MIZ1 regulates ECA1 to generate a slow, long-distance phloem-transmitted Ca2+ signal essential for root water tracking in Arabidopsis.

Ever since Darwin postulated that the tip of the root is sensitive to moisture differences and that it "transmits an influence to the upper adjoining part, which bends towards the source of moisture" [Darwin C, Darwin F (1880) The Power of Movement in Plants, pp 572-574], the signal underlying this tropic response has remained elusive. Using the FRET-based Cameleon Ca2+ sensor in planta, we show that a water potential gradient applied across the root tip generates a slow, long-distance asymmetric cytosolic Ca2+ signal in the phloem, which peaks at the elongation zone, where it is dispersed laterally and asymmetrically to peripheral cells, where cell elongation occurs. In addition, the MIZ1 protein, whose biochemical function is unknown but is required for root curvature toward water, is indispensable for generating the slow, long-distance Ca2+ signal. Furthermore, biochemical and genetic manipulations that elevate cytosolic Ca2+ levels, including mutants of the endoplasmic reticulum (ER) Ca2+-ATPase isoform ECA1, enhance root curvature toward water. Finally, coimmunoprecipitation of plant proteins and functional complementation assays in yeast cells revealed that MIZ1 directly binds to ECA1 and inhibits its activity. We suggest that the inhibition of ECA1 by MIZ1 changes the balance between cytosolic Ca2+ influx and efflux and generates the cytosolic Ca2+ signal required for water tracking.

CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6: A Key Regulator of Na+ Homeostasis during Germination.

Salinity impairs seed germination and seedling establishment. We investigated the role of Arabidopsis (Arabidopsis thaliana) CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6 (CAMTA6) in salinity stress responses during early germination. Compared with the wild type, the camta6-4 and camta6-5 mutants were more tolerant to NaCl and abscisic acid (ABA) and accumulated less Na+ In contrast, 4- to 11-d-old camta6 seedlings were more sensitive to NaCl. In camta6, expression of HIGH-AFFINITY K+ TRANSPORTER1 (AtHKT1;1), encoding an Na+/K+ transporter, was restricted to the radicles and was not enhanced by NaCl or ABA. During germination, the camta6 hkt1 double mutant was as sensitive as the wild type and hkt1 to NaCl, suggesting that HKT1;1 is crucial for the salt tolerance of camta6 An ABA response element in the HKT1;1 promoter was found to be indispensable for the enhanced expression of the gene in response to NaCl and to ABA. Transcriptome analysis of the wild type and camta6-5 with and without salt treatment revealed 1,020 up-regulated and 1,467 down-regulated salt-responsive genes in the wild type. Among these, 638 up-regulated and 1,242 down-regulated genes were classified as CAMTA6-dependent. Expression of several known salt stress-associated genes, including SALT OVERLY SENSITIVE1 and Na+/H+ ANTIPORTER, was impaired in camta6 mutants. Bioinformatics analysis of the 5' upstream sequences of the salt-responsive CAMTA6-dependent up-regulated genes revealed the CACGTGTC motif as the most prominent element, representing an ABA response element and a potential CAMTA-binding site. We suggest that CAMTA6 regulates, directly or indirectly, the expression of most of the salt-responsive genes in germinating seeds, including genes that are crucial for Na+ homeostasis and salt stress tolerance.

GABA signaling in plants: targeting the missing pieces of the puzzle.

The adaptation of plants to unstable environments relies on their ability to sense their surroundings and to generate and transmit corresponding signals to different parts of the plant to evoke changes necessary for optimizing growth and defense. Plants, like animals, contain a huge repertoire of intra- and intercellular signals, including organic and inorganic molecules. The occurrence of neurotransmitter-like signaling molecules in plants has been an intriguing field of research. Among these, γ-aminobutyric acid (GABA) was discovered in plants over half a century ago, and studies of its roles as a primary metabolite have been well documented, particularly in the context of stress responses. In contrast, evidence of the potential mechanism by which GABA acts as a signaling molecule in plants has only recently been reported. In spite of this breakthrough, the roles of GABA as a signaling molecule in plants have yet to be established and several aspects of the complexity of the GABA signaling system remain obscure. This review summarizes the uncertainties in GABA signaling in plants and suggests research directions and technologies that would help in answering unsolved questions.

Reactive Oxygen Species Tune Root Tropic Responses.

The default growth pattern of primary roots of land plants is directed by gravity. However, roots possess the ability to sense and respond directionally to other chemical and physical stimuli, separately and in combination. Therefore, these root tropic responses must be antagonistic to gravitropism. The role of reactive oxygen species (ROS) in gravitropism of maize and Arabidopsis (Arabidopsis thaliana) roots has been previously described. However, which cellular signals underlie the integration of the different environmental stimuli, which lead to an appropriate root tropic response, is currently unknown. In gravity-responding roots, we observed, by applying the ROS-sensitive fluorescent dye dihydrorhodamine-123 and confocal microscopy, a transient asymmetric ROS distribution, higher at the concave side of the root. The asymmetry, detected at the distal elongation zone, was built in the first 2 h of the gravitropic response and dissipated after another 2 h. In contrast, hydrotropically responding roots show no transient asymmetric distribution of ROS Decreasing ROS levels by applying the antioxidant ascorbate, or the ROS-generation inhibitor diphenylene iodonium attenuated gravitropism while enhancing hydrotropism. Arabidopsis mutants deficient in Ascorbate Peroxidase 1 showed attenuated hydrotropic root bending. Mutants of the root-expressed NADPH oxidase RBOH C, but not rbohD, showed enhanced hydrotropism and less ROS in their roots apices (tested in tissue extracts with Amplex Red). Finally, hydrostimulation prior to gravistimulation attenuated the gravistimulated asymmetric ROS and auxin signals that are required for gravity-directed curvature. We suggest that ROS, presumably H2O2, function in tuning root tropic responses by promoting gravitropism and negatively regulating hydrotropism.

Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity, and fiber differentiation in tobacco stems.

The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C(19)-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling.

Transcriptomic analysis of Sorghum bicolor responding to combined heat and drought stress.

BACKGROUND: Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination. RESULTS: Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these 'unique' transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response. CONCLUSIONS: These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism.

In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

Calmodulin-binding transcription activator 1 mediates auxin signaling and responds to stresses in Arabidopsis.

Auxin is a key plant hormone that regulates various aspects of plant development. However, the mechanisms integrating auxin growth effects with stress responses are not fully understood. In this study, we investigated the possible role of calmodulin-binding transcription activator 1 (CAMTA1), an Arabidopsis thaliana calcium/calmodulin-binding transcription activator, in auxin signaling and its responses to different stresses. Plants harboring the AtCAMTA1 promoter fused to the GUS reporter gene revealed cell-specific expression patterns reminiscent of auxin responses. The responsiveness of CAMTA1 to auxin was further assessed by chemical disturbances in polar auxin transport, and by RT-PCR analysis of gene expression of dissected leaf sections from plants exposed to the auxin transport inhibitor NPA. Furthermore, the intensity and cell-specific expression patterns of CAMTA1 changed significantly and differentially on exposure to increasing salt concentrations and heat. Transcriptome analysis of a camta1 T-DNA insertion mutant revealed 63 up-regulated genes, of which 17 are associated with auxin signaling. Finally, analysis of hypocotyl elongation in the presence and absence of auxin revealed that camta1 T-DNA insertion mutants and CAMTA1-repressor lines are hyper-responsive to auxin compared to wild-type seedlings. Thus, CAMTA1 participates in auxin signaling and responds to abiotic stresses.

Plant-specific calmodulin-binding proteins.

Calmodulin CaM is the most prominent Ca2+ transducer in eukaryotic cells, regulating the activity of numerous proteins with diverse cellular functions. Many features of CaM and its downstream targets are similar in plants and other eukaryotes. However, plants possess a unique set of CaM-related proteins, and several unique CaM target proteins. This review discusses recent progress in identifying plant-specific CaM-binding proteins and their roles in response to biotic and abiotic stresses and development. The review also addresses aspects emerging from recent structural studies of CaM interactions with target proteins relevant to plants.

Highway or byway: the metabolic role of the GABA shunt in plants.

Much of the recent work on the gamma-aminobutyrate (GABA) shunt in plants has concentrated on stress/pest-associated and signalling roles. However, fifty years after the structural elucidation of the pathway, aspects of its regulation and even of its biological significance remain largely obscure. Here, we assess the importance of GABA metabolism in plants, reviewing relevant biological circumstances and taking advantage of high-throughput data accessibility and computational approaches. We discuss the premise that GABA metabolism plays a major role in carbon and nitrogen primary metabolism. We further evaluate technological developments that will likely allow us to address the quantitative importance of this shunt within the biological processes to which it contributes.

GABA signaling: a conserved and ubiquitous mechanism.

In plants, research on gamma-aminobutyric acid (GABA) has focused on its role as a metabolite, mainly in the context of responses to biotic and abiotic stresses. By contrast, studies of GABA in vertebrates have concentrated mainly on its role as a neurotransmitter and signaling molecule. Here, we discuss recent findings that point towards a possible role for GABA as a signaling molecule in plants.

Root Plasticity in the Pursuit of Water.

One of the greatest challenges of terrestrial vegetation is to acquire water through soil-grown roots. Owing to the scarcity of high-quality water in the soil and the environment's spatial heterogeneity and temporal variability, ranging from extreme flooding to drought, roots have evolutionarily acquired tremendous plasticity regarding their geometric arrangement of individual roots and their three-dimensional organization within the soil. Water deficiency has also become an increasing threat to agriculture and dryland ecosystems due to climate change. As a result, roots have become important targets for genetic selection and modification in an effort to improve crop resilience under water-limiting conditions. This review addresses root plasticity from different angles: Their structures and geometry in response to the environment, potential genetic control of root traits suitable for water-limiting conditions, and contemporary and future studies of the principles underlying root plasticity post-Darwin's 'root-brain' hypothesis. Our increasing knowledge of different disciplines of plant sciences and agriculture should contribute to a sustainable management of natural and agricultural ecosystems for the future of mankind.

A Ca2+/CaM-regulated transcriptional switch modulates stomatal development in response to water deficit.

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis.

Calmodulin-binding transcription activator (CAMTA) 3 (also called SR1) is a calmodulin-binding transcription factor in Arabidopsis. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) showed enhanced spontaneous lesions. Transcriptome analysis of both mutants revealed 6 genes with attenuated expression and 99 genes with elevated expression. Of the latter, 32 genes are related to defense against pathogens (e.g. WRKY33, PR1 and chitinase). Propagation of a virulent strain of the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea were attenuated in both mutants. Moreover, both mutants accumulated high levels of H2O2. We suggest that CAMTA3 regulates the expression of a set of genes involved in biotic defense responses.

SELENOPROTEIN O is a chloroplast protein involved in ROS scavenging and its absence increases dehydration tolerance in Arabidopsis thaliana.

The evolutionary conserved family of Selenoproteins performs redox-regulatory functions in bacteria, archaea and eukaryotes. Among them, members of the SELENOPROTEIN O (SELO) subfamily are located in mammalian and yeast mitochondria, but their functions are thus far enigmatic. Screening of T-DNA knockout mutants for resistance to the proline analogue thioproline (T4C), identified mutant alleles of the plant SELO homologue in Arabidopsis thaliana. Absence of SELO resulted in a stress-induced transcriptional activation instead of silencing of mitochondrial proline dehydrogenase, and also high elevation of Δ(1)-pyrroline-5-carboxylate dehydrogenase involved in degradation of proline, thereby alleviating T4C inhibition and lessening drought-induced proline accumulation. Unlike its animal homologues, SELO was localized to chloroplasts of plants ectopically expressing SELO-GFP. The protein was co-fractionated with thylakoid membrane complexes, and co-immunoprecipitated with FNR, PGRL1 and STN7, all involved in regulating PSI and downstream electron flow. The selo mutants displayed extended survival under dehydration, accompanied by longer photosynthetic activity, compared with wild-type plants. Enhanced expression of genes encoding ROS scavenging enzymes in the unstressed selo mutant correlated with higher oxidant scavenging capacity and reduced methyl viologen damage. The study elucidates SELO as a PSI-related component involved in regulating ROS levels and stress responses.

A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters.

C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin.

Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.

CAMTAs: calmodulin-binding transcription activators from plants to human.

Recently, a novel family of calmodulin-binding transcription activators (CAMTAs) was reported in various eukaryotes. All CAMTAs share a similar domain organization, with a novel type of sequence-specific DNA-binding domain (designated CG-1). This domain could bind DNA directly and activate transcription, or interact with other transcription factors, not through DNA binding, thus acting as a co-activator of transcription. Investigations of CAMTAs in various organisms imply a broad range of functions from sensory mechanisms to embryo development and growth control, highlighted by the apparent involvement of mammalian CAMTA2 in cardiac growth, and of CAMTA1 in tumor suppression and memory performance.

Cyclic nucleotide-gated channels in plants.

Until recently the role of cyclic nucleotide monophosphates (cNMPs) in plants had been controversial, with equivocal data about their concentrations, biosynthetic and degrading enzymes, and cellular targets. This review discusses the current knowledge in this field, with focus on the largest class of cNMP targets in plant cells, the cyclic nucleotide-gated channels (CNGCs). Aspects of structure and function are addressed, with reference to studies in heterologous systems and in planta. The picture emerging, albeit still fragmented, is of proteins with diverse functions in the control of ion homeostasis, development, and defense against biotic and abiotic threats.

The Cholodny-Went theory does not explain hydrotropism.

Optimization of water foraging by plants is partially achieved by the ability of roots to direct growth towards high water potential, a phenomenon termed hydrotropism. In contrast to gravitropism and phototropism, which require auxin redistribution, as suggested by the Cholodny-Went theory, hydrotropism is not mediated by the phytohormone auxin, which raises questions about the mechanism underlying this tropic response. Here we specify the open questions in this field of research and discuss the possible interactions of abscisic acid, calcium and reactive oxygen species as part of a dynamic system of sensing water potential in the root tip, transmission of the signal to the root elongation zone and promoting root curvature towards water. We conclude that root hydrotropism is mediated by inter-cellular signals that are not explained by the Cholodny-Went theory.