Denise Scuffi

Hydrogen Sulfide Generated by L-Cysteine Desulfhydrase Acts Upstream of Nitric Oxide to Modulate Abscisic Acid-Dependent Stomatal Closure

An l-cysteine desulfhydrase is a unique component of ABA signaling in guard cells, mediating H2S production and acting upstream of nitric oxide to induce stomatal closure. Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H2O2 and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H2O2 levels as the wild type in response to ABA. However, ABA- or H2O2-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H2O2 in ABA-induced stomatal closure.

Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis.

Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well-described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb-4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.

Phospholipase D δ knock-out mutants are tolerant to severe drought stress

Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance.

Gasotransmitters and Stomatal Closure: Is There Redundancy, Concerted Action, or Both?

The epidermis of the aerial part of land plants is pierced by pores through which plants perform gas exchange with environment. The guard cells (GCs), the specialized cells that surround the pore, have the capacity to sense diverse environmental and endogenous stimuli and integrate them into a single output which is the regulation of the stomatal pore width. The stomatal pore size is modulated by changes of the guard cell volume, driven by variations in the osmotic potential of the GCs. The stress hormone abscisic acid (ABA), the master regulator of stomatal movement, induces stomatal closure by the inhibition of H+-ATPases and activation of rapid and slow anion channels, producing the depolarization of the plasma membrane (PM) in GCs, and by an increase in the cytosolic Ca2+ concentratrion [Ca2+]cyt. While the rise of the [Ca2+]cyt blocks the influx of K+ by the inactivation of the inward rectifying K+ channels (Kin+), the depolarization of the PM, in turn promotes K+ efflux driven by outward rectifying K+ channels (Kout+; Blatt, 2000). This process is closely regulated by a complex signaling network that involves the participation of numerous ubiquitous signaling components like ROS, protein kinases, phospholipases, and protein phosphatases (Kim et al., 2010; Song et al., 2014); and by other signaling components that are emerging as active players in this signaling network, such is the case of gasotransmitters (Garcia-Mata and Lamattina, 2013). A gasotransmitter is a small gas molecule that: (i) can freely permeates biological membranes; (ii) it is endogenously generated by specific enzymes; (iii) it has specific functions at physiologically relevant concentrations; (iv) it functions can be mimicked by exogenous application of a donor; and (v) it has specific cellular and molecular targets (Wang, 2002). The group of gasotransmitters is, so far, composed by Nitric Oxide (NO), Carbon Monoxide (CO), and Hydrogen Sulfide (H2S) and the three of them have been reported to participate in the promotion of stomatal closure (Garcia-Mata and Lamattina, 2013), however, the biology of CO in this physiological process is less known than that of NO and H2S. Therefore, this opinion will be focused mainly on the action and interaction of NO and H2S. The two of them are accepted as active players in the regulation of stomatal movement, however there are still obscure points and some of them will be discussed in this opinion article: (i) their specific molecular targets; (ii) the molecular mechanisms underpinning their action; (iii) the interplay between them during the stomatal closure induction; and (iv) the crossed-regulation of their metabolism. All the three gasotransmitters are synthesized during the promotion of stomatal closure. CO is synthesized via the activity of heme oxygenase (Shekhawat and Verma, 2010). In Vicia faba CO induces stomatal closure in a dose-dependent manner and acts upstream of the production of NO during ABA-dependent stomatal closure (Cao et al., 2007; She and Song, 2008). It has been reported that the hormones ABA and ethylene (Eth) require the production of NO for the regulation of stomatal movement (Garcia-Mata and Lamattina, 2002; Neill et al., 2002; He et al., 2011; Song et al., 2011). NO can be synthesized either from NO2 by two genes, NIA1 and NIA2, that code for a nitrate reductase (NR), or from L-arginine in a reaction catalyzed by an enzyme with nitric oxide synthase (NOS)-like activity, even though the involved enzyme named AtNOA1 possesses GTPase activity (Moreau et al., 2008). However, it was reported that the Arabidopsis triple mutant nia1/nia2/atnoa1, which produces very low levels of NO, is hypersensitive to ABA (Lozano-Juste and Leon, 2010), suggesting that NO could have a dual role in ABA-dependent responses. Moreover, it has been found that increased levels of NO are dependent on the NADPHox-dependent production of H2O2 (Bright et al., 2006). NO levels can also be modified by an Alternative Oxidase (AOX). Tobacco plants lacking AOX show high NO levels that impacts in stomatal function (Cvetkovska et al., 2014). Recently Garcia et al. (2010) have shown that Arabidopsis mutant plants in β-Cyanoalanine synthase (Cys-C1), a mitochondrial enzymatic source of H2S, show higher AOX1a transcript levels than wild type, however exogenous application of H2S to rice cell culture induced AOX expression (Xiao et al., 2010). Further physiological studies are needed to clarify this interaction. H2S is produced during the passage of L-cysteine to pyruvate and ammonia in a reaction catalyzed by L-cysteine desulfhydrase (DES1; Alvarez et al., 2010). In Arabidopsis there are three different genes involved in this reaction: the DES1 gene (Alvarez et al., 2010; Scuffi et al., 2014), the At-LCDES gene (Jin et al., 2011), and L-CDes gene (Hou et al., 2013). Recently, it has been shown that the expression of these genes is upregulated in response to ABA, Eth, JA, and SA, all hormones that modulate stomatal movement (Hou et al., 2013). Even if sequence analysis has shown that the promoter region of the DES1 gene contains ABA-responsive elements (Scuffi et al., 2014), further work is needed in order to have a better understanding about the mechanism by which these hormones induce the expression of those genes. Although, DES1 was reported to mediate ABA-dependent stomatal closure (Scuffi et al., 2014), it was recently reported that H2S regulates the activity of Kin+ channel mostly in an ABA- and Ca2+-independent manner, suggesting the existence of ABA-regulated signaling pathways that can be, alternatively, activated in response to other stimuli (Papanatsiou et al., 2015).

Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D-Derived Phosphatidic Acid in Guard Cell Signaling

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in plants that participates in stress responses and development. l-Cys desulfhydrase 1, one of the enzymatic sources of H2S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H2S in stomatal closure and the interplay between H2S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H2O2) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (Arabidopsis thaliana). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H2S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H2S stimulates H2O2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H2S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H2S-induced stomatal closure, and most of the H2S-dependent H2O2 production required the activity of PLDα1. Finally, we showed that H2S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H2S in the signaling network that controls stomatal closure, and suggest that H2S regulates NADPH oxidase and PLD activity in guard cells.

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production

Arabidopsis Phospholipase C2 (PLC2) participates in a branch of microbe-associated molecular pattern-triggered immunity that involves reactive oxygen species-regulated processes. The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC− and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi. However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLCs role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLCs function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-β-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plants tolerance to drought. Our results provide genetic evidence for PLCs involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

Decoding the Interaction Between Nitric Oxide and Hydrogen Sulfide in Stomatal Movement

Stomatal pore regulation is a key process for carbon and water homeostasis of terrestrial plants. The pore size is modulated through changes in the guard cell volume, driven by variations in the osmotic potential of the guard cells. This process is closely regulated by a complex signaling network that involves the participation of several second messengers including gasotransmitters. The importance of gaseous molecules in signaling has been highlighted in the last decade and, in plants, has been reported to modulate many adaptive responses to different biotic and abiotic stresses including the regulation of stomatal movement in response to drought stress. Nitric oxide (NO) and hydrogen sulfide (H2S) have been reported to close the stomata in different plant species, and the production, mode of action, and interplay between them and with other molecules are under constant review. Recently, the interest in animal research has drifted to the functional role of nitroxyl (HNO/NO−), an alternative redox form of NO which is formed by biochemical reactions between H2S and NO in vivo. It has been reported that HNO has effects in different processes, and several works have studied the interaction between H2S and different NO donors demonstrating the formation of new chemical species. One of them is the formation of HNO from the mixture of NaHS, a H2S donor, and SNP, a NO donor. In our lab, we have preliminary data showing that HNO blocks H2S-dependent stomatal closure in Vicia faba. Interestingly, no stomatal closure induction was evidenced when SNP and NaHS were added together, supporting the interaction between these two gasotransmitters.

Knock-Down of Arabidopsis PLC5 Reduces Primary Root Growth and Secondary Root Formation While Overexpression Improves Drought Tolerance and Causes Stunted Root Hair Growth

Phospholipase C (PLC) is a well-known signaling enzyme in metazoans that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate and diacylglycerol as second messengers involved in mutiple processes. Plants contain PLC too, but relatively little is known about its function there. The model system Arabidopsis thaliana contains nine PLC genes. Reversed genetics have implicated several roles for PLCs in plant development and stress signaling. Here, PLC5 is functionally addressed. Promoter-β-glucuronidase (GUS) analyses revealed expression in roots, leaves and flowers, predominantly in vascular tissue, most probably phloem companion cells, but also in guard cells, trichomes and root apical meristem. Only one plc5-1 knock-down mutant was obtained, which developed normally but grew more slowly and exhibited reduced primary root growth and decreased lateral root numbers. These phenotypes could be complemented by expressing the wild-type gene behind its own promoter. Overexpression of PLC5 (PLC5-OE) using the UBQ10 promoter resulted in reduced primary and secondary root growth, stunted root hairs, decreased stomatal aperture and improved drought tolerance. PLC5-OE lines exhibited strongly reduced phosphatidylinositol 4-monophosphate (PIP) and PIP2 levels and increased amounts of phosphatidic acid, indicating enhanced PLC activity in vivo. Reduced PIP2 levels and stunted root hair growth of PLC5-OE seedlings could be recovered by inducible overexpression of a root hair-specific PIP 5-kinase, PIP5K3. Our results show that PLC5 is involved in primary and secondary root growth and that its overexpression improves drought tolerance. Independently, we provide new evidence that PIP2 is essential for the polar tip growth of root hairs.