PIF4 enhances the expression of SAUR genes to promote growth in response to nitrate.

Nitrate supply is fundamental to support shoot growth and crop performance, but the associated increase in stem height exacerbates the risks of lodging and yield losses. Despite their significance for agriculture, the mechanisms involved in the promotion of stem growth by nitrate remain poorly understood. Here, we show that the elongation of the hypocotyl of Arabidopsis thaliana, used as a model, responds rapidly and persistently to upshifts in nitrate concentration, rather than to the nitrate level itself. The response occurred even in shoots dissected from their roots and required NITRATE TRANSPORTER 1.1 (NRT1.1) in the phosphorylated state (but not NRT1.1 nitrate transport capacity) and NIN-LIKE PROTEIN 7 (NLP7). Nitrate increased PHYTOCHROME INTERACTING FACTOR 4 (PIF4) nuclear abundance by posttranscriptional mechanisms that depended on NRT1.1 and phytochrome B. In response to nitrate, PIF4 enhanced the expression of numerous SMALL AUXIN-UP RNA (SAUR) genes in the hypocotyl. The growth response to nitrate required PIF4, positive and negative regulators of its activity, including AUXIN RESPONSE FACTORs, and SAURs. PIF4 integrates cues from the soil (nitrate) and aerial (shade) environments adjusting plant stature to facilitate access to light.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide.

The evolutionarily conserved POLYMERASE-ASSOCIATED FACTOR1 complex (Paf1C) participates in transcription, and research in animals and fungi suggests that it facilitates RNA POLYMERASE II (RNAPII) progression through chromatin. We examined the genomic distribution of the EARLY FLOWERING7 (ELF7) and VERNALIZATION INDEPENDENCE3 subunits of Paf1C in Arabidopsis (Arabidopsis thaliana). The occupancy of both subunits was confined to thousands of gene bodies and positively associated with RNAPII occupancy and the level of gene expression, supporting a role as a transcription elongation factor. We found that monoubiquitinated histone H2B, which marks most transcribed genes, was strongly reduced genome wide in elf7 seedlings. Genome-wide profiling of RNAPII revealed that in elf7 mutants, RNAPII occupancy was reduced throughout the gene body and at the transcription end site of Paf1C-targeted genes, suggesting a direct role for the complex in transcription elongation. Overall, our observations suggest a direct functional link between Paf1C activity, monoubiquitination of histone H2B, and the transition of RNAPII to productive elongation. However, for several genes, Paf1C may also act independently of H2Bub deposition or occupy these genes more stable than H2Bub marking, possibly reflecting the dynamic nature of Paf1C association and H2Bub turnover during transcription.

PICLN modulates alternative splicing and light/temperature responses in plants.

Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and transcription start site determination in Arabidopsis.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5'-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5' transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Spatial regulation of plant hormone action.

Although many plant cell types are capable of producing hormones, and plant hormones can in most cases act in the same cells in which they are produced, they also act as signaling molecules that coordinate physiological responses between different parts of the plant, indicating that their action is subject to spatial regulation. Numerous publications have reported that all levels of plant hormonal pathways, namely metabolism, transport, and perception/signal transduction, can help determine the spatial ranges of hormone action. For example, polar auxin transport or localized auxin biosynthesis contribute to creating a differential hormone accumulation across tissues that is instrumental for specific growth and developmental responses. On the other hand, tissue specificity of cytokinin actions has been proposed to be regulated by mechanisms operating at the signaling stages. Here, we review and discuss current knowledge about the contribution of the three levels mentioned above in providing spatial specificity to plant hormone action. We also explore how new technological developments, such as plant hormone sensors based on FRET (fluorescence resonance energy transfer) or single-cell RNA-seq, can provide an unprecedented level of resolution in defining the spatial domains of plant hormone action and its dynamics.

COP1 destabilizes DELLA proteins in Arabidopsis.

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

A genetic approach reveals different modes of action of prefoldins.

The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.

Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors.

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

Prefoldins contribute to maintaining the levels of the spliceosome LSM2-8 complex through Hsp90 in Arabidopsis.

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2-8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2-8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2-8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.

Regulation of xylem fiber differentiation by gibberellins through DELLA-KNAT1 interaction.

The thickening of plant organs is supported by secondary growth, a process by which new vascular tissues (xylem and phloem) are produced. Xylem is composed of several cell types, including xylary fibers, parenchyma and vessel elements. In Arabidopsis, it has been shown that fibers are promoted by the class-I KNOX gene KNAT1 and the plant hormones gibberellins, and are repressed by a small set of receptor-like kinases; however, we lack a mechanistic framework to integrate their relative contributions. Here, we show that DELLAs, negative elements of the gibberellin signaling pathway, physically interact with KNAT1 and impair its binding to KNAT1-binding sites. Our analysis also indicates that at least 37% of the transcriptome mobilized by KNAT1 is potentially dependent on this interaction, and includes genes involved in secondary cell wall modifications and phenylpropanoid biosynthesis. Moreover, the promotion by constitutive overexpression of KNAT1 of fiber formation and the expression of genes required for fiber differentiation were still reverted by DELLA accumulation, in agreement with post-translational regulation of KNAT1 by DELLA proteins. These results suggest that gibberellins enhance fiber development by promoting KNAT1 activity.

Auxin methylation is required for differential growth in Arabidopsis.

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.

Regulation of DELLA Proteins by Post-translational Modifications.

DELLA proteins are the negative regulators of the gibberellin (GA) signaling pathway. GAs have a pervasive effect on plant physiology, influencing processes that span the entire life cycle of the plant. All the information encoded by GAs, either environmental or developmental in origin, is canalized through DELLAs, which modulate the activity of many transcription factors and transcriptional regulators. GAs unlock the signaling pathway by triggering DELLA polyubiquitination and degradation by the 26S proteasome. Recent reports indicate, however, that there are other pathways that trigger DELLA polyubiquitination and degradation independently of GAs. Moreover, results gathered during recent years indicate that other post-translational modifications (PTMs), namely phosphorylation, SUMOylation and glycosylation, modulate DELLA function. The convergence of several PTMs in DELLA therefore highlights the strict regulation to which these proteins are subject. In this review, we summarize these discoveries and discuss DELLA PTMs from an evolutionary perspective and examine the possibilities these and other post-translational regulations offer to improve DELLA-dependent agronomic traits.

Long-Day Photoperiod Enhances Jasmonic Acid-Related Plant Defense.

Agricultural crops are exposed to a range of daylengths, which act as important environmental cues for the control of developmental processes such as flowering. To explore the additional effects of daylength on plant function, we investigated the transcriptome of Arabidopsis (Arabidopsis thaliana) plants grown under short days (SD) and transferred to long days (LD). Compared with that under SD, the LD transcriptome was enriched in genes involved in jasmonic acid-dependent systemic resistance. Many of these genes exhibited impaired expression induction under LD in the phytochrome A (phyA), cryptochrome 1 (cry1), and cry2 triple photoreceptor mutant. Compared with that under SD, LD enhanced plant resistance to the necrotrophic fungus Botrytis cinerea This response was reduced in the phyA cry1 cry2 triple mutant, in the constitutive photomorphogenic1 (cop1) mutant, in the myc2 mutant, and in mutants impaired in DELLA function. Plants grown under SD had an increased nuclear abundance of COP1 and decreased DELLA abundance, the latter of which was dependent on COP1. We conclude that growth under LD enhances plant defense by reducing COP1 activity and enhancing DELLA abundance and MYC2 expression.

Reduction of indole-3-acetic acid methyltransferase activity compensates for high-temperature male sterility in Arabidopsis.

High temperature is a general stress factor that causes a decrease in crop yield. It has been shown that auxin application reduces the male sterility caused by exposure to higher temperatures. However, widespread application of a hormone with vast effects on plant physiology may be discouraged in many cases. Therefore, the generation of new plant varieties that locally enhance auxin in reproductive organs may represent an alternative strategy. We have explored the possibility of increasing indole-3-acetic acid (IAA) in ovaries by reducing IAA methyltransferase1 (IAMT1) activity in Arabidopsis thaliana. The iamt1 mutant showed increased auxin signalling in funiculi, which correlated with a higher growth rate of wild-type pollen in contact with mutant ovaries and premature ovule fertilization. While the production of seeds per fruit was similar in the wild type and the mutant at 20 °C, exposure to 29 °C caused a more severe decrease in fertility in the wild type than in the mutant. Loss of IAMT1 activity was also associated with the production of more nodes after flowering and higher tolerance of the shoot apical meristem to higher temperatures. As a consequence, the productivity of the iamt1 mutant under higher temperatures was more than double of that of the wild type, with almost no apparent trade-off.

SMZ/SNZ and gibberellin signaling are required for nitrate-elicited delay of flowering time in Arabidopsis thaliana.

The reproductive success of plants largely depends on the correct programming of developmental phase transitions, particularly the shift from vegetative to reproductive growth. The timing of this transition is finely regulated by the integration of an array of environmental and endogenous factors. Nitrogen is the mineral macronutrient that plants require in the largest amount, and as such its availability greatly impacts on many aspects of plant growth and development, including flowering time. We found that nitrate signaling interacts with the age-related and gibberellic acid pathways to control flowering time in Arabidopsis thaliana. We revealed that repressors of flowering time belonging to the AP2-type transcription factor family including SCHLAFMUTZE (SMZ) and SCHNARCHZAPFEN (SNZ) are important regulators of flowering time in response to nitrate. Our results support a model whereby nitrate activates SMZ and SNZ via the gibberellin pathway to repress flowering time in Arabidopsis thaliana.

Genome Wide Binding Site Analysis Reveals Transcriptional Coactivation of Cytokinin-Responsive Genes by DELLA Proteins.

The ability of plants to provide a plastic response to environmental cues relies on the connectivity between signaling pathways. DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis.

AUXIN BINDING PROTEIN1 links cell wall remodeling, auxin signaling, and cell expansion in arabidopsis.

Cell expansion is an increase in cell size and thus plays an essential role in plant growth and development. Phytohormones and the primary plant cell wall play major roles in the complex process of cell expansion. In shoot tissues, cell expansion requires the auxin receptor AUXIN BINDING PROTEIN1 (ABP1), but the mechanism by which ABP1 affects expansion remains unknown. We analyzed the effect of functional inactivation of ABP1 on transcriptomic changes in dark-grown hypocotyls and investigated the consequences of gene expression on cell wall composition and cell expansion. Molecular and genetic evidence indicates that ABP1 affects the expression of a broad range of cell wall-related genes, especially cell wall remodeling genes, mainly via an SCF(TIR/AFB)-dependent pathway. ABP1 also functions in the modulation of hemicellulose xyloglucan structure. Furthermore, fucosidase-mediated defucosylation of xyloglucan, but not biosynthesis of nonfucosylated xyloglucan, rescued dark-grown hypocotyl lengthening of ABP1 knockdown seedlings. In muro remodeling of xyloglucan side chains via an ABP1-dependent pathway appears to be of critical importance for temporal and spatial control of cell expansion.

Large-scale identification of gibberellin-related transcription factors defines group VII ETHYLENE RESPONSE FACTORS as functional DELLA partners.

DELLA proteins are the master negative regulators in gibberellin (GA) signaling acting in the nucleus as transcriptional regulators. The current view of DELLA action indicates that their activity relies on the physical interaction with transcription factors (TFs). Therefore, the identification of TFs through which DELLAs regulate GA responses is key to understanding these responses from a mechanistic point of view. Here, we have determined the TF interactome of the Arabidopsis (Arabidopsis thaliana) DELLA protein GIBBERELLIN INSENSITIVE and screened a collection of conditional TF overexpressors in search of those that alter GA sensitivity. As a result, we have found RELATED TO APETALA2.3, an ethylene-induced TF belonging to the group VII ETHYLENE RESPONSE FACTOR of the APETALA2/ethylene responsive element binding protein superfamily, as a DELLA interactor with physiological relevance in the context of apical hook development. The combination of transactivation assays and chromatin immunoprecipitation indicates that the interaction with GIBBERELLIN INSENSITIVE impairs the activity of RELATED TO APETALA2.3 on the target promoters. This mechanism represents a unique node in the cross regulation between the GA and ethylene signaling pathways controlling differential growth during apical hook development.