Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries.

Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood. We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP-l-fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-l-Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l-fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant-water relations.

A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2 , abscisic acid, darkness, vapor pressure deficit and ozone.

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO2 , light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO2 , light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO2 induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5-2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO2 and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).

Ozone responses in Arabidopsis: beyond stomatal conductance.

Tropospheric ozone (O3) is a major air pollutant that decreases yield of important crops worldwide. Despite long-lasting research of its negative effects on plants, there are many gaps in our knowledge on how plants respond to O3. In this study, we used natural variation in the model plant Arabidopsis (Arabidopsis thaliana) to characterize molecular and physiological mechanisms underlying O3 sensitivity. A key parameter in models for O3 damage is stomatal uptake. Here we show that the extent of O3 damage in the sensitive Arabidopsis accession Shahdara (Sha) does not correspond with O3 uptake, pointing toward stomata-independent mechanisms for the development of O3 damage. We compared tolerant (Col-0) versus sensitive accessions (Sha, Cvi-0) in assays related to photosynthesis, cell death, antioxidants, and transcriptional regulation. Acute O3 exposure increased cell death, development of lesions in the leaves, and decreased photosynthesis in sensitive accessions. In both Sha and Cvi-0, O3-induced lesions were associated with decreased maximal chlorophyll fluorescence and low quantum yield of electron transfer from Photosystem II to plastoquinone. However, O3-induced repression of photosynthesis in these two O3-sensitive accessions developed in different ways. We demonstrate that O3 sensitivity in Arabidopsis is influenced by genetic diversity given that Sha and Cvi-0 developed accession-specific transcriptional responses to O3. Our findings advance the understanding of plant responses to O3 and set a framework for future studies to characterize molecular and physiological mechanisms allowing plants to respond to high O3 levels in the atmosphere as a result of high air pollution and climate change.

A role for calcium-dependent protein kinases in differential CO2 - and ABA-controlled stomatal closing and low CO2 -induced stomatal opening in Arabidopsis.

Low concentrations of CO2 cause stomatal opening, whereas [CO2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2 -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+ -binding proteins that function in the stomatal CO2 response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2 -induced stomatal closure and, unexpectedly, also in low CO2 -induced stomatal opening. Furthermore, K+ -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2 -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

Adenosine 5' phosphosulfate reductase and sulfite oxidase regulate sulfite-induced water loss in Arabidopsis.

Chloroplast-localized adenosine-5'-phosphosulphate reductase (APR) generates sulfite and plays a pivotal role in reduction of sulfate to cysteine. The peroxisome-localized sulfite oxidase (SO) oxidizes excess sulfite to sulfate. Arabidopsis wild type, SO RNA-interference (SO Ri) and SO overexpression (SO OE) transgenic lines infiltrated with sulfite showed increased water loss in SO Ri plants, and smaller stomatal apertures in SO OE plants compared with wild-type plants. Sulfite application also limited sulfate and abscisic acid-induced stomatal closure in wild type and SO Ri. The increases in APR activity in response to sulfite infiltration into wild type and SO Ri leaves resulted in an increase in endogenous sulfite, indicating that APR has an important role in sulfite-induced increases in stomatal aperture. Sulfite-induced H2O2 generation by NADPH oxidase led to enhanced APR expression and sulfite production. Suppression of APR by inhibiting NADPH oxidase and glutathione reductase2 (GR2), or mutation in APR2 or GR2, resulted in a decrease in sulfite production and stomatal apertures. The importance of APR and SO and the significance of sulfite concentrations in water loss were further demonstrated during rapid, harsh drought stress in root-detached wild-type, gr2 and SO transgenic plants. Our results demonstrate the role of SO in sulfite homeostasis in relation to water consumption in well-watered plants.

The Receptor-like Pseudokinase GHR1 Is Required for Stomatal Closure.

Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion currents in Xenopus laevis oocytes and present supporting in silico and in vitro evidence suggesting that GHR1 is an inactive pseudokinase. Biochemical analyses suggested that GHR1-mediated activation of SLAC1 occurs via interacting proteins and that CALCIUM-DEPENDENT PROTEIN KINASE3 interacts with GHR1. We propose that GHR1 acts in stomatal closure as a scaffolding component.

Stomatal VPD Response: There Is More to the Story Than ABA.

Guard cells shrink and close stomatal pores when air humidity decreases (i.e. when the difference between the vapor pressures of leaf and atmosphere [VPD] increases). The role of abscisic acid (ABA) in VPD-induced stomatal closure has been studied using ABA-related mutants that respond to VPD in some studies and not in others. The importance of ABA biosynthesis in guard cells versus vasculature for whole-plant stomatal regulation is unclear as well. Here, we show that Arabidopsis (Arabidopsis thaliana) lines carrying mutations in different steps of ABA biosynthesis as well as pea (Pisum sativum) wilty and tomato (Solanum lycopersicum) flacca ABA-deficient mutants had higher stomatal conductance compared with wild-type plants. To characterize the role of ABA production in different cells, we generated transgenic plants where ABA biosynthesis was rescued in guard cells or phloem companion cells of an ABA-deficient mutant. In both cases, the whole-plant stomatal conductance, stunted growth phenotype, and leaf ABA level were restored to wild-type values, pointing to the redundancy of ABA sources and to the effectiveness of leaf ABA transport. All ABA-deficient lines closed their stomata rapidly and extensively in response to high VPD, whereas plants with mutated protein kinase OST1 showed stunted VPD-induced responses. Another strongly ABA-insensitive mutant, defective in the six ABA PYR/RCAR receptors, responded to changes in VPD in both directions strongly and symmetrically, indicating that its VPD-induced closure could be passive hydraulic. We discuss that both the VPD-induced passive hydraulic stomatal closure and the stomatal VPD regulation of ABA-deficient mutants may be conditional on the initial pretreatment stomatal conductance.

Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification.

The Arabidopsis (Arabidopsis thaliana) aldehyde oxidases are a multigene family of four oxidases (AAO1-AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification.

Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants.

Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5'-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves.

Sulfite reductase protects plants against sulfite toxicity.

Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum 'Rheinlands Ruhm') and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances.

Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.

A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants.

Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.

Stomatal CO2/bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12/MPK4.

The continuing rise in the atmospheric carbon dioxide (CO2) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO2 sensor remains unknown. Here, we show that elevated CO2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO2, HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO2. Dominant HT1 mutants abrogate the CO2/bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO2 sensor function and CO2-triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO2/bicarbonate sensor upstream of the CBC1 kinase in plants.

Differential role of MAX2 and strigolactones in pathogen, ozone, and stomatal responses.

Strigolactones are a group of phytohormones that control developmental processes including shoot branching and various plant-environment interactions in plants. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria. Here we show that both strigolactone biosynthesis (max3 and max4) and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. Further analysis of gene expression revealed no major role for strigolactone in regulation of defense gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4, and d14 mutants. We analyzed stomatal responses to stimuli that cause stomatal closure. While the response to abscisic acid (ABA) was not impaired in any of the mutants, the response to darkness and high CO2 was impaired in max2 and d14-1 mutants, and to CO2 also in strigolactone synthesis (max3, max4) mutants. To position the role of MAX2 in the guard cell signaling network, max2 was crossed with mutants defective in ABA biosynthesis or signaling. This revealed that MAX2 acts in a signaling pathway that functions in parallel to the guard cell ABA signaling pathway. We propose that the impaired defense responses of max2 are related to higher stomatal conductance that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signaling (related to CO2 signaling), this protein could be one of the components that allow guard cells to distinguish between different environmental conditions.

Reactive Oxygen Species, Photosynthesis, and Environment in the Regulation of Stomata.

SIGNIFICANCE: Stomata sense the intercellular carbon dioxide (CO2) concentration (Ci) and water availability under changing environmental conditions and adjust their apertures to maintain optimal cellular conditions for photosynthesis. Stomatal movements are regulated by a complex network of signaling cascades where reactive oxygen species (ROS) play a key role as signaling molecules. Recent Advances: Recent research has uncovered several new signaling components involved in CO2- and abscisic acid-triggered guard cell signaling pathways. In addition, we are beginning to understand the complex interactions between different signaling pathways. CRITICAL ISSUES: Plants close their stomata in reaction to stress conditions, such as drought, and the subsequent decrease in Ci leads to ROS production through photorespiration and over-reduction of the chloroplast electron transport chain. This reduces plant growth and thus drought may cause severe yield losses for agriculture especially in arid areas. FUTURE DIRECTIONS: The focus of future research should be drawn toward understanding the interplay between various signaling pathways and how ROS, redox, and hormonal balance changes in space and time. Translating this knowledge from model species to crop plants will help in the development of new drought-resistant crop species with high yields.

5-arylethynyl-2'-deoxyuridines: energy transfer and SNP-detection.

Energy transfer between different fluorescent 5-alkynyl-2' -deoxyuridines in complementary and mismatched duplexes was studied.

Isolation of Guard-cell Enriched Tissue for RNA Extraction.

This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes.

Determination of Enzymes Associated with Sulfite Toxicity in Plants: Kinetic Assays for SO, APR, SiR, and In-Gel SiR Activity.

The amino acid cysteine plays a major role in plant response to abiotic stress by being the donor of elemental sulfur for the sulfuration of the molybdenum cofactor, otherwise the last step of ABA biosynthesis, the oxidation of abscisic aldehyde, is inactivated. Additionally, cysteine serves as a precursor for the biosynthesis of glutathione, the reactive oxygen species scavenger essential for redox status homeostasis during stress. Cysteine is generated by the sulfate reductive pathway where sulfite oxidase (SO; EC 1.8.3.1) is an important enzyme in the homeostasis of sulfite levels (present either as a toxic intermediate in the pathway or as a toxic air pollutant that has penetrated the plant tissue via the stomata). SO is localized to the peroxisomes and detoxifies excess sulfite by catalyzing its oxidation to sulfate. Here we show a kinetic assay that relies on fuchsin colorimetric detection of sulfite, a substrate of SO activity. This SO assay is highly specific, technically simple, and readily performed in any laboratory.5'-adenylylsulfate (APS) reductase (APR, E.C. 1.8.4.9) enzyme regulates a crucial step of sulfate assimilation in plants, algae and some human pathogens. The enzyme is upregulated in response to oxidative stress induced by abiotic stresses, such as salinity and hydrogen peroxide, to generate sulfite an intermediate for cysteine generation essential for the biosynthesis of glutathione, the hydrogen peroxide scavenger. Here we present two robust, sensitive, and simple colorimetric methods of APR activity based on sulfite determination by fuchsin.Sulfite reductase (SiR) is one of the key enzymes in the primary sulfur assimilation pathway. It has been shown that SiR is an important plant enzyme for protection plant against sulfite toxicity and premature senescence. Here we describe two methods for SiR activity determination: a kinetic assay using desalted extract and an in-gel assay using crude extract.Due to the energetically favorable equilibrium, sulfurtransferase (ST) activity measured as sulfite generation or consumption. Sulfite-generating ST activity is determined by colorimetric detection of SCN- formation at 460 nm as the red Fe(SCN)3 complex from cyanide and thiosulfate using acidic iron reagent. Sulfite-consuming (MST) activity is detected as sulfite disappearance in the presence of thiocyanate (SCN-) or as SCN- disappearance. To abrogate interfering SO activity, total ST activities is detected by inhibiting SO activity with tungstate.