Browsing by Author "Loake, Gary J."

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Detection of nitric oxide from chickpea using DAF fluorescence and chemiluminescence methods
(John Wiley & Sons, 2022) Kumari, Aprajita; Bhatoee, Manbir; Singh, Pooja; Kaladhar, Vemula Chandra; Yadav, Nidhi; Paul, Debarati; Loake, Gary J.; Gupta, Kapuganti Jagadis
The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half-life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell-permeable forms (DAF-FM-DA). In this method, the tissues are immersed in DAF-FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF-FM release. The resulting DAF-FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF-FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non-invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF-FM-DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF-FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings
The emerging roles of nitric oxide and its associated scavengers-phytoglobins-in plant symbiotic interactions
(Oxford University Press, 2024) Pathak, Pradeep Kumar; Yadav, Nidhi; Kaladhar, Vemula Chandra; Jaiswal, Rekha; Kumari, Aprajita; Igamberdiev, Abir U.; Loake, Gary J.; Gupta, Kapuganti Jagadis
A key feature in the establishment of symbiosis between plants and microbes is the maintenance of the balance between the production of the small redox-related molecule, nitric oxide (NO), and its cognate scavenging pathways. During the establishment of symbiosis, a transition from a normoxic to a microoxic environment often takes place, triggering the production of NO from nitrite via a reductive production pathway. Plant hemoglobins [phytoglobins (Phytogbs)] are a central tenant of NO scavenging, with NO homeostasis maintained via the Phytogb–NO cycle. While the first plant hemoglobin (leghemoglobin), associated with the symbiotic relationship between leguminous plants and bacterial Rhizobium species, was discovered in 1939, most other plant hemoglobins, identified only in the 1990s, were considered as non-symbiotic. From recent studies, it is becoming evident that the role of Phytogbs1 in the establishment and maintenance of plant–bacterial and plant–fungal symbiosis is also essential in roots. Consequently, the division of plant hemoglobins into symbiotic and non-symbiotic groups becomes less justified. While the main function of Phytogbs1 is related to the regulation of NO levels, participation of these proteins in the establishment of symbiotic relationships between plants and microorganisms represents another important dimension among the other processes in which these key redox-regulatory proteins play a central role.
Expanding roles for S-nitrosylation in the regulation of plant immunity
(Elsevier B.V., 2023) Borrowman, Sam; Gupta, Kapuganti Jagadis; Loake, Gary J.
Following pathogen recognition, plant cells produce a nitrosative burst resulting in a striking increase in nitric oxide (NO), altering the redox state of the cell, which subsequently helps orchestrate a plethora of immune responses. NO is a potent redox cue, efficiently relayed between proteins through its co-valent attachment to highly specific, powerfully reactive protein cysteine (Cys) thiols, resulting in formation of protein S-nitrosothiols (SNOs). This process, known as S-nitrosylation, can modulate the function of target proteins, enabling responsiveness to cellular redox changes. Key targets of S-nitrosylation control the production of reactive oxygen species (ROS), the transcription of immune-response genes, the triggering of the hypersensitive response (HR) and the establishment of systemic acquired resistance (SAR). Here, we bring together recent advances in the control of plant immunity by S-nitrosylation, furthering our appreciation of how changes in cellular redox status reprogramme plant immune function.
New insights into nitric oxide biosynthesis underpin lateral root development
(Elsevier B.V., 2024) Gupta, Kapuganti Jagadis; Yadav, Nidhi; Kumari, Aprajita; Loake, Gary J.
In recent years, nitric oxide (NO) has emerged as a key redox signaling molecule in plants functioning in the regulation of key developmental programs and the orchestration of responses to a plethora of environmental cues (Kolbert et al., 2019). The predominant route for the transfer of NO bioactivity is through S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (Yun et al., 2011). Specificity for this process is established by the structural constraints imposed by tertiary protein structure in gating access to given cysteine redox switches and associated proteins that can either facilitate the addition or removal of the NO moiety at these residues (Umbreen et al., 2018).
Nitric oxide accelerates germination via the regulation of respiration in chickpea
(Oxford University Press, 2019) Pandey, Sonika; Kumari, Aprajita; Shree, Manu; Kumar, Vinod; Singh, Pooja; Bharadwaj, Chellapilla; Loake, Gary J.; Parida, Swarup K.; Masakapalli, Shyam Kumar; Gupta, Kapuganti Jagadis
Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1 H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.
Nitric oxide-releasing nanomaterials: from basic research to potential biotechnological applications in agriculture
(John Wiley & Sons, 2022) Seabra, Amedea B.; Silveira, Neidiquele M.; Ribeiro, Rafael V.; Pieretti, Joana C.; Barroso, Juan B.; Corpas, Francisco J.; Palma, José M.; Hancock, John T.; Petřivalský, Marek; Gupta, Kapuganti Jagadis; Wendehenne, David; Loake, Gary J.; Durner, Jorg; Lindermayr, Christian; Molnár, Árpád; Kolbert, Zsuzsanna; Oliveira, Halley C.
Nitric oxide (NO) is a multifunctional gaseous signal that modulates the growth, development and stress tolerance of higher plants. NO donors have been used to boost plant endogenous NO levels and to activate NO-related responses, but this strategy is often hindered by the relative instability of donors. Alternatively, nanoscience offers a new, promising way to enhance NO delivery to plants, as NO-releasing nanomaterials (e.g., S-nitrosothiol-containing chitosan nanoparticles) have many beneficial physicochemical and biochemical properties compared to non-encapsulated NO donors. Nano NO donors are effective in increasing tissue NO levels and enhancing NO effects both in animal and human systems. The authors believe, and would like to emphasize, that new trends and technologies are essential for advancing plant NO research and nanotechnology may represent a breakthrough in traditional agriculture and environmental science. Herein, we aim to draw the attention of the scientific community to the potential of NO-releasing nanomaterials in both basic and applied plant research as alternatives to conventional NO donors, providing a brief overview of the current knowledge and identifying future research directions. We also express our opinion about the challenges for the application of nano NO donors, such as the environmental footprint and stakeholder’s acceptance of these materials.
Novel and conserved functions of S-nitrosoglutathione reductase in tomato
(Oxford University Press, 2019) Hussain, Adil; Yun, Byung-Wook; Kim, Ji Hyun; Gupta, Kapuganti Jagadis; Hyung, Nam-In; Loake, Gary J.
Nitric oxide (NO) is emerging as a key signalling molecule in plants. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by depleting S-nitrosoglutathione (GSNO), the major cellular NO donor. Here we show that depletion of GSNOR function impacts tomato (Solanum lycopersicum. L) fruit development. Thus, reduction of GSNOR expression through RNAi modulated both fruit formation and yield, establishing a novel function for GSNOR. Further, depletion of S. lycopersicum GSNOR (SlGSNOR) additionally impacted a number of other developmental processes, including seed development, which also has not been previously linked with GSNOR activity. In contrast to Arabidopsis, depletion of GSNOR function did not influence root development. Further, reduction of GSNOR transcript abundance compromised plant immunity. Surprisingly, this was in contrast to previous data in Arabidopsis that reported that reducing Arabidopsis thaliana GSNOR (AtGSNOR) expression by antisense technology increased disease resistance. We also show that increased SlGSNOR expression enhanced pathogen protection, uncovering a potential strategy to enhance disease resistance in crop plants. Collectively, our findings reveal, at the genetic level, that some but not all GSNOR activities are conserved outside the Arabidopsis reference system. Thus, manipulating the extent of GSNOR expression may control important agricultural traits in tomato and possibly other crop plants.
The PHYTOGLOBIN-NO cycle regulates plant mycorrhizal symbiosis
(Elsevier B.V., 2019) Kumari, Aprajita; Pathak, Pradeep Kumar; Loake, Gary J.; Gupta, Kapuganti Jagadis
The production of the redox-active signaling molecule, NO, has long been associated with interactions between microbes and their host plants. Emerging evidence now suggests that specific NO signatures and cognate patterns of PHYTOGLOBIN1 (PHYTOGB1) expression, a key regulator of cellular NO homeostasis, may help determine either symbiosis or pathogenicity.
Recommendations on terminology and experimental best practice associated with plant nitric oxide research
(John Wiley & Sons, 2020) Gupta, Kapuganti Jagadis; Hancock, John T.; Petrivalsky, Marek; Kolbert, Zsuzsanna; Lindermayr, Christian; Durner, Jorg; Barroso, Juan B.; Palma, José M.; Brouquisse, Renaud; Wendehenne, David; Corpas, Francisco J.; Loake, Gary J.
Nitric oxide (NO) emerged as a key signal molecule in plants. During the last two decades impressive progress has been made in plant NO research. This small, redox‐active molecule is now known to play an important role in plant immunity, stress responses, environmental interactions, plant growth and development. To more accurately and robustly establish the full spectrum of NO bioactivity in plants, it will be essential to apply methodological best practice. In addition, there are some instances of conflicting nomenclature within the field, which would benefit from standardisation. In this context, we attempt to provide some helpful guidance for best practice associated with NO research and also suggestions for the cognate terminology.
Regulating the regulator: nitric oxide control of post-translational modifications
(John Wiley & Sons, Inc., 2020) Gupta, Kapuganti Jagadis; Kolbert, Zsuzsanna; Durner, Jorg; Lindermayr, Christian; Corpas, Francisco J.; Brouquisse, Renaud; Barroso, Juan B.; Saima, Umbreen; Palma, José M; Hancock, John T.; Petrivalsky, Marek; Wendehenne, David; Loake, Gary J.
Nitric oxide (NO) is perfectly suited for duties as a redox signalling molecule. A key route for NO bioactivity occurs via protein S‐nitrosation, the addition of a NO moiety to a protein cysteine (Cys) thiol (‐SH) to form a S‐nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post‐translational modifications (PTMs) including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.