Department of Plant Sciences

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Browsing Department of Plant Sciences by Author "Agurla, Srinivas"
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    Convergence and divergence of signaling events in guard cells during stomatal closure by plant hormones or microbial elicitors
    ( 2016-08-24) Agurla, Srinivas ; Raghavendra, Agepati S.
    Dynamic regulation of stomatal aperture is essential for plants to optimize water use and CO2 uptake. Stomatal opening or closure is accompanied by the modulation of guard cell turgor. Among the events leading to stomatal closure by plant hormones or microbial elicitors, three signaling components stand out as the major converging points. These are reactive oxygen species (ROS), cytosolic free Ca2+, and ion channels. Once formed, the ROS and free Ca2+ of guard cells regulate both downstream and upstream events. A major influence of ROS is to increase the levels of NO and cytosolic free Ca2+ in guard cells. Although the rise in NO is an important event during stomatal closure, the available evidences do not support the description of NO as the point of convergence. The rise in ROS and NO would cause an increase of free Ca2+ and modulate ion channels, through a network of events, in such a way that the guard cells lose K+/Cl−/anions. The efflux of these ions decreases the turgor of guard cells and leads to stomatal closure. Thus, ROS, NO, and cytosolic free Ca2+ act as points of divergence. The other guard cell components, which are modulated during stomatal closure are G-proteins, cytosolic pH, phospholipids, and sphingolipids. However, the current information on the role of these components is not convincing so as to assign them as the points of convergence or divergence. The interrelationships and interactions of ROS, NO, cytosolic pH, and free Ca2+ are quite complex and need further detailed examination. Our review is an attempt to critically assess the current status of information on guard cells, while emphasizing the convergence and divergence of signaling components during stomatal closure. The existing gaps in our knowledge are identified to stimulate further research.
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    Mechanism of stomatal closure in plants exposed to drought and cold stress
    ( 2018-01-01) Agurla, Srinivas ; Gahir, Shashibhushan ; Munemasa, Shintaro ; Murata, Yoshiyuki ; Raghavendra, Agepati S.
    Drought is one of the abiotic stresses which impairs the plant growth/development and restricts the yield of many crops throughout the world. Stomatal closure is a common adaptation response of plants to the onset of drought condition. Stomata are microscopic pores on the leaf epidermis, which regulate the transpiration/CO 2 uptake by leaves. Stomatal guard cells can sense various abiotic and biotic stress stimuli from the internal and external environment and respond quickly to initiate closure under unfavorable conditions. Stomata also limit the entry of pathogens into leaves, restricting their invasion. Drought is accompanied by the production and/or mobilization of the phytohormone, abscisic acid (ABA), which is well-known for its ability to induce stomatal closure. Apart from the ABA, various other factors that accumulate during drought and affect the stomatal function are plant hormones (auxins, MJ, ethylene, brassinosteroids, and cytokinins), microbial elicitors (salicylic acid, harpin, Flg 22, and chitosan), and polyamines. The role of various signaling components/secondary messengers during stomatal opening or closure has been a matter of intense investigation. Reactive oxygen species (ROS), nitric oxide (NO), cytosolic pH, and calcium are some of the well-documented signaling components during stomatal closure. The interrelationship and interactions of these signaling components such as ROS, NO, cytosolic pH, and free Ca 2+ are quite complex and need further detailed examination. Low temperatures can have deleterious effects on plants. However, plants evolved protection mechanisms to overcome the impact of this stress. Cold temperature inhibits stomatal opening and causes stomatal closure. Cold-acclimated plants often exhibit marked changes in their lipid composition, particularly of the membranes. Cold stress often leads to the accumulation of ABA, besides osmolytes such as glycine betaine and proline. The role of signaling components such as ROS, NO, and Ca 2+ during cold acclimation is yet to be established, though the effects of cold stress on plant growth and development are studied extensively. The information on the mitigation processes is quite limited. We have attempted to describe consequences of drought and cold stress in plants, emphasizing stomatal closure. Several of these factors trigger signaling components in roots, shoots, and atmosphere, all leading to stomatal closure. A scheme is presented to show the possible signaling events and their convergence and divergence of action during stomatal closure. The possible directions for future research are discussed.
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    Methyl salicylate is the most effective natural salicylic acid ester to close stomata while raising reactive oxygen species and nitric oxide in Arabidopsis guard cells
    ( 2020-12-01) Agurla, Srinivas ; Sunitha, Vaidya ; Raghavendra, Agepati S.
    Modulation by salicylic acid (SA) and its six esters of stomatal closure was evaluated in Arabidopsis thaliana. The seven compounds tested are salicylic acid (SA), acetylsalicylate (ASA), methyl salicylate (MeSA), propyl salicylate (PrSA), amyl salicylate, benzyl salicylate, and salicin. Among these, MeSA was the most effective to induce stomatal closure, followed by salicin and SA, while ASA was the least effective. Since SA, ASA, and MeSA could modulate plant function, the effects of these three compounds on the levels of reactive oxygen species (ROS) or nitric oxide (NO) in guard cells were studied. MeSA and SA raised the content of ROS or NO in as with ABA. The extent of ROS/NO production in response to ASA was the lowest. Reversal by cPTIO or catalase of stomatal closure by MeSA indicated the essentiality of NO and ROS for stomatal closure. Further studies revealed peroxidase as the ROS source during stomatal closure by MeSA, unlike the dominant role of NADPH oxidase in ROS production induced by ABA. The rise in NO production by ABA or MeSA was dependent on nitrate reductase and NO synthase-like enzyme. Given its most effective nature, MeSA can be an excellent tool to examine the signaling components in guard cells and other plant tissues. The ability of MeSA to induce stomatal closure is physiologically relevant because of its volatile nature, stability, and systemic action.
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    Nitric oxide (NO) measurements in stomatal guard cells
    ( 2016-04-01) Agurla, Srinivas ; Gayatri, Gunja ; Raghavendra, Agepati S.
    The quantitative measurement of nitric oxide (NO) in plant cells acquired great importance, in view of the multifaceted function and involvement of NO as a signal in various plant processes. Monitoring of NO in guard cells is quite simple because of the large size of guard cells and ease of observing the detached epidermis under microscope. Stomatal guard cells therefore provide an excellent model system to study the components of signal transduction. The levels and functions of NO in relation to stomatal closure can be monitored, with the help of an inverted fluorescence or confocal microscope. We can measure the NO in guard cells by using flouroprobes like 4,5-diamino fluorescein diacetate (DAF-2DA). This fluorescent dye, DAF-2DA, is cell permeable and after entry into the cell, the diacetate group is removed by the cellular esterases. The resulting DAF-2 form is membrane impermeable and reacts with NO to generate the highly fluorescent triazole (DAF-2T), with excitation and emission wavelengths of 488 and 530 nm, respectively. If time-course measurements are needed, the epidermis can be adhered to a cover-glass or glass slide and left in a small petri dishes. Fluorescence can then be monitored at required time intervals; with a precaution that excitation is done minimally, only when a fluorescent image is acquired. The present method description is for the epidermis of Arabidopsis thaliana and Pisum sativum and should work with most of the other dicotyledonous plants.
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    Nitric oxide as a secondary messenger during stomatal closure as a part of plant immunity response against pathogens
    ( 2014-12-01) Agurla, Srinivas ; Gayatri, Gunja ; Raghavendra, Agepati S.
    Stomata facilitate the loss of water, as well as CO2 uptake for photosynthesis. In addition, stomatal closure restricts the entry of pathogens into leaves and forms a part of plant defense response. Plants have evolved ways to modulate stomata by plant hormones as well as microbial elicitors, including pathogen/microbe associated molecular patterns. Stomatal closure initiated by signals of either abiotic or biotic factors results from the loss of guard cell turgor due mainly to K+/anion efflux. Nitric oxide (NO) is a key element among the signaling elements leading to stomatal closure, hypersensitive response and programmed cell death. Due to the growing importance of NO as signaling molecule in plants, and the strong relation between stomata and pathogen resistance, we attempted to present a critical overview of plant innate immunity, in relation to stomatal closure. The parallel role of NO during plant innate immunity and stomatal closure is highlighted. The cross-talk between NO and other signaling components, such as reactive oxygen species (ROS) is discussed. The possible sources of NO and mechanisms of NO action, through post-translational modification of proteins are discussed. The mini-review is concluded with remarks on the existing gaps in our knowledge and suggestions for future research.
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    Nitric oxide in guard cells as an important secondary messenger during stomatal closure
    ( 2013-10-29) Gayatri, Gunja ; Agurla, Srinivas ; Raghavendra, Agepati S.
    The modulation of guard cell function is the basis of stomatal closure, essential for optimizing water use and CO2 uptake by leaves. Nitric oxide (NO) in guard cells plays a very important role as a secondary messenger during stomatal closure induced by effectors, including hormones. For example, exposure to abscisic acid (ABA) triggers a marked increase in NO of guard cells, well before stomatal closure. In guard cells of multiple species, like Arabidopsis, Vicia and pea, exposure to ABA or methyl jasmonate or even microbial elicitors (e.g., chitosan) induces production of NO as well as reactive oxygen species (ROS). The role of NO in stomatal closure has been confirmed by using NO donors (e.g., SNP) and NO scavengers (like cPTIO) and inhibitors of NOS (L-NAME) or NR (tungstate). Two enzymes: a L-NAME-sensitive, nitric oxide synthase (NOS)-like enzyme and a tungstate-sensitive nitrate reductase (NR), can mediate ABA-induced NO rise in guard cells. However, the existence of true NOS in plant tissues and its role in guard cell NO-production are still a matter of intense debate. Guard cell signal transduction leading to stomatal closure involves the participation of several components, besides NO, such as cytosolic pH, ROS, free Ca2+, and phospholipids. Use of fluorescent dyes has revealed that the rise in NO of guard cells occurs after the increase in cytoplasmic pH and ROS. The rise in NO causes an elevation in cytosolic free Ca2+ and promotes the efflux of cations as well as anions from guard cells. Stomatal guard cells have become a model system to study the signaling cascade mechanisms in plants, particularly with NO as a dominant component. The interrelationships and interactions of NO with cytosolic pH, ROS, and free Ca2+ are quite complex and need further detailed examination. While assessing critically the available literature, the present review projects possible areas of further work related to NO-action in stomatal guard cells. © 2013. Gayatri, Agurla and Raghavendra.
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    Polyamines increase nitric oxide and reactive oxygen species in guard cells of Arabidopsis thaliana during stomatal closure
    ( 2018-01-01) Agurla, Srinivas ; Gayatri, Gunja ; Raghavendra, Agepati S.
    A comprehensive study which was undertaken on the effect of three polyamines (PAs) on stomatal closure was examined in relation to nitric oxide (NO) and reactive oxygen species (ROS) levels in guard cells of Arabidopsis thaliana. Three PAs—putrescine (Put), spermidine (Spd), and spermine (Spm)—induced stomatal closure, while increasing the levels of NO as well as ROS in guard cells. The roles of NO and ROS were confirmed by the reversal of closure by cPTIO (NO scavenger) and catalase (ROS scavenger). The presence of L-NAME (NOS-like enzyme inhibitor) reversed PA-induced stomatal closure, suggesting that NOS-like enzyme played a significant role in NO production during stomatal closure. The reversal of stomatal closure by diphenylene iodonium (DPI, NADPH oxidase inhibitor) or 2-bromoethylamine (BEA, copper amine oxidase inhibitor) or 1,12 diaminododecane (DADD, polyamine oxidase inhibitor) was partial. In contrast, the presence of DPI along with BEA/DADD reversed completely the closure by PAs. We conclude that both NO and ROS are essential signaling components during Put-, Spd-, and Spm-induced stomatal closure. The PA-induced ROS production is mediated by both NADPH oxidase and amine oxidase. The rise in ROS appears to be upstream of NO. Ours is the first detailed study on the role of NO and its dependence on ROS during stomatal closure by three major PAs.
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    Stomatal closure and rise in ROS/NO of arabidopsis guard cells by tobacco microbial elicitors: Cryptogein and harpin
    ( 2017-06-21) Gayatri, Gunja ; Agurla, Srinivas ; Kuchitsu, Kazuyuki ; Anil, Kondreddy ; Podile, Appa R. ; Raghavendra, Agepati S.
    Plants use stomatal closure mediated by elicitors as the first step of the innate immune response to restrict the microbial entry. We present a comprehensive study of the effect of cryptogein and harpin, two elicitors from microbial pathogens of tobacco, on stomatal closure and guard cell signaling components in Arabidopsis thaliana, a model plant. Cryptogein as well as harpin induced stomatal closure, while elevating the levels of reactive oxygen species (ROS) and nitric oxide (NO) in the guard cells of A. thaliana. Kinetic studies with fluorescent dyes revealed that the rise in ROS levels preceded that of NO in guard cells, when treated with these two elicitors. The restriction of NO levels in guard cells, even by ROS modulators indicates the essentiality of ROS for NO production during elicitor-triggered stomatal closure. The signaling events during elicitor-induced stomatal closure appear to converge at NADPH oxidase and ROS production. Our results provide the first report on stomatal closure associated with rise in ROS/NO of guard cells by cryptogein and harpin in A. thaliana. Our results establish that A. thaliana can be used to study stomatal responses to the typical elicitors from microbial pathogens of other plants. The suitability of Arabidopsis opens up an excellent scope for further studies on signaling events leading to stomatal closure by microbial elicitors.
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    Stomatal closure and rise in ROS/NO of arabidopsis guard cells by tobacco microbial elicitors: Cryptogein and harpin
    ( 2017-06-21) Gayatri, Gunja ; Agurla, Srinivas ; Kuchitsu, Kazuyuki ; Anil, Kondreddy ; Podile, Appa R. ; Raghavendra, Agepati S.
    Plants use stomatal closure mediated by elicitors as the first step of the innate immune response to restrict the microbial entry. We present a comprehensive study of the effect of cryptogein and harpin, two elicitors from microbial pathogens of tobacco, on stomatal closure and guard cell signaling components in Arabidopsis thaliana, a model plant. Cryptogein as well as harpin induced stomatal closure, while elevating the levels of reactive oxygen species (ROS) and nitric oxide (NO) in the guard cells of A. thaliana. Kinetic studies with fluorescent dyes revealed that the rise in ROS levels preceded that of NO in guard cells, when treated with these two elicitors. The restriction of NO levels in guard cells, even by ROS modulators indicates the essentiality of ROS for NO production during elicitor-triggered stomatal closure. The signaling events during elicitor-induced stomatal closure appear to converge at NADPH oxidase and ROS production. Our results provide the first report on stomatal closure associated with rise in ROS/NO of guard cells by cryptogein and harpin in A. thaliana. Our results establish that A. thaliana can be used to study stomatal responses to the typical elicitors from microbial pathogens of other plants. The suitability of Arabidopsis opens up an excellent scope for further studies on signaling events leading to stomatal closure by microbial elicitors.
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    Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in Pisum sativum
    ( 2016-10-01) Puli, Mallikarjuna Rao ; Rajsheel, Pidakala ; Aswani, Vetcha ; Agurla, Srinivas ; Kuchitsu, Kazuyuki ; Raghavendra, Agepati S.
    Main conclusion: Phyto-S1P and S1P induced stomatal closure in epidermis of pea (Pisum sativum) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N’,N’-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.