A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM.

Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation. We demonstrate that NPF2.14 is a subcellular GA/ABA transporter, presumably the first to be identified in plants, facilitating GA and ABA accumulation in the root endodermis to regulate suberization. Further, NPF2.12 and NPF2.13, closely related proteins, are plasma membrane-localized GA and ABA importers that facilitate shoot-to-root GA12 translocation, regulating endodermal hormone accumulation. This work reveals that GA is required for root suberization and that GA and ABA can act non-antagonistically. We demonstrate how the clade of transporters mediates hormone flow with cell-file-specific vacuolar storage at the phloem unloading zone, and slow release of hormone to induce suberin formation in the maturation zone.

COP1 dynamics integrate conflicting seasonal light and thermal cues in the control of Arabidopsis elongation.

As the summer approaches, plants experience enhanced light inputs and warm temperatures, two environmental cues with an opposite morphogenic impact. Key components of this response are PHYTOCHROME B (phyB), EARLY FLOWERING 3 (ELF3), and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). Here, we used single and double mutant/overexpression lines to fit a mathematical model incorporating known interactions of these regulators. The fitted model recapitulates thermal growth of all lines used and correctly predicts thermal behavior of others not used in the fit. While thermal COP1 function is accepted to be independent of diurnal timing, our model shows that it acts at temperature signaling only during daytime. Defective response of cop1-4 mutants is epistatic to phyB-9 and elf3-8, indicating that COP1 activity is essential to transduce phyB and ELF3 thermosensory function. Our thermal model provides a unique toolbox to identify best allelic combinations enhancing climate change resilience of crops adapted to different latitudes.

Nitrate signaling promotes plant growth by upregulating gibberellin biosynthesis and destabilization of DELLA proteins.

Nitrate, one of the main nitrogen (N) sources for crops, acts as a nutrient and key signaling molecule coordinating gene expression, metabolism, and various growth processes throughout the plant life cycle. It is widely accepted that nitrate-triggered developmental programs cooperate with hormone synthesis and transport to finely adapt plant architecture to N availability. Here, we report that nitrate, acting through its signaling pathway, promotes growth in Arabidopsis and wheat, in part by modulating the accumulation of gibberellin (GA)-regulated DELLA growth repressors. We show that nitrate reduces the abundance of DELLAs by increasing GA contents through activation of GA metabolism gene expression. Consistently, the growth restraint conferred by nitrate deficiency is partially rescued in global-DELLA mutant that lacks all DELLAs. At the cellular level, we show that nitrate enhances both cell proliferation and elongation in a DELLA-dependent and -independent manner, respectively. Our findings establish a connection between nitrate and GA signaling pathways that allow plants to adapt their growth to nitrate availability.

Outgrowth of the axillary bud in rose is controlled by sugar metabolism and signalling.

Shoot branching is a pivotal process during plant growth and development, and is antagonistically orchestrated by auxin and sugars. In contrast to extensive investigations on hormonal regulatory networks, our current knowledge on the role of sugar signalling pathways in bud outgrowth is scarce. Based on a comprehensive stepwise strategy, we investigated the role of glycolysis/the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (OPPP) in the control of bud outgrowth. We demonstrated that these pathways are necessary for bud outgrowth promotion upon plant decapitation and in response to sugar availability. They are also targets of the antagonistic crosstalk between auxin and sugar availability. The two pathways act synergistically to down-regulate the expression of BRC1, a conserved inhibitor of shoot branching. Using Rosa calluses stably transformed with GFP-fused promoter sequences of RhBRC1 (pRhBRC1), glycolysis/TCA cycle and the OPPP were found to repress the transcriptional activity of pRhBRC1 cooperatively. Glycolysis/TCA cycle- and OPPP-dependent regulations involve the -1973/-1611 bp and -1206/-709 bp regions of pRhBRC1, respectively. Our findings indicate that glycolysis/TCA cycle and the OPPP are integrative parts of shoot branching control and can link endogenous factors to the developmental programme of bud outgrowth, likely through two distinct mechanisms.

Inhibition of Arabidopsis thaliana CIN-like TCP transcription factors by Agrobacterium T-DNA-encoded 6B proteins.

Agrobacterium T-DNA-encoded 6B proteins cause remarkable growth effects in plants. Nicotiana otophora carries two cellular T-DNAs with three slightly divergent 6b genes (TE-1-6b-L, TE-1-6b-R and TE-2-6b) originating from a natural transformation event. In Arabidopsis thaliana, expression of 2×35S:TE-2-6b, but not 2×35S:TE-1-6b-L or 2×35S:TE-1-6b-R, led to plants with crinkly leaves, which strongly resembled mutants of the miR319a/TCP module. This module is composed of MIR319A and five CIN-like TCP (TEOSINTHE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR) genes (TCP2, TCP3, TCP4, TCP10 and TCP24) targeted by miR319a. The CIN-like TCP genes encode transcription factors and are required for cell division arrest at leaf margins during development. MIR319A overexpression causes excessive growth and crinkly leaves. TE-2-6b plants did not show increased miR319a levels, but the mRNA levels of the TCP4 target gene LOX2 were decreased, as in jaw-D plants. Co-expression of green fluorescent protein (GFP)-tagged TCPs with native or red fluorescent protein (RFP)-tagged TE-6B proteins led to an increase in TCP protein levels and formation of numerous cytoplasmic dots containing 6B and TCP proteins. Yeast double-hybrid experiments confirmed 6B/TCP binding and showed that TE-1-6B-L and TE-1-6B-R bind a smaller set of TCP proteins than TE-2-6B. A single nucleotide mutation in TE-1-6B-R enlarged its TCP-binding repertoire to that of TE-2-6B and caused a crinkly phenotype in Arabidopsis. Deletion analysis showed that TE-2-6B targets the TCP4 DNA-binding domain and directly interferes with transcriptional activation. Taken together, these results provide detailed insights into the mechanism of action of the N. otophora TE-encoded 6b genes.

Root-derived GA12 contributes to temperature-induced shoot growth in Arabidopsis.

Plants are able to sense a rise in temperature of several degrees, and appropriately adapt their metabolic and growth processes. To this end, plants produce various signalling molecules that act throughout the plant body. Here, we report that root-derived GA12, a precursor of the bioactive gibberellins, mediates thermo-responsive shoot growth in Arabidopsis. Our data suggest that root-to-shoot translocation of GA12 enables a flexible growth response to ambient temperature changes.

BRANCHED1: A Key Hub of Shoot Branching.

Shoot branching is a key process for plant growth and fitness. Newly produced axes result from axillary bud outgrowth, which is at least partly mediated through the regulation of BRANCHED1 gene expression (BRC1/TB1/FC1). BRC1 encodes a pivotal bud-outgrowth-inhibiting transcription factor belonging to the TCP family. As the regulation of BRC1 expression is a hub for many shoot-branching-related mechanisms, it is influenced by endogenous (phytohormones and nutrients) and exogenous (light) inputs, which involve so-far only partly identified molecular networks. This review highlights the central role of BRC1 in shoot branching and its responsiveness to different stimuli, and emphasizes the different knowledge gaps that should be addressed in the near future.

INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana.

INDETERMINATE DOMAIN (IDD)/ BIRD proteins are a highly conserved plant-specific family of transcription factors which play multiple roles in plant development and physiology. Here, we show that mutation in IDD4/IMPERIAL EAGLE increases resistance to the hemi-biotrophic pathogen Pseudomonas syringae, indicating that IDD4 may act as a repressor of basal immune response and PAMP-triggered immunity. Furthermore, the idd4 mutant exhibits enhanced plant-growth indicating IDD4 as suppressor of growth and development. Transcriptome comparison of idd4 mutants and IDD4ox lines aligned to genome-wide IDD4 DNA-binding studies revealed major target genes related to defense and developmental-biological processes. IDD4 is a phospho-protein that interacts and becomes phosphorylated on two conserved sites by the MAP kinase MPK6. DNA-binding studies of IDD4 after flg22 treatment and with IDD4 phosphosite mutants show enhanced binding affinity to ID1 motif-containing promoters and its function as a transcriptional regulator. In contrast to the IDD4-phospho-dead mutant, the IDD4 phospho-mimicking mutant shows altered susceptibility to PstDC3000, salicylic acid levels and transcriptome reprogramming. In summary, we found that IDD4 regulates various hormonal pathways thereby coordinating growth and development with basal immunity.

Tissue-Specific Regulation of Gibberellin Signaling Fine-Tunes Arabidopsis Iron-Deficiency Responses.

Iron is an essential element for most living organisms. Plants acquire iron from the rhizosphere and have evolved different biochemical and developmental responses to adapt to a low-iron environment. In Arabidopsis, FIT encodes a basic helix-loop-helix transcription factor that activates the expression of iron-uptake genes in root epidermis upon iron deficiency. Here, we report that the gibberellin (GA)-signaling DELLA repressors contribute substantially in the adaptive responses to iron-deficient conditions. When iron availability decreases, DELLAs accumulate in the root meristem, thereby restraining root growth, while being progressively excluded from epidermal cells in the root differentiation zone. Such DELLA exclusion from the site of iron acquisition relieves FIT from DELLA-dependent inhibition and therefore promotes iron uptake. Consistent with this mechanism, expression of a non-GA-degradable DELLA mutant protein in root epidermis interferes with iron acquisition. Hence, spatial distribution of DELLAs in roots is essential to fine-tune the adaptive responses to iron availability.

A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals.

Plant phenotypic plasticity is controlled by diverse hormone pathways, which integrate and convey information from multiple developmental and environmental signals. Moreover, in plants many processes such as growth, development, and defense are regulated in similar ways by multiple hormones. Among them, gibberellins (GAs) are phytohormones with pleiotropic actions, regulating various growth processes throughout the plant life cycle. Previous work has revealed extensive interplay between GAs and other hormones, but the molecular mechanism became apparent only recently. Molecular and physiological studies have demonstrated that DELLA proteins, considered as master negative regulators of GA signaling, integrate multiple hormone signaling pathways through physical interactions with transcription factors or regulatory proteins from different families. In this review, we summarize the latest progress in GA signaling and its direct crosstalk with the main phytohormone signaling, emphasizing the multifaceted role of DELLA proteins with key components of major hormone signaling pathways.

Long-distance transport of endogenous gibberellins in Arabidopsis.

Gibberellins (GAs) are phytohormones controlling major aspects of plant growth and development. Although previous studies suggested the existence of a transport of GAs in plants, the nature and properties associated with this transport were unknown. We recently showed through micrografting and biochemical approaches that the GA12 precursor is the chemical form of GA undergoing long-distance transport across plant organs in Arabidopsis. Endogenous GA12 moves through the plant vascular system from production sites to recipient tissues, in which GA12 can be converted to bioactive forms to support growth via the activation of GA-dependent processes. GAs are also essential to promote seed germination; hence GA biosynthesis mutants do not germinate without exogenous GA treatment. Our results suggest that endogenous GAs are not (or not sufficiently) transmitted to the offspring to successfully complete the germination under permissive conditions.

ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex.

The circadian clock plays a pivotal role in the control of Arabidopsis hypocotyl elongation by regulating rhythmic expression of the bHLH factors PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and 5). Coincidence of increased PIF4/PIF5 transcript levels with the dark period allows nuclear accumulation of these factors, and in short days it phases maximal hypocotyl growth at dawn. During early night, PIF4 and PIF5 transcription is repressed by the Evening Complex (EC) proteins EARLY FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRHYTHMO (LUX). While ELF3 has an essential role in EC complex assembly, several lines of evidence indicate that this protein controls plant growth via other mechanisms that are presently unknown. Here, we show that the ELF3 and PIF4 proteins interact in an EC-independent manner, and that this interaction prevents PIF4 from activating its transcriptional targets. We also show that PIF4 overexpression leads to ELF3 protein destabilization, and that this effect is mediated indirectly by negative feedback regulation of photoactive PHYTOCHROME B (phyB). Physical interaction of the phyB photoreceptor with ELF3 has been reported, but its functional relevance remains poorly understood. Our findings establish that phyB is needed for ELF3 accumulation in the light, most likely by competing for CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-mediated ubiquitination and the proteasomal degradation of ELF3. Our results explain the short hypocotyl phenotype of ELF3 overexpressors, despite their normal clock function, and provide a molecular framework for understanding how warm temperatures promote hypocotyl elongation and affect the endogenous clock.

The gibberellin precursor GA12 acts as a long-distance growth signal in Arabidopsis.

The gibberellin (GA) phytohormones play important roles in plant growth and development, promoting seed germination, elongation growth and reproductive development(1). Over the years, substantial progress has been made in understanding the regulation of GA signalling and metabolism, which ensures appropriate levels of GAs for growth and development(2). Moreover, an additional level of regulation may reside in the transport of GAs from production sites to recipient tissues that require GAs for growth. Although there is considerable evidence suggesting the existence of short- and long-distance movement of GAs in plants(3-8), the nature and the biological properties of this transport are not yet understood. Here, we combine biochemical and conventional micrografting experiments in Arabidopsis thaliana to show that the GA precursor GA12, although biologically inactive by itself, is the major mobile GA signal over long distances. Quantitative analysis of endogenous GAs in xylem and phloem exudates further indicates that GA12 moves through the plant vascular system. Finally, we demonstrate that GA12 is functional in recipient tissues, supporting growth via the activation of the GA signalling cascade. Collectively, these results reveal the existence of long-range transport of endogenous GA12 in plants that may have implications for the control of developmental phase transitions and the adaptation to adverse environments.

The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development.

Ent-kaurenoic acid oxidase (KAO), a class of cytochrome P450 monooxygenases of the subfamily CYP88A, catalyzes the conversion of ent-kaurenoic acid (KA) to gibberellin (GA) GA12 , the precursor of all GAs, thereby playing an important role in determining GA concentration in plants. Past work has demonstrated the importance of KAO activity for growth in various plant species. In Arabidopsis, this enzyme is encoded by two genes designated KAO1 and KAO2. In this study, we used various approaches to determine the physiological roles of KAO1 and KAO2 throughout plant development. Analysis of gene expression pattern reveals that both genes are mainly expressed in germinating seeds and young developing organs, thus suggesting functional redundancy. Consistent with this, kao1 and kao2 single mutants are indistinguishable from wild-type plants. By contrast, the kao1 kao2 double mutant exhibits typical non-germinating GA-dwarf phenotypes, similar to those observed in the severely GA-deficient ga1-3 mutant. Phenotypic characterization and quantitative analysis of endogenous GA contents of single and double kao mutants further confirm an overlapping role of KAO1 and KAO2 throughout Arabidopsis development.

Class I TCP-DELLA interactions in inflorescence shoot apex determine plant height.

Regulation of plant height, one of the most important agronomic traits, is the focus of intensive research for improving crop performance. Stem elongation takes place as a result of repeated cell divisions and subsequent elongation of cells produced by apical and intercalary meristems. The gibberellin (GA) phytohormones have long been known to control stem and internodal elongation by stimulating the degradation of nuclear growth-repressing DELLA proteins; however, the mechanism allowing GA-responsive growth is only slowly emerging. Here, we show that DELLAs directly regulate the activity of the plant-specific class I TCP transcription factor family, key regulators of cell proliferation. Our results demonstrate that class I TCP factors directly bind the promoters of core cell-cycle genes in Arabidopsis inflorescence shoot apices while DELLAs block TCP function by binding to their DNA-recognition domain. GAs antagonize such repression by promoting DELLA destruction and therefore cause a concomitant accumulation of TCP factors on promoters of cell-cycle genes. Consistent with this model, the quadruple mutant tcp8 tcp14 tcp15 tcp22 exhibits severe dwarfism and reduced responsiveness to GA action. Altogether, we conclude that GA-regulated DELLA-TCP interactions in inflorescence shoot apex provide a novel mechanism to control plant height.

BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.

Signaling by the hormones brassinosteroid (BR) and gibberellin (GA) is critical to normal plant growth and development and is required for hypocotyl elongation in response to dark and elevated temperatures. Active BR signaling is essential for GA promotion of hypocotyl growth and suppresses the dwarf phenotype of GA mutants. Cross-talk between these hormones occurs downstream from the DELLAs, as GA-induced destabilization of these GA signaling repressors is not affected by BRs. Here we show that the light-regulated PIF4 (phytochrome-interacting factor 4) factor is a phosphorylation target of the BR signaling kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which marks this transcriptional regulator for proteasome degradation. Expression of a mutated PIF41A protein lacking a conserved BIN2 phosphorylation consensus causes a severe elongated phenotype and strongly up-regulated expression of the gene targets. However, PIF41A is not able to suppress the dwarf phenotype of the bin2-1 mutant with constitutive activation of this kinase. PIFs were shown to be required for the constitutive BR response of bes1-D and bzr1-1D mutants, these factors acting in an interdependent manner to promote cell elongation. Here, we show that bes1-D seedlings are still repressed by the inhibitor BRZ in the light and that expression of the nonphosphorylatable PIF41A protein makes this mutant fully insensitive to brassinazole (BRZ). PIF41A is preferentially stabilized at dawn, coinciding with the diurnal time of maximal growth. These results uncover a main role of BRs in antagonizing light signaling by inhibiting BIN2-mediated destabilization of the PIF4 factor. This regulation plays a prevalent role in timing hypocotyl elongation to late night, before light activation of phytochrome B (PHYB) and accumulation of DELLAs restricts PIF4 transcriptional activity.