Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Stress-induced F-Box protein-coding gene OsFBX257 modulates drought stress adaptations and ABA responses in rice.

F-box (FB) proteins that form part of SKP1-CUL1-F-box (SCF) type of E3 ubiquitin ligases are important components of plant growth and development. Here we characterized OsFBX257, a rice FB protein-coding gene that is differentially expressed under drought conditions and other abiotic stresses. Population genomics analysis suggest that OsFBX257 shows high allelic diversity in aus accessions and has been under positive selection in some japonica, aromatic and indica cultivars. Interestingly, allelic variation at OsFBX257 in aus cultivar Nagina22 is associated with an alternatively spliced transcript. Conserved among land plants, OsFBX257 is a component of the SCF complex, can form homomers and interact molecularly with the 14-3-3 rice proteins GF14b and GF14c. OsFBX257 is co-expressed in a network involving protein kinases and phosphatases. We show that OsFBX257 can bind the kinases OsCDPK1 and OsSAPK2, and that its phosphorylation can be reversed by phosphatase OsPP2C08. OsFBX257 expression level modulates root architecture and drought stress tolerance in rice. OsFBX257 knockdown (OsFBX257KD ) lines show reduced total root length and depth, crown root number, panicle size and survival under stress. In contrast, its overexpression (OsFBX257OE ) increases root depth, leaf and grain length, number of panicles, and grain yield in rice. OsFBX257 is a promising breeding target for alleviating drought stress-induced damage in rice.

New fluorescent auxin probes visualise tissue-specific and subcellular distributions of auxin in Arabidopsis.

In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non-invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state-of-the-art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography-mass spectrometry (LC-MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin-related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.

Conservation Agriculture Affects Grain and Nutrient Yields of Maize (Zea Mays L.) and Can Impact Food and Nutrition Security in Sub-Saharan Africa.

Maize is a major staple and plays an essential role in food and nutrition security in Sub-Saharan Africa (SSA). Conservation agriculture (CA), a climate-smart agriculture practise based on minimum soil disturbance, crop residue retention, and crop diversification, has been widely advocated but without extensive research on the impact it may have on maize nutrient composition, and food and nutrition security. This study assessed the grain yield, macro- and micronutrient mineral content, and nutrient yield of eight maize varieties grown in Malawi, and how these are affected by CA practises over two seasons. The minerals were analysed by inductively coupled plasma (ICP) coupled to optical emission spectroscopy (OES) and to mass spectroscopy (MS). Grain yield and Se content differed among the varieties, while C, N, Fe, K, Mg, Mn, P, and Zn were similar. The local variety Kanjerenjere showed lowest grain and nutrient yields. The open-pollinated varieties (OPVs) concentrated more minerals than the F1 hybrids, but the latter showed higher yields for both grain and nutrients. Typical consumption of the eight maize varieties could fully meet the protein and Mg dietary reference intake (DRIs) of Malawian children (1-3 years), as well as Mg and Mn needs of adult women (19-50 years), but their contribution to dietary requirements was low for Fe (39-41%) and K (13-21%). The trials showed that CA increased grain yield (1.2- to 1.8-fold) and Se content (1.1- to 1.7-fold), but that it had no effect on C, K, Mg, P, and Zn, and that N (1.1- to 1.2-fold), Mn (1.1- to 1.8-fold), and Fe (1.3- to 3.4-fold) were reduced. The high increase in grain yield under CA treatments resulted in increased yields of protein and Se, no effect on the yields of K, Mg, Mn, P, Zn, and reduced Fe yield. Conservation agriculture could contribute in reducing the risk of Se deficiency in Malawian women and children but exacerbates the risk of Fe deficiency. A combination of strategies will be needed to mitigate some of the foreseen effects of climate change on agriculture, and food and nutrition security, and improve nutrient intake.

Selective auxin agonists induce specific AUX/IAA protein degradation to modulate plant development.

Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.

The Tetrazole Analogue of the Auxin Indole-3-acetic Acid Binds Preferentially to TIR1 and Not AFB5.

Indole-3-acetic acid (auxin) is considered one of the cardinal hormones in plant growth and development. It regulates a wide range of processes throughout the plant. Synthetic auxins exploit the auxin-signaling pathway and are valuable as herbicidal agrochemicals. Currently, despite a diversity of chemical scaffolds all synthetic auxins have a carboxylic acid as the active core group. By applying bio-isosteric replacement we discovered that indole-3-tetrazole was active by surface plasmon resonance spectrometry, showing that the tetrazole could initiate assembly of the Transport Inhibitor Resistant 1 (TIR1) auxin coreceptor complex. We then tested the tetrazole's efficacy in a range of whole plant physiological assays and in protoplast reporter assays, which all confirmed auxin activity, albeit rather weak. We then tested indole-3-tetrazole against the AFB5 homologue of TIR1, finding that binding was selective against TIR1, absent with AFB5. The kinetics of binding to TIR1 are contrasted to those for the herbicide picloram, which shows the opposite receptor preference, as it binds to AFB5 with far greater affinity than to TIR1. The basis of the preference of indole-3-tetrazole for TIR1 was revealed to be a single residue substitution using molecular docking, and assays using tir1 and afb5 mutant lines confirmed selectivity in vivo. Given the potential that a TIR1-selective auxin might have for unmasking receptor-specific actions, we followed a rational design, lead optimization campaign, and a set of chlorinated indole-3-tetrazoles was synthesized. Improved affinity for TIR1 and the preference for binding to TIR1 was maintained for 4- and 6-chloroindole-3-tetrazoles, coupled with improved efficacy in vivo. This work expands the range of auxin chemistry for the design of receptor-selective synthetic auxins.

The Arabidopsis JAGGED LATERAL ORGANS (JLO) gene sensitizes plants to auxin.

Plant growth and development of new organs depend on the continuous activity of the meristems. In the shoot, patterns of organ initiation are determined by PINFORMED (PIN)-dependent auxin distribution, while the undifferentiated state of meristem cells requires activity of KNOTTED LIKE HOMEOBOX (KNOX) transcription factors. Cell proliferation and differentiation of the root meristem are regulated by the largely antagonistic functions of auxin and cytokinins. It has previously been shown that the transcription factor JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) family, coordinates KNOX and PIN expression in the shoot and promotes root meristem growth. Here we show that JLO is required for the establishment of the root stem cell niche, where it interacts with the auxin/PLETHORA pathway. Auxin signaling involves the AUX/IAA co-repressor proteins, ARF transcription factors and F-box receptors of the TIR1/AFB1-5 family. Because jlo mutants fail to degrade the AUX/IAA protein BODENLOS, root meristem development is inhibited. We also demonstrate that the expression levels of two auxin receptors, TIR1 and AFB1, are controlled by JLO dosage, and that the shoot and root defects of jlo mutants are alleviated in jlo plants expressing TIR1 and AFB1 from a transgene. The finding that the auxin sensitivity of a plant can be differentially regulated through control of auxin receptor expression can explain how different developmental processes can be integrated by the activity of a key transcription factor.

The developmental and environmental regulation of gravitropic setpoint angle in Arabidopsis and bean.

Root and shoot branches are major determinants of plant form and critical for the effective capture of resources below and above ground. These branches are often maintained at specific angles with respect to gravity, known as gravitropic set point angles (GSAs). We have previously shown that the mechanism permitting the maintenance of non-vertical GSAs is highly auxin-dependent and here we investigate the developmental and environmental regulation of root and shoot branch GSA. We show that nitrogen and phosphorous deficiency have opposing, auxin signalling-dependent effects on lateral root GSA in Arabidopsis: while low nitrate induces less vertical lateral root GSA, phosphate deficiency results in a more vertical lateral root growth angle, a finding that contrasts with the previously reported growth angle response of bean adventitious roots. We find that this root-class-specific discrepancy in GSA response to low phosphorus is mirrored by similar differences in growth angle response to auxin treatment between these root types. Finally we show that both shaded, low red/far-red light conditions and high temperature induce more vertical growth in Arabidopsis shoot branches. We discuss the significance of these findings in the context of efforts to improve crop performance via the manipulation of root and shoot branch growth angle.

HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1.

Recent studies have revealed that a mild increase in environmental temperature stimulates the growth of Arabidopsis seedlings by promoting biosynthesis of the plant hormone auxin. However, little is known about the role of other factors in this process. In this report, we show that increased temperature promotes rapid accumulation of the TIR1 auxin co-receptor, an effect that is dependent on the molecular chaperone HSP90. In addition, we show that HSP90 and the co-chaperone SGT1 each interact with TIR1, confirming that TIR1 is an HSP90 client. Inhibition of HSP90 activity results in degradation of TIR1 and interestingly, defects in a range of auxin-mediated growth processes at lower as well as higher temperatures. Our results indicate that HSP90 and SGT1 integrate temperature and auxin signalling in order to regulate plant growth in a changing environment.

Auxin controls gravitropic setpoint angle in higher plant lateral branches.

Lateral branches in higher plants are often maintained at specific angles with respect to gravity, a quantity known as the gravitropic setpoint angle (GSA) [1]. Despite the importance of GSA control as a fundamental determinant of plant form, the mechanisms underlying gravity-dependent angled growth are not known. Here we address the central questions of how stable isotropic growth of a branch at a nonvertical angle is maintained and of how the value of that angle is set. We show that nonvertical lateral root and shoot branches are distinguished from the primary axis by the existence of an auxin-dependent antigravitropic offset mechanism that operates in tension with gravitropic response to generate angled isotropic growth. Further, we show that the GSA of lateral roots and shoots is dependent upon the magnitude of the antigravitropic offset component. Finally, we show that auxin specifies GSA values dynamically throughout development by regulating the magnitude of the antigravitropic offset component via TIR1/AFB-Aux/IAA-ARF-dependent auxin signaling within the gravity-sensing cells of the root and shoot. The involvement of auxin in controlling GSA is yet another example of auxin's remarkable capacity to self-organize in development [2] and provides a conceptual framework for understanding the specification of GSA throughout nature.

In vitro propagation of cauliflower using curd microexplants.

Cauliflower (Brassica oleracea var. botrytis) with its distinctive pre-inflorescence or curd is a remarkable member of the Brassica cabbage group. During curd development, intense and repetitive branching leads to a spectacular increase in size and the accumulation of millions of meristems at its surface. Although destined to produce flowers, most of these meristems are capable of regenerating vegetative shoots in vitro, making curd fragments an excellent material for the micropropagation of cauliflower. Most reported methods using these tissues were devised for the production of small clones of vitroplants as the true potential of curd fragments remained highly underestimated. We describe a technique exploiting fully this abundance of meristems and optimized for the large-scale in vitro propagation of cauliflower. The curd surface is first mechanically disrupted to break up the meristem clusters and generate microexplants carrying 1-3 meristems. These microexplants are then cultured at high density 1:100 (v:v) (microexplants:medium) in liquid medium containing Kinetin and indole-3-butyric acid (IBA) and produce thousands of microshoots in 12 days. After selecting the best quality microshoots on a sucrose pad, they are transferred en masse to a rooting medium supplemented with IBA. Four weeks later, rooted microshoots are carefully acclimatized before transfer to the field. This semi-automated protocol is rapid, cost effective, and well adapted for the production of clones of several thousands of plants by a single worker in a short space of time.

The Arabidopsis O-linked N-acetylglucosamine transferase SPINDLY interacts with class I TCPs to facilitate cytokinin responses in leaves and flowers.

O-linked N-acetylglucosamine (O-GlcNAc) modifications regulate the posttranslational fate of target proteins. The Arabidopsis thaliana O-GlcNAc transferase (OGT) SPINDLY (SPY) suppresses gibberellin signaling and promotes cytokinin (CK) responses by unknown mechanisms. Here, we present evidence that two closely related class I TCP transcription factors, TCP14 and TCP15, act with SPY to promote CK responses. TCP14 and TCP15 interacted with SPY in yeast two-hybrid and in vitro pull-down assays and were O-GlcNAc modified in Escherichia coli by the Arabidopsis OGT, SECRET AGENT. Overexpression of TCP14 severely affected plant development in a SPY-dependent manner and stimulated typical CK morphological responses, as well as the expression of the CK-regulated gene RESPONSE REGULATOR5. TCP14 also promoted the transcriptional activity of the CK-induced mitotic factor CYCLIN B1;2. Whereas TCP14-overexpressing plants were hypersensitive to CK, spy and tcp14 tcp15 double mutant leaves and flowers were hyposensitive to the hormone. Reducing CK levels by overexpressing CK OXIDASE/DEHYDROGENASE3 suppressed the TCP14 overexpression phenotypes, and this suppression was reversed when the plants were treated with exogenous CK. Taken together, we suggest that responses of leaves and flowers to CK are mediated by SPY-dependent TCP14 and TCP15 activities.

TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis.

TCP transcription factors constitute a small family of plant-specific bHLH-containing, DNA-binding proteins that have been implicated in the control of cell proliferation in plants. Despite the significant role that is likely to be played by genes that control cell division in the elaboration of plant architecture, functional analysis of this family by forward and reverse genetics has been hampered by genetic redundancy. Here we show that mutants in two related class I TCP genes display a range of growth-related phenotypes, consistent with their dynamic expression patterns; these phenotypes are enhanced in the double mutant. Together, the two genes influence plant stature by promoting cell division in young internodes. Reporter gene analysis and use of SRDX fusions suggested that TCP14 and TCP15 modulate cell proliferation in the developing leaf blade and specific floral tissues; a role that was not apparent in our phenotypic analysis of single or double mutants. However, when the relevant mutants were subjected to computer-aided morphological analysis of the leaves, the consequences of loss of either or both genes became obvious. The effects on cell proliferation of perturbing the function of TCP14 and TCP15 vary with tissue, as has been suggested for other TCP factors. These findings indicate that the precise elaboration of plant form is dependent on the cumulative influence of many TCP factors acting in a context-dependent fashion. The study highlights the need for advanced methods of phenotypic analysis in order to characterize phenotypes and to construct a dynamic model for TCP gene function.

Defining auxin response contexts in plant development.

The apparent domination of so much of plant development by the hormone auxin raises important questions about how this simple, generic signalling molecule can give rise to such an enormous range of very specific developmental outputs. What is becoming increasingly clear is that alongside the tight control of auxin distribution within the plant, the cellular and developmental context in which the auxin signal is received is of utmost importance. Recent work has highlighted that these distinct auxin response contexts are defined by complex and inter-dependent relationships between auxin metabolism, transport and response that can be modulated at many levels by inputs from both endogenous and environmental signals.

UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in Arabidopsis.

Aberrant mRNAs containing premature termination codons (PTCs) have the potential to be translated into truncated proteins, which could act to the detriment of the organism by interfering with normal cellular processes. Eukaryotes have mechanisms of mRNA quality control that identify PTC-containing transcripts and target them for destruction, a process known as nonsense-mediated mRNA decay (NMD). Surprising differences have been reported in the mechanisms of NMD between different organisms. UPF1 and UPF3 are structurally unrelated proteins, which function in the NMD pathway in yeast, mammals, Drosophila and Caenorhabditis elegans. Here we show that NMD in plants requires UPF1, as mRNAs containing PTCs become stabilized in upf1-5 mutants. However, in contrast to NMD in mammals, UPF1-dependent NMD is capable of targeting both spliced and unspliced PTC-containing mRNAs. An allelic series of upf1 mutants exhibits a range of unexpected vegetative and floral abnormalities, including jagged leaves, late flowering, fused flowers and seedling lethality. We also show that mutants in UPF3 share these abnormalities. As both UPF1 and UPF3 are required for NMD, the similar phenotypes of the upf1 and upf3 mutants suggest that NMD regulates a common set of genes required for plant development and survival. Finally, gene silencing by an inverted repeat transgene is impaired in upf1-5 mutants, indicating a connection between UPF1 and RNA interference in plants.

Analysis of the transcription factor WUSCHEL and its functional homologue in Antirrhinum reveals a potential mechanism for their roles in meristem maintenance.

One of the most significant features of plant development is the way in which it can be elaborated and modulated throughout the life of the plant, an ability that is conferred by meristems. The Arabidopsis thaliana WUSCHEL gene (WUS), which encodes a homeodomain transcription factor, is required to maintain the stem cells in the shoot apical meristem in an undifferentiated state. The mechanism by which WUS prevents the differentiation of stem cells is unknown. We have characterized a meristem maintenance mutant in Antirrhinum majus and shown that it arises from a defect in the WUS orthologue ROSULATA (ROA). Detailed characterization of a semidominant roa allele revealed an essential role for the conserved C-terminal domain. Expression of either ROA or WUS lacking this domain causes a failure of meristem maintenance. The conserved domain mediates an interaction between WUS and two members of a small family of corepressor-like proteins in Arabidopsis. Our results suggest that WUS functions by recruiting transcriptional corepressors to repress target genes that promote differentiation, thereby ensuring stem cell maintenance.

Comprehensive interaction map of the Arabidopsis MADS Box transcription factors.

Interactions between proteins are essential for their functioning and the biological processes they control. The elucidation of interaction maps based on yeast studies is a first step toward the understanding of molecular networks and provides a framework of proteins that possess the capacity and specificity to interact. Here, we present a comprehensive plant protein-protein interactome map of nearly all members of the Arabidopsis thaliana MADS box transcription factor family. A matrix-based yeast two-hybrid screen of >100 members of this family revealed a collection of specific heterodimers and a few homodimers. Clustering of proteins with similar interaction patterns pinpoints proteins involved in the same developmental program and provides valuable information about the participation of uncharacterized proteins in these programs. Furthermore, a model is proposed that integrates the floral induction and floral organ formation networks based on the interactions between the proteins involved. Heterodimers between flower induction and floral organ identity proteins were observed, which point to (auto)regulatory mechanisms that prevent the activity of flower induction proteins in the flower.

Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world.

MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, Malpha, Mbeta, Mgamma, and Mdelta) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.