The emerging roles of nitric oxide and its associated scavengers-phytoglobins-in plant symbiotic interactions.

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.

Nitric oxide regulates mitochondrial biogenesis in plants.

The site of nitric oxide (NO) production in mitochondrial cytochrome c oxidase and the role of NO in mitochondrial biogenesis are not known in plants. By imposing osmotic stress and recovery on Arabidopsis seedlings we investigated the site of NO production and its role in mitochondrial biogenesis. Osmotic stress reduced growth and mitochondrial number while increasing NO production. During the recovery phase the mitochondrial number increased and this increase was higher in wild type and the high NO-producing Pgb1 silencing line in comparison to the NO-deficient nitrate reductase double mutant (nia1/nia2). Application of nitrite stimulated NO production and mitochondrial number in the nia1/nia2 mutant. Osmotic stress induced COX6b- 3 and COA6-L genes encoding subunits of COX. The mutants cox6b-3 and coa6-l were impaired both in NO production and mitochondrial number during stress to recovery suggesting the involvement of these subunits in nitrite-dependent NO production. Transcripts encoding the mitochondrial protein import machinery showed reduced expression in cox6b-3 and coa6-l mutants. Finally, COX6b-3 and COA6-L interacted with the VQ27 motif-containing protein in the presence of NO. The vq27 mutant was impaired in mitochondrial biogenesis. Our results suggest the involvement of COX derived NO in mitochondrial biogenesis.

A small cysteine-rich fungal effector, BsCE66 is essential for the virulence of Bipolaris sorokiniana on wheat plants.

The Spot Blotch (SB) caused by hemibiotrophic fungal pathogen Bipolaris sorokiniana is one of the most devastating wheat diseases leading to 15-100% crop loss. However, the biology of Triticum-Bipolaris interactions and host immunity modulation by secreted effector proteins remain underexplored. Here, we identified a total of 692 secretory proteins including 186 predicted effectors encoded by B. sorokiniana genome. Gene Ontology categorization showed that these proteins belong to cellular, metabolic and signaling processes, and exhibit catalytic and binding activities. Further, we functionally characterized a cysteine-rich, B. sorokiniana Candidate Effector 66 (BsCE66) that was induced at 24-96 hpi during host colonization. The Δbsce66 mutant did not show vegetative growth defects or stress sensitivity compared to wild-type, but developed drastically reduced necrotic lesions upon infection in wheat plants. The loss-of-virulence phenotype was rescued upon complementing the Δbsce66 mutant with BsCE66 gene. Moreover, BsCE66 does not form homodimer and conserved cysteine residues form intra-molecular disulphide bonds. BsCE66 localizes to the host nucleus and cytosol, and triggers a strong oxidative burst and cell death in Nicotiana benthamiana. Overall, our findings demonstrate that BsCE66 is a key virulence factor that is necessary for host immunity modulation and SB disease progression. These findings would significantly improve our understanding of Triticum-Bipolaris interactions and assist in the development of SB resistant wheat varieties.

Detection of Nitric Oxide from Chickpea Using DAF Fluorescence and Chemiluminescence Methods.

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.

Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice.

An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.

Nitric oxide regulation of plant metabolism.

Nitric oxide (NO) has emerged as an important signal molecule in plants, having myriad roles in plant development. In addition, NO also orchestrates both biotic and abiotic stress responses, during which intensive cellular metabolic reprogramming occurs. Integral to these responses is the location of NO biosynthetic and scavenging pathways in diverse cellular compartments, enabling plants to effectively organize signal transduction pathways. NO regulates plant metabolism and, in turn, metabolic pathways reciprocally regulate NO accumulation and function. Thus, these diverse cellular processes are inextricably linked. This review addresses the numerous redox pathways, located in the various subcellular compartments that produce NO, in addition to the mechanisms underpinning NO scavenging. We focus on how this molecular dance is integrated into the metabolic state of the cell. Within this context, a reciprocal relationship between NO accumulation and metabolite production is often apparent. We also showcase cellular pathways, including those associated with nitrate reduction, that provide evidence for this integration of NO function and metabolism. Finally, we discuss the potential importance of the biochemical reactions governing NO levels in determining plant responses to a changing environment.

Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading.

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading.IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.

mQTL-seq and classical mapping implicates the role of an AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family gene in Ascochyta blight resistance of chickpea.

Ascochyta blight (AB) caused by the fungal pathogen Ascochyta rabiei is a serious foliar disease of chickpea (Cicer arietinum L.). Despite many genetic studies on chickpea-Ascochyta interaction, genome-wide scan of chickpea for the identification of AB-associated quantitative trait loci (QTLs) and their gene(s) has not been accomplished. To elucidate narrow QTLs for AB resistance, here, we report the use of multiple QTL-sequencing approach on 2 sets of extreme AB phenotype bulks derived from Cicer intraspecific and interspecific crosses. Two major QTLs, qABR4.1 and qABR4.2, and a minor QTL, qABR4.3, were identified on assembled chickpea pseudomolecule 4. We narrowed qABR4.1 to a "robust region" at 4.568-4.618 Mb through mapping on a larger intraspecific cross-derived population and comparative analysis. Among 4 genes, the CaAHL18 gene showed higher expression under Ascochyta stress in AB resistant parent suggesting that it is the candidate gene under "robust qABR4.1." Dual-luciferase assay with CaAHL18 polymorphic cis-regulatory sequences showed that allelic variation is associated with higher expression. Thus, our findings on chickpea-Ascochyta interaction have narrowed down AB resistance associated QTLs on chickpea physical map. The narrowed QTLs and gene-associated markers will help in biotechnological and breeding programs for chickpea improvement.

Phylogenomic analysis of MKKs and MAPKs from 16 legumes and detection of interacting pairs in chickpea divulge MAPK signalling modules.

The mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascade is a vital component of plant cellular signalling. Despite this, MAPK signalling cascade is less characterized in crop legumes. To fill this void, we present here a comprehensive phylogeny of MAPK kinases (MKKs) and MAPKs identified from 16 legume species belonging to genistoid (Lupinus angustifolius), dalbergioid (Arachis spp.), phaseoloid (Glycine max, Cajanus cajan, Phaseolus vulgaris, and Vigna spp.), and galegoid (Cicer arietinum, Lotus japonicus, Medicago truncatula, Pisum sativum, Trifolium spp., and Vicia faba) clades. Using the genes of the diploid crop chickpea (C. arietinum), an exhaustive interaction analysis was performed between MKKs and MAPKs by split-ubiquitin based yeast two-hybrid (Y2H). Twenty seven interactions of varying strengths were identified between chickpea MKKs and MAPKs. These interactions were verified in planta by bimolecular fluorescence complementation (BiFC). As a first report in plants, four intra-molecular interactions of weak strength were identified within chickpea MKKs. Additionally; two TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors of class I were identified as novel down-stream interacting partners of seven MAPKs. We propose that this highly reliable MAPK interaction network, presented here for chickpea, can be utilized as a reference for legumes and thus will help in deciphering their role in legume-specific events.