization (ISH) method using Invitrogen™ ViewRNA™ ISH Tissue Assay (ThermoFisher Scientific) optimized for Medicago root and nodules sections. The method is based on branched (b)DNA signal amplification technology originally developed for use in microplate format and further adapted for detection of (m)RNAs in mammalian tissue sections. Signal amplification is achieved via a series of sequential hybridizations of linking sequences which are anchored to complementary sequences present on specific oligonucleotide probes. The typical (m)RNA probe set contains ~20 synthetic adjacent oligonucleotide pairs. Each probe is composed of a 20bp primary sequence designed to target sequence of interest and a secondary extended sequence serving as a template for hybridization of a preamplifier oligonucleotide. The preamplifier forms a stable hybrid only if it hybridizes to two adjacent probes. By this principle, background is reduced. Other regions on the preamplifier are designed to hybridize to multiple bDNA amplifier molecules that create a branched structure. Finally, alkaline phosphatase (AP)-labeled oligonucleotides, which are complementary to bDNA amplifier sequences, bind to the bDNA molecule by hybridization. By adding Fast Red substrate, red punctuated precipitates are formed that can be detected by light bright and/or fluorescent microscope. ThermoFisher Scientific (https://www.thermofisher.com/nl/en/home.html) designs and synthesizes probe sets for a gene of interest and Invitrogen™ ViewRNA™ ISH Tissue Assay kits include all components required for pretreatment of plant tissues, hybridization and signal amplification.</p

Cell-surface receptors enable perception of extracellular cytokinins

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses

In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples

High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is approx7 d, depending on the tissue used

Analysis of 3D gene expression patterns in plants using whole-mount RNA <em>in situ</em> hybridization

International audienceIn situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall-digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition