Plant growth promotion by the interaction of a novel synthetic small molecule with GA-DELLA function.

Synthesized small molecules are useful as tools to investigate hormonal signaling involved in plant growth and development. They are also important as agrochemicals to promote beneficial properties of crops in the field. We describe here the synthesis and mode of action of a novel growth-promoting chemical, A1. A1 stimulates enhanced growth in both shoot and root tissues of plants, acting by increasing both dry and fresh weight. This suggests that A1 not only promotes uptake of water but also increases production of cellular material. A1 treatment of Arabidopsisleads to the degradation of DELLA growth-inhibitory proteins suggesting that A1-mediated growth promotion is dependent upon this mechanism. We performed genetic analysis to confirm this and further dissect the mechanism of A1 action upon growth in Arabidopsis. A quintuple dellamutant was insensitive to A1, confirming that the mode of action was indeed via a DELLA-dependent mechanism. The ga1-5gibberellin synthesis mutant was similarly insensitive, suggesting that to promote growth in ArabidopsisA1 requires the presence of endogenous gibberellins. This was further suggested by the observation that double mutants of GID1 gibberellin receptor genes were insensitive to A1. Taken together, our data suggest that A1 acts to enhance sensitivity to endogenous gibberellins thus leading to observed enhanced growth via DELLA degradation. A1 and related compounds will be useful to identify novel signaling components involved in plant growth and development, and as agrochemicals suitable for a wide range of crop species.

Chloroplast calcium signalling regulates thermomemory.

After an episode of heat stress plants retain a cellular "memory" of this event, a phenomenon known as thermomemory. This mechanism allows plants to better cope against a subsequent heat event. Thermomemory occurs through the persistence of heat shock proteins (HSPs) synthesized after the first "priming" event. Maintenance of this thermomemory comes at the cost to growth though, therefore it is vital that the memory is reset when no longer required. Recently, it has been reported that autophagy is important for resetting the thermomemory. It has also been shown recently that in response to heat, Arabidopsis displays an increase in chloroplast free calcium concentration which is partially dependent on calcium sensing receptor (CAS) protein. It is not known what the purpose of this heat-activated calcium signal is. Therefore, we compared downstream responses to heat in wild type (WT) and cas mutants, as the latter produce a reduced chloroplast calcium signal to heat. We found that after thermopriming the cas mutants displayed a greater biomass and a reduced level of the small heat shock protein HSP 17.6 degradation compared to WT. cas mutants did not show an increase in free amino acid levels after thermopriming, suggesting reduced autophagy. These results suggest that heat-induced chloroplast calcium elevation is a positive signal for resetting of the thermomemory.

Mediator Subunits MED16, MED14, and MED2 Are Required for Activation of ABRE-Dependent Transcription in Arabidopsis.

The Mediator complex controls transcription of most eukaryotic genes with individual subunits required for the control of particular gene regulons in response to various perturbations. In this study, we reveal the roles of the plant Mediator subunits MED16, MED14, and MED2 in regulating transcription in response to the phytohormone abscisic acid (ABA) and we determine which cis elements are under their control. Using synthetic promoter reporters we established an effective system for testing relationships between subunits and specific cis-acting motifs in protoplasts. Our results demonstrate that MED16, MED14, and MED2 are required for the full transcriptional activation by ABA of promoters containing both the ABRE (ABA-responsive element) and DRE (drought-responsive element). Using synthetic promoter motif concatamers, we showed that ABA-responsive activation of the ABRE but not the DRE motif was dependent on these three Mediator subunits. Furthermore, the three subunits were required for the control of water loss from leaves but played no role in ABA-dependent growth inhibition, highlighting specificity in their functions. Our results identify new roles for three Mediator subunits, provide a direct demonstration of their function and highlight that our experimental approach can be utilized to identify the function of subunits of plant transcriptional regulators.

The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

Basal stomatal aperture is regulated by GA-DELLAs in Arabidopsis.

Stomatal aperture is tightly regulated in order to achieve the best compromise between gas exchange and water conservation. Steady-state (basal) stomatal aperture is therefore understandably a key component in plant fitness. It has been shown previously in tomato that DELLA proteins act as positive regulators of closure of stomata, and their action is enhanced by the hormone ABA, which is itself important in mediating drought stress tolerance. DELLAs are regulated by a variety of signals which promote plant growth, most notably the hormones gibberellins, which have been shown to promote stomatal opening. We have found that DELLA proteins are also used in Arabidopsis for regulating basal stomatal aperture. We also discovered that the perception of endogenous gibberellins via the GID1 receptors is necessary for optimal basal stomatal aperture.

Design Principle for Decoding Calcium Signals to Generate Specific Gene Expression Via Transcription.

The second messenger calcium plays a key role in conveying specificity of signaling pathways in plant cells. Specific calcium signatures are decoded to generate correct gene expression responses and amplification of calcium signatures is vital to this process. (1) It is not known if this amplification is an intrinsic property of all calcium-regulated gene expression responses and whether all calcium signatures have the potential to be amplified, or (2) how a given calcium signature maintains specificity in cells containing a great number of transcription factors (TFs) and other proteins with the potential to be calcium-regulated. The work presented here uncovers the design principle by which it is possible to decode calcium signals into specific changes in gene transcription in plant cells. Regarding the first question, we found that the binding mechanism between protein components possesses an intrinsic property that will nonlinearly amplify any calcium signal. This nonlinear amplification allows plant cells to effectively distinguish the kinetics of different calcium signatures to produce specific and appropriate changes in gene expression. Regarding the second question, we found that the large number of calmodulin (CaM)-binding TFs or proteins in plant cells form a buffering system such that the concentration of an active CaM-binding TF is insensitive to the concentration of any other CaM-binding protein, thus maintaining specificity. The design principle revealed by this work can be used to explain how any CaM-binding TF decodes calcium signals to generate specific gene expression responses in plant cells via transcription.

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE-TO-FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination-based mapping and whole-genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC-MS and polyacrylamide gel electrophoresis to measure cell-wall fucose and boron (B)-dependent dimerization of the cell-wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate-bridging of RGII, we assessed freeze-induced damage in wild-type and sfr8 plants by measuring electrolyte leakage from freeze-thawed leaf discs. We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP-d-mannose-4,6-dehydratase. sfr8 cell walls exhibited low cell-wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing-sensitive. Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.

The identification of small molecule inhibitors of the plant inositol phosphorylceramide synthase which demonstrate herbicidal activity.

Resistance to 157 different herbicides and 88% of known sites of action has been observed, with many weeds resistant to two or more modes. Coupled with tighter environmental regulation, this demonstrates the need to identify new modes of action and novel herbicides. The plant sphingolipid biosynthetic enzyme, inositol phosphorylceramide synthase (IPCS), has been identified as a novel, putative herbicide target. The non-mammalian nature of this enzyme offers the potential of discovering plant specific inhibitory compounds with minimal impact on animals and humans, perhaps leading to the development of new non-toxic herbicides. The best characterised and most highly expressed isoform of the enzyme in the model-dicot Arabidopsis, AtIPCS2, was formatted into a yeast-based assay which was then utilized to screen a proprietary library of over 11,000 compounds provided by Bayer AG. Hits from this screen were validated in a secondary in vitro enzyme assay. These studies led to the identification of a potent inhibitor that showed selectivity for AtIPCS2 over the yeast orthologue, and activity against Arabidopsis seedlings. This work highlighted the use of a yeast-based screening assay to discover herbicidal compounds and the status of the plant IPCS as a novel herbicidal target.

Expression levels of inositol phosphorylceramide synthase modulate plant responses to biotic and abiotic stress in Arabidopsis thaliana.

This research was undertaken to investigate the global role of the plant inositol phosphorylceramide synthase (IPCS), a non-mammalian enzyme previously shown to be associated with the pathogen response. RNA-Seq analyses demonstrated that over-expression of inositol phosphorylceramide synthase isoforms AtIPCS1, 2 or 3 in Arabidopsis thaliana resulted in the down-regulation of genes involved in plant response to pathogens. In addition, genes associated with the abiotic stress response to salinity, cold and drought were found to be similarly down-regulated. Detailed analyses of transgenic lines over-expressing AtIPCS1-3 at various levels revealed that the degree of down-regulation is specifically correlated with the level of IPCS expression. Singular enrichment analysis of these down-regulated genes showed that AtIPCS1-3 expression affects biological signaling pathways involved in plant response to biotic and abiotic stress. The up-regulation of genes involved in photosynthesis and lipid localization was also observed in the over-expressing lines.

Leaves of isoprene-emitting tobacco plants maintain PSII stability at high temperatures.

At high temperatures, isoprene-emitting plants display a higher photosynthetic rate and a lower nonphotochemical quenching (NPQ) compared with nonemitting plants. The mechanism of this phenomenon, which may be very important under current climate warming, is still elusive. NPQ was dissected into its components, and chlorophyll fluorescence lifetime imaging microscopy (FLIM) was used to analyse the dynamics of excited chlorophyll relaxation in isoprene-emitting and nonemitting plants. Thylakoid membrane stiffness was also measured using atomic force microscope (AFM) to identify a possible mode of action of isoprene in improving photochemical efficiency and photosynthetic stability. We show that, when compared with nonemitters, isoprene-emitting tobacco plants exposed at high temperatures display a reduced increase of the NPQ energy-dependent component (qE) and stable (1) chlorophyll fluorescence lifetime; (2) amplitude of the fluorescence decay components; and (3) thylakoid membrane stiffness. Our study shows for the first time that isoprene maintains PSII stability at high temperatures by preventing the modifications of the surrounding environment, namely providing a more steady and homogeneous distribution of the light-absorbing centres and a stable thylakoid membrane stiffness. Isoprene photoprotects leaves with a mechanism alternative to NPQ, enabling plants to maintain a high photosynthetic rate at rising temperatures.

Increases in Absolute Temperature Stimulate Free Calcium Concentration Elevations in the Chloroplast.

Plants need to sense increases in temperature to be able to adapt their physiology and development to survive; however, the mechanisms of heat perception are currently relatively poorly understood. Here we demonstrate that in response to elevated temperature, the free calcium concentration of the stroma of chloroplasts increases. This response is specific to the chloroplast, as no corresponding increase in calcium is seen in the cytosol. The chloroplast calcium response is dose dependent above a threshold. The magnitude of this calcium response is dependent upon absolute temperature, not the rate of heating. This response is dynamic: repeated stimulation leads to rapid attenuation of the response, which can be overcome by sensitization at a higher temperature. More long-term acclimation to different temperatures resets the basal sensitivity of the system, such that plants acclimated to lower temperatures are more sensitive than those acclimated to higher temperatures. The heat-induced chloroplast calcium response was partially dependent upon the calcium-sensing receptor CAS which has been shown previously to regulate other chloroplast calcium signaling responses. Taken together, our data demonstrate the ability of chloroplasts to sense absolute high temperature and produce commensurately quantitative stromal calcium response, the magnitude of which is a function of both current temperature and stress history.

OXI1 kinase plays a key role in resistance of Arabidopsis towards aphids (Myzus persicae).

Plants have co-evolved with a diverse array of pathogens and insect herbivores and so have evolved an extensive repertoire of constitutive and induced defence mechanisms activated through complex signalling pathways. OXI1 kinase is required for activation of mitogen-activated protein kinases (MAPKs) and is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. Furthermore, changes in the levels of OXI1 appear to be crucial for appropriate signalling. Callose deposition also plays a key role in defence. Here we demonstrate, for the first time, that OXI1 plays an important role in defence against aphids. The Arabidopsis mutant, oxi1-2, showed significant resistance both in terms of population build-up (p < 0.001) and the rate of build-up (p < 0.001). Arabidopsis mutants for ß-1,3-glucanase, gns2 and gns3, showed partial aphid resistance, significantly delaying developmental rate, taking two-fold longer to reach adulthood. Whilst ß-1,3-glucanase genes GNS1, GNS2, GNS3 and GNS5 were not induced in oxi1-2 in response to aphid feeding, GNS2 was expressed to high levels in the corresponding WT (Col-0) in response to aphid feeding. Callose synthase GSL5 was up-regulated in oxi1-2 in response to aphids. The results suggest that resistance in oxi1-2 mutants is through induction of callose deposition via MAPKs resulting in ROS induction as an early response. Furthermore, the results suggest that the ß-1,3-glucanase genes, especially GNS2, play an important role in host plant susceptibility to aphids. Better understanding of signalling cascades underpinning tolerance to biotic stress will help inform future breeding programmes for enhancing crop resilience.

Predicting plant immunity gene expression by identifying the decoding mechanism of calcium signatures.

Calcium plays a key role in determining the specificity of a vast array of signalling pathways in plants. Cellular calcium elevations with different characteristics (calcium signatures) carry information on the identity of the primary stimulus, ensuring appropriate downstream responses. However, the mechanism for decoding calcium signatures is unknown. To determine this, decoding of the salicylic acid (SA)-mediated plant immunity signalling network controlling gene expression was examined. A dynamic mathematical model of the SA-mediated plant immunity network was developed. This model was used to predict responses to different calcium signatures; these were validated empirically using quantitative real-time PCR to measure gene expression. The mechanism for decoding calcium signatures to control expression of plant immunity genes enhanced disease susceptibility 1 (EDS1) and isochorismate synthase 1 (ICS1) was identified. Calcium, calmodulin, calmodulin-binding transcription activators (CAMTA)3 and calmodulin binding protein 60g (CBP60g) together amplify each calcium signature into three active signals, simultaneously regulating expression. The time required for calcium to return to steady-state level also quantitatively regulates gene expression. Decoding of calcium signatures occurs via nonlinear interactions between these active signals, producing a unique response in each case. Key properties of the calcium signatures are not intuitive, exemplifying the importance of mathematical modelling approaches. This approach can be applied to identifying the decoding mechanisms of other plant calcium signalling pathways.

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.

Transcriptomic analysis comparing stay-green and senescent Sorghum bicolor lines identifies a role for proline biosynthesis in the stay-green trait.

Sorghum bicolor is an important cereal crop grown on the arid and semi-arid regions of >98 different countries. These regions are such that this crop is often subjected to low water conditions, which can compromise yields. Stay-green sorghum plants are able to retain green leaf area for longer under drought conditions and as such have higher yields than their senescent counterparts. However, the molecular and physiological basis of this drought tolerance is yet to be fully understood. Here, a transcriptomic approach was used to compare gene expression between stay-green (B35) and senescent (R16) sorghum varieties. Ontological analysis of the differentially expressed transcripts identified an enrichment of genes involved with the 'response to osmotic stress' Gene Ontology (GO) category. In particular, delta1-pyrroline-5-carboxylate synthase 2 (P5CS2) was highly expressed in the stay-green line compared with the senescent line, and this high expression was correlated with higher proline levels. Comparisons of the differentially expressed genes with those that lie in known stay-green qualitative trait loci (QTLs) revealed that P5CS2 lies within the Stg1 QTL. Polymorphisms in known cis-elements were identified in the putative promoter region of P5CS2 and these could be responsible for the differences in the expression of this gene. This study provides greater insight into the stay-green trait in sorghum. This will be greatly beneficial not only to improve our understanding of drought tolerance mechanisms in sorghum, but also to facilitate the improvement of future sorghum cultivars by marker-assisted selection (MAS).

Combining modelling and experimental approaches to explain how calcium signatures are decoded by calmodulin-binding transcription activators (CAMTAs) to produce specific gene expression responses.

Experimental data show that Arabidopsis thaliana is able to decode different calcium signatures to produce specific gene expression responses. It is also known that calmodulin-binding transcription activators (CAMTAs) have calmodulin (CaM)-binding domains. Therefore, the gene expression responses regulated by CAMTAs respond to calcium signals. However, little is known about how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. A dynamic model of Ca(2+) -CaM-CAMTA binding and gene expression responses is developed following thermodynamic and kinetic principles. The model is parameterized using experimental data. Then it is used to analyse how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. Modelling analysis reveals that: calcium signals in the form of cytosolic calcium concentration elevations are nonlinearly amplified by binding of Ca(2+) , CaM and CAMTAs; amplification of Ca(2+) signals enables calcium signatures to be decoded to give specific CAMTA-regulated gene expression responses; gene expression responses to a calcium signature depend upon its history and accumulate all the information during the lifetime of the calcium signature. Information flow from calcium signatures to CAMTA-regulated gene expression responses has been established by combining experimental data with mathematical modelling.

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.

The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

A C-repeat binding factor transcriptional activator (CBF/DREB1) from European bilberry (Vaccinium myrtillus) induces freezing tolerance when expressed in Arabidopsis thaliana.

Freezing stress affects all plants from temperate zones to the poles. Global climate change means such freezing events are becoming less predictable. This in turn reduces the ability of plants to predict the approaching low temperatures and cold acclimate. This has consequences for crop yields and distribution of wild plant species. C-repeat binding factors (CBFs) are transcription factors previously shown to play a vital role in the acclimation process of Arabidopsis thaliana, controlling the expression of hundreds of genes whose products are necessary for freezing tolerance. Work in other plant species cements CBFs as key determinants in the trait of freezing tolerance in higher plants. To test the function of CBFs from highly freezing tolerant plants species we cloned and sequenced CBF transcription factors from three Vaccinium species (Vaccinium myrtillus, Vaccinium uliginosum and Vaccinium vitis-idaea) which we collected in the Arctic. We tested the activity of CBF transcription factors from the three Vaccinium species by producing transgenic Arabidopsis lines overexpressing them. Only the Vaccinium myrtillus CBF was able to substantially activate COR (CBF-target) gene expression in the absence of cold. Correspondingly, only the lines expressing the Vaccinium myrtillus CBF were constitutively freezing tolerant. The basis for the differences in potency of the three Vaccinium CBFs was tested by observing cellular localisation and protein levels. All three CBFs were correctly targeted to the nucleus, but Vaccinium uliginosum CBF appeared to be relatively unstable. The reasons for lack of potency for Vaccinium vitis-idaea CBF were not due to stability or targeting, and we speculate that this was due to altered transcription factor function.