m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here, we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.

All the information necessary to build the proteins that perform the biological processes required for life is encoded in the DNA of an organism. Making these proteins requires the DNA sequence of a gene to be transcribed into a 'messenger RNA' (mRNA), which is then processed into a final, mature form. This blueprint is then translated to assemble the corresponding protein. When an mRNA is processed, segments of the sequence that do not code for protein are removed and the remaining coding sequences are joined together in the right order. An intricate molecular machine known as the spliceosome controls this mechanism by recognising the 'splice sites' where coding and non-coding sequences meet. Depending on external conditions, the spliceosome can 'pick-and-mix' the coding sequences to create different processed mRNAs (and therefore proteins) from a single gene. This alternative splicing mechanism is often used to regulate when certain biological processes take place based on environmental cues; for example, the splicing of genes which control the timing of plant flowering is sensitive to ambient temperatures. To investigate this mechanism, Parker et al. focused on Arabidopsis thaliana, a plant that blooms later when temperatures are low. This precise timing partly relies on a gene whose mRNA is efficiently spliced in the cold, resulting in an active form of its protein that blocks blooming. Parker et al. grew and screened many A. thaliana plants to find individuals that could flower early in the cold, in which splicing of this gene was disrupted. A mutant fitting these criteria was identified and subjected to further investigation, which revealed that it could not produce FIONA1. In non-mutant plants, this enzyme chemically modifies one of the components of the spliceosome, a small nuclear RNA known as U6. Parker et al found that there are two types of splice site ­ one more likely to interact with U6 and another that preferentially interacts with another small nuclear RNA, U5. When FIONA1 is inactive (such as in the mutant identified by Parker et al.), splice sites that tend to strongly interact with U5 are selected. However, when the enzyme is active, splice sites that tend to bind with the chemically modified U6 are used instead. Further work by Parker et al. showed that these two types of splice sites ('preferring' either U5 or U6) are found in equal proportions in the genomes of many species, including humans. This suggests that Parker et al. have uncovered an essential feature of how genomes are organised and splicing is controlled.

Transcriptional reprogramming during floral fate acquisition.

Coordinating growth and patterning is essential for eukaryote morphogenesis. In plants, auxin is a key regulator of morphogenesis implicated throughout development. Despite this central role, our understanding of how auxin coordinates cell fate and growth changes is still limited. Here, we addressed this question using a combination of genomic screens to delve into the transcriptional network induced by auxin at the earliest stage of flower development, prior to morphological changes. We identify a shoot-specific network suggesting that auxin initiates growth through an antagonistic regulation of growth-promoting and growth-repressive hormones, quasi-synchronously to floral fate specification. We further identify two DNA-binding One Zinc Finger (DOF) transcription factors acting in an auxin-dependent network that could interface growth and cell fate from the early stages of flower development onward.

Repressor for hire! The vital roles of TOPLESS-mediated transcriptional repression in plants.

Transcriptional corepressors play important roles in establishing the appropriate levels of gene expression during growth and development. The TOPLESS (TPL) family of corepressors are critical for all plant life. TPLs are involved in numerous developmental processes and in the response to extrinsic challenges. As such these proteins have been the focus of intense study since Long and colleagues first described the TPL corepressor in 2006. In this review we will explore the evolutionary history of these essential plant-specific proteins, their mechanism of action based on recent structural analyses, and the myriad of pathways in which they function. We speculate how relatively minor changes in the peptide sequence of transcriptional regulators allowed them to recruit TPL into new processes, driving innovation and resulting in TPL becoming vital for plant development.

The molecular signatures of compatible and incompatible pollination in Arabidopsis.

BACKGROUND: Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. Deciphering the specific transcriptomic response of both pollen and stigmatic cells during their interaction constitutes an important challenge to better our understanding of this cell recognition event. RESULTS: Here we describe a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. This strategy allowed us to distinguish 80% of transcripts according to their parental origins. We also developed a tool which predicts male/female specific expression for genes without SNP. We report an unanticipated transcriptional activity triggered in stigma upon incompatible pollination and show that following compatible interaction, components of the pattern-triggered immunity (PTI) pathway are induced on the female side. CONCLUSIONS: Our work unveils the molecular signatures of compatible and incompatible pollinations both at the male and female side. We provide invaluable resource and tools to identify potential new molecular players involved in pollen-stigma interaction.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

Use of Fluorescent Reporters to Analyse Dynamic and Spatial Responses to Mechanical Wounding.

Mechanical wounding of plant tissues triggers many different responses (Savatin DV, Gramegna G, Modesti V, Front Plant Sci 5:470, 2014). These are primarily mediated by the plant hormone Jasmonic Acid Isoleucine (JA-Ile). Recently, a fluorescent biosensor for JA-Ile showed that sample preparation (i.e., handling of samples) for fluorescent microscopy very often triggers wound response, even without apparent damage to the seedling, affecting downstream analyses (Larrieu A, Champion A, Legrand J, Nat Commun 6:6043, 2015). In this chapter, we describe how to overcome this technical limitation to monitor any fluorescent reporter or dye in response to wounding, using any type of fluorescent or confocal (inverted or upright, laser scanning or spinning disc) microscopes. Pharmacological or wound treatments can easily be performed and responses monitored over long periods of time. We further describe a simple method to extract and analyse quantitative data from confocal images using the open source software Fiji (Fiji Is Just ImageJ (Schindelin J, Arganda-Carreras I, Frise E, Nat Methods 9:676-682, 2012)) and OpenOffice.

Wounding-Induced Stomatal Closure Requires Jasmonate-Mediated Activation of GORK K+ Channels by a Ca2+ Sensor-Kinase CBL1-CIPK5 Complex.

Guard cells integrate various hormone signals and environmental cues to balance plant gas exchange and transpiration. The wounding-associated hormone jasmonic acid (JA) and the drought hormone abscisic acid (ABA) both trigger stomatal closure. In contrast to ABA however, the molecular mechanisms of JA-induced stomatal closure have remained largely elusive. Here, we identify a fast signaling pathway for JA targeting the K+ efflux channel GORK. Wounding triggers both local and systemic stomatal closure by activation of the JA signaling cascade followed by GORK phosphorylation and activation through CBL1-CIPK5 Ca2+ sensor-kinase complexes. GORK activation strictly depends on plasma membrane targeting and Ca2+ binding of CBL1-CIPK5 complexes. Accordingly, in gork, cbl1, and cipk5 mutants, JA-induced stomatal closure is specifically abolished. The ABA-coreceptor ABI2 counteracts CBL1-CIPK5-dependent GORK activation. Hence, JA-induced Ca2+ signaling in response to biotic stress converges with the ABA-mediated drought stress pathway to facilitate GORK-mediated stomatal closure upon wounding.

The Rosa genome provides new insights into the domestication of modern roses.

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

Author Correction: Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate.

The original version of this Article omitted the following from the Acknowledgements:'We also thank DBT-CREST BT/HRD/03/01/2002.'This has been corrected in both the PDF and HTML versions of the Article.

Transcriptional induction of cell wall remodelling genes is coupled to microtubule-driven growth isotropy at the shoot apex in Arabidopsis.

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.

Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate.

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice.

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression.

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

Q&A: How does jasmonate signaling enable plants to adapt and survive?

Jasmonates (JAs) are a class of plant hormones that play essential roles in response to tissue wounding. They act on gene expression to slow down growth and to redirect metabolism towards producing defense molecules and repairing damage. These responses are systemic and have dramatic impacts on yields, making JAs a very active research area. JAs interact with many other plant hormones and therefore also have essential functions throughout development, notably during plant reproduction, leaf senescence and in response to many biotic and abiotic stresses.

Lateral root emergence in Arabidopsis is dependent on transcription factor LBD29 regulation of auxin influx carrier LAX3.

Lateral root primordia (LRP) originate from pericycle stem cells located deep within parental root tissues. LRP emerge through overlying root tissues by inducing auxin-dependent cell separation and hydraulic changes in adjacent cells. The auxin-inducible auxin influx carrier LAX3 plays a key role concentrating this signal in cells overlying LRP. Delimiting LAX3 expression to two adjacent cell files overlying new LRP is crucial to ensure that auxin-regulated cell separation occurs solely along their shared walls. Multiscale modeling has predicted that this highly focused pattern of expression requires auxin to sequentially induce auxin efflux and influx carriers PIN3 and LAX3, respectively. Consistent with model predictions, we report that auxin-inducible LAX3 expression is regulated indirectly by AUXIN RESPONSE FACTOR 7 (ARF7). Yeast one-hybrid screens revealed that the LAX3 promoter is bound by the transcription factor LBD29, which is a direct target for regulation by ARF7. Disrupting auxin-inducible LBD29 expression or expressing an LBD29-SRDX transcriptional repressor phenocopied the lax3 mutant, resulting in delayed lateral root emergence. We conclude that sequential LBD29 and LAX3 induction by auxin is required to coordinate cell separation and organ emergence.

Comparison of plant hormone signalling systems.

Plant growth and development are controlled by nine structurally distinct small molecules termed phytohormones. Over the last 20 years, the molecular basis of their signal transduction, from receptors to transcription factors, has been dissected using mainly Arabidopsis thaliana and rice as model systems. Phytohormones can be broadly classified into two distinct groups on the basis of whether the subcellular localization of their receptors is in the cytoplasm or nucleus, and hence soluble, or membrane-bound, and hence insoluble. Soluble receptors, which control the responses to auxin, jasmonates, gibberellins, strigolactones and salicylic acid, signal either directly or indirectly via the destruction of regulatory proteins. Responses to abscisic acid are primarily mediated by soluble receptors that indirectly regulate the phosphorylation of targeted proteins. Insoluble receptors, which control the responses to cytokinins, brassinosteroids and ethylene, transduce their signal through protein phosphorylation. This chapter provides a comparison of the different components of these signalling systems, and discusses the similarities and differences between them.

A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants.

Activated forms of jasmonic acid (JA) are central signals coordinating plant responses to stresses, yet tools to analyse their spatial and temporal distribution are lacking. Here we describe a JA perception biosensor termed Jas9-VENUS that allows the quantification of dynamic changes in JA distribution in response to stress with high spatiotemporal sensitivity. We show that Jas9-VENUS abundance is dependent on bioactive JA isoforms, the COI1 co-receptor, a functional Jas motif and proteasome activity. We demonstrate the utility of Jas9-VENUS to analyse responses to JA in planta at a cellular scale, both quantitatively and dynamically. This included using Jas9-VENUS to determine the cotyledon-to-root JA signal velocities on wounding, revealing two distinct phases of JA activity in the root. Our results demonstrate the value of developing quantitative sensors such as Jas9-VENUS to provide high-resolution spatiotemporal data about hormone distribution in response to plant abiotic and biotic stresses.

Time-profiling fluorescent reporters in the Arabidopsis root.

Confocal laser scanning microscopy is a useful nondestructive approach for the visualization of fluorescent reporters in planta. Samples are usually placed between a slide and a cover slip which, although suited to single time-point imaging, does not allow long-term observation. Here, we describe a technique to monitor changes in fluorescence in the Arabidopsis root over a long period of time. Treatment can easily be performed, and this approach is suitable for use in low-throughput chemical screens. We also present a rapid method to analyze fluorescence intensity profiles generated using this protocol.

From jellyfish to biosensors: the use of fluorescent proteins in plants.

The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors.

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell-wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three-dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required--later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.