Guard cells surround stomatal pores in the epidermis of plant leaves and stems. Stomatal pore opening is essential for CO2 influx into leaves for photosynthetic carbon fixation. In exchange, plants lose over 95% of their water via transpiration to the atmosphere. Signal transduction mechanisms in guard cells integrate hormonal stimuli, light signals, water status, CO2, temperature, and other environmental conditions to modulate stomatal apertures for regulation of gas exchange and plant survival under diverse conditions. Stomatal guard cells have become a highly developed model system for characterizing early signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single cell. In this review we focus on recent advances in understanding signal transduction mechanisms in guard cells.

The plant hormone abscisic acid (ABA) regulates important stress and developmental responses. We have isolated a recessive ABA hypersensitive mutant, abh1, that shows hormone specificity to ABA. ABH1 encodes the Arabidopsis homolog of a nuclear mRNA cap binding protein and functions in a heterodimeric complex to bind the mRNA cap structure. DNA chip analyses show that only a few transcripts are down-regulated in abh1, several of which are implicated in ABA signaling. Consistent with these results, abh1 plants show ABA-hypersensitive stomatal closing and reduced wilting during drought. Interestingly, ABA-hypersensitive cytosolic calcium increases in abh1 guard cells demonstrate amplification of early ABA signaling. Thus, ABH1 represents a modulator of ABA signaling proposed to function by transcript alteration of early ABA signaling elements.

Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life, yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS. This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.

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To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker ␣-mannosidase, the enrichment factor of the vacuoles was estimated at ϳ42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca 2ϩ influx current in Arabidopsis guard cells termed "I Ca " (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klü sener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734 Calcium acts as an intracellular second messenger, coupling extracellular stimuli to intracellular and whole-plant responses (Hepler and Wayne, 1985; Sanders et al., 1999). Guard cells have been developed as a model system for dissecting early signal transduction processes in plant cells. Guard cells respond to a great variety of external stimuli, including abscisic acid (ABA; McAinsh et al., 1990; Schroeder and Hagiwara, 1990), auxin (Gehring et al., 1990(Gehring et al., , 1998, ozone (Clayton et al., 1999), and reactive oxygen species (ROS; McAinsh et al., 1996; Pei et al., 2000) with an increase in the cytoplasmic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) and subsequent stomatal movements (for review, see Blatt, 2000; Schroeder et al., 2001a). Cytosolic Ca 2ϩ increases down-regulate inward-rectifying K ϩ channels and activate anion channels, providing mechanisms for Ca 2ϩ -dependent stomatal closure (Schroeder and Hagiwara, 1989). Particularly well analyzed is the Ca 2ϩ response of guard cells to the phytohormone ABA (McAinsh et al., 1990; Schroeder and Hagiwara, 1990; Blatt and Armstrong, 1993; Schmidt et al., 1995; Leckie et al., 1998; Allen et al., 1999a; Staxén et al., 1999; MacRobbie, 2000; Hugouvieux et al., 2001; for review, see Blatt, 2000; Schroeder et al., 2001b).ABA has been shown to activate a hyperpolarization-dependent Ca 2ϩ -permeable current in the plasma membrane of guard cells, leading to Ca 2ϩ influx and an increase in the cytoplasmic free Ca 2ϩ concentration (Hamilton et al., 2000; Pei et al., 2000). Furthermore, it has been demonstrated that ABA elevates levels of ROS, and that elevated ROS levels stimulate Ca 2ϩ -permeable cation currents in the plasma membrane termed "I Ca " (Pei et al., 2000). I Ca channels have been shown to be permeable to several cations including Mg 2ϩ (Pei et al., 2000). The ABA-insensitive mutants gca2, abi1-1, and abi2-1 disrupt I Ca channel activation at 2 This paper is dedicated to the memory of Gethyn Allen. 3 These authors contributed equally to the paper. 4 Present address: Lehrstuhl fü r Pflanzenphysiologie, RuhrUniversität Bochum, D-44780 Bochum, Germany.5 Present address: Department of Agriculture, Okayama University, Okayama 700, Japan.6 Present address: Laboratoire de Physiologie Cellulaire Vég-étale, Département Réponse et Dynamique Cellulaires, CEA Grenoble, 38054 Grenoble cedex 9, France.Corresponding author; e-mail julian@biomail.ucsd.edu; fax 858 -534 -7108.Article, publication date, and citation...

Monitoring molecular dynamics of an organism upon stress is probably the best approach to decipher physiological mechanisms involved in the stress response. Quantitative analysis of proteins and metabolites is able to provide accurate information about molecular changes allowing the establishment of a range of more or less specific mechanisms, leading to the identification of major players in the considered pathways. Such tools have been successfully used to analyze the plant response to cadmium (Cd), a major pollutant capable of causing severe health issues as it accumulates in the food chain. We present a summary of proteomics and metabolomics works that contributed to a better understanding of the molecular aspects involved in the plant response to Cd. This work allowed us to provide a finer picture of general signaling, regulatory and metabolic pathways that appeared to be affected upon Cd stress. In particular, we conclude on the advantage of employing different approaches of global proteome- and metabolome-wide techniques, combined with more targeted analysis to answer molecular questions and unravel biological networks. Finally, we propose possible directions and methodologies for future prospectives in this field, as many aspects of the plant-Cd interaction remain to be discovered.