The complexity of Ca2+ patterns observed in eukaryotic cells, including plants, has led to the hypothesis that specific patterns of Ca2+ propagation, termed Ca2+ signatures, encode information and relay it to downstream elements (effectors) for translation into appropriate cellular responses. Ca2+ -binding proteins (sensors) play a key role in decoding Ca2+ signatures and transducing signals by activating specific targets and pathways. Calmodulin is a Ca2+ sensor known to modulate the activity of many mammalian proteins, whose targets in plants are now being actively characterized. Plants possess an interesting and rapidly growing list of calmodulin targets with a variety of cellular roles. Nevertheless, many targets appear to be unique to plants and remain uncharacterized, calling for a concerted effort to elucidate their functions. Moreover, the extended family of calmodulin-related proteins in plants consists of evolutionarily divergent members, mostly of unknown function, although some have recently been implicated in stress responses. It is hoped that advances in functional genomics, and the research tools it generates, will help to explain themultiplicity of calmodulin genes in plants, and to identify their downstream effectors. This review summarizes current knowledge of the Ca2+ -calmodulin messenger system in plants and presents suggestions for future areas of research. Contents I. Introduction 36 II. CaM isoforms and CaM-like proteins 37 III. CaM-target proteins 42 IV. CaM and nuclear functions 46 V. Regulation of ion transport 49 VI. CaM and plant responses to environmental stimuli 52 VII. Conclusions and future studies 58 Acknowledgements 59 References 59.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1469-8137.2001.00154.x

Calmodulin as a versatile calcium signal transducer in plants.

The role of γ-aminobutyric acid (GABA) as a signal in animals has been documented for over 60 years. In contrast, evidence that GABA is a signal in plants has only emerged in the last 15 years, and it was not until last year that a mechanism by which this could occur was identified—a plant ‘GABA receptor’ that inhibits anion passage through the aluminium-activated malate transporter family of proteins (ALMTs). ALMTs are multigenic, expressed in different organs and present on different membranes. We propose GABA regulation of ALMT activity could function as a signal that modulates plant growth, development, and stress response. In this review, we compare and contrast the plant ‘GABA receptor’ with mammalian GABAA receptors in terms of their molecular identity, predicted topology, mode of action, and signalling roles. We also explore the implications of the discovery that GABA modulates anion flux in plants, its role in signal transduction for the regulation of plant physiology, and predict the possibility that there are other GABA interaction sites in the N termini of ALMT proteins through in silico evolutionary coupling analysis; we also explore the potential interactions between GABA and other signalling molecules.

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γ-Aminobutyric acid (GABA) signalling in plants

Four-day-old rice (Oryza sativa L.) seedlings were subjected to varying temperatures of 30/20, 35/25, and 42/37 °C [light/dark (15/9 h); light intensity: 350 μmol m−2 s−1, RH 65–70 %] in glass Petri dishes for 10 days in the absence (control) or the presence of γ-aminobutyric acid (GABA) 1 mM under the controlled conditions of a growth chamber. With rise in temperature, the length of both shoots and roots was inhibited severely and there was a marked decrease in survival, especially at 42/37 °C. Endogenous GABA content increased more than twofold in moderately stressed (MS) 35/25 °C plants, whereas it decreased sevenfold in severely stressed (SS) 42/37 °C plants compared to MS plants, and this decrease was associated with marked reduction in growth and survival. Exogenous application of GABA to the heat-stressed plants significantly improved growth as well as survival. It was linked to reduction in damage to membranes, improvement in cellular reducing ability, chlorophyll content, and photochemical efficiency in shoots. Relative leaf water content and stomatal conductance were also improved with the application of GABA and their improvement was related to increased accumulation of the osmolytes proline and trehalose. In the presence of GABA, the shoots suffered less oxidative damage in terms of malondialdehyde and hydrogen peroxide contents. The activities of enzymatic antioxidants such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase were severely inhibited in plants growing at 42/37 °C compared to those growing at 35/25 °C. The nonenzymatic antioxidants like ascorbate and glutathione followed a similar pattern. GABA-treated SS plants showed enhanced levels of enzymatic and nonenzymatic antioxidants compared to untreated controls. Thus, GABA appears to impart partial protection from heat stress to rice plants by elevating leaf turgor due to increased accumulation of osmolytes and reduction of oxidative damage by stimulation of antioxidants. These findings provided evidence about the involvement of GABA in governing heat sensitivity in rice.

γ-Aminobutyric Acid (GABA) Imparts Partial Protection from Heat Stress Injury to Rice Seedlings by Improving Leaf Turgor and Upregulating Osmoprotectants and Antioxidants

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Stress responses are largely conserved in eukaryotic cells, but with plants having certain distinctive reactions to specific stresses, e.g. the induction of pathogenesis-related proteins. General responses to stress involve signaling stress detection via the redox system, checkpoints arresting the cell cycle and DNA repair processes stimulated in response to DNA damage. Specific responses to stress include the induction of protective metabolites, such as betaines, and protective proteins, for example, heat shock proteins. Chemical signals, e.g. reactive oxygen species, Ca2+ and plant hormones, acting through signal transduction cascades activate genomic re-programming. Genome plasticity in plants allows adaptation to environmental conditions and includes genomic or epigenetic changes (histone acetylation, methylation, chromatin remodeling etc.) and possibly directed mutation. In plants, recent research has indicated that intricate stress response mechanisms and `cross talk' between stress responses exist. Here, changes in the plant genome and in genomic expression in development and as a response to environmental stress are reviewed as background to a discussion of the basis of aberrant genomic expression in vitro. Markers are discussed which may be used to characterize the stress exposure of in vitro tissues.

Stress and aberrant phenotypes in vitro culture

GABA (4-Aminobutyric acid) is a non-protein amino acid that is made in response to a number of environmental stresses in plants including high temperature stress. Glutamate decarboxylase (GAD) is the enzyme responsible for catalyzing this increase in GABA, and it has been shown to be a calmodulin binding protein in plant systems. It is our hypothesis that high temperature stresses generate an increase in cytoplasmic calcium ([Ca’+]y,), that combines with calmodulin to activate GAD, producing the high-temperature induced accumulation of GABA in Arabidopsis roots but not in shoot tissues. We have previously shown that the calmodulin inhibitors chlorpromazine and trifluoperazine inhibit heat-induced GABA accumulation. We have further examined the effectiveness of calcium transport inhibitors (EGTA, BAPTA, verapamil, methoxyverapamil, ruthenium red, dantrolene, thapsigargin, nifedipine, and 3,4,5trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMBA) on the heat-induced accumulation of GABA in Arabidopsis seedlings. Only ruthenium red and dantrolene were found to be effective in blocking heat-induced GABA accumulation. Ca’ ionophore stimulated GABA accumulation even in the absence of heat stress. All of the above agents that inhibited heat-induced GABA accumulation were also shown to increase the sensitivity of Arabidopsis seedlings to lethal temperature treatments, while Ca’+ ionophore decreased this sensitivity. We have also utilized transgenic Arabidopsis plants expressing the chemiluminescent calcium sensing protein aequorin in an attempt to directly demonstrate the effects of the calcium transport inhibitors and Ca’ ionophore on calcium accumulation in Arabidopsis roots and shoots. Western blot analysis further suggests that the levels of GAD protein present in tissues does not change in response to heat stress. These data collectively demonstrate that cytoplasmic calcium sensed during heat stress is sufficient to account for the accumulation of GABA during heat stress, and that other mechanisms of regulating GABA accumulation may not be involved in heat-stress regulation.

The Regulation of GABA Accumulation by Heat Stress in Arabidopsis

GABA (4-aminobutyric acid) is a non-protein amino acid widely found in plant tissues that accumulates in response to various environmental stresses including heat, cold, drought, salinity, and anaerobic stress. Although there is limited information on its biological function in plants, some studies suggest that GABA acts as a carbon and/or nitrogen source in microorganisms, and it has been suggested that GABA metabolism under stress is related to excessive glutamate accumulation and/or cytoplasmic pH regulation. We found that exogenously applied GABA had an effect on root growth and development in Arabidopsis thaliana L. seedlings. This effect was modulated between inhibition of root elongation when seedlings were grown on full strength Murashige and Skoog salts (1/1X MS) to stimulation of root elongation when plants were grown on 1/8 strength MS salts (1/8X MS). When the concentration of single ions in MS salts was varied, the primary effect was found to be a direct interaction between GABA and the level of nitrate (NO3) in the growing medium. At NO3 concentrations below 40 mM, root growth was stimulated by the addition of GABA to the growth medium, whereas at concentrations above 40 mM NO3 addition of GABA to the growth medium inhibited root elongation. The uptake of NO; and tissue levels of nitrate were also investigated at high (40 mM NO3) and low (5 mM NO,) with and without GABA in the growing medium. In correlation with the growth results GABA promotes NO3 uptake at low NO3, while GABA inhibits NO3 uptake at high NO3. However, tissue levels of NO3 are increased by GABA at high NO3 while they are decreased by GABA at low NO3. These results can be interpreted to indicate that GABA produced during stress is capable of regulating NO3 uptake and metabolism during stress.

T. W. Barger

Papers

The Regulation of GABA Accumulation by Heat Stress in Arabidopsis

GABA Increases the Rate of Nitrate Uptake and Utilization in Arabidopsis Roots