Aprajita Kumari

Moving nitrogen to the centre of plant defence against pathogens

Background Plants require nitrogen (N) for growth, development and defence against abiotic and biotic stresses. The extensive use of artificial N fertilizers has played an important role in the Green Revolution. N assimilation can involve a reductase series (Symbol → Symbol → Symbol) followed by transamination to form amino acids. Given its widespread use, the agricultural impact of N nutrition on disease development has been extensively examined. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Scope When a pathogen first comes into contact with a host, it is usually nutrient starved such that rapid assimilation of host nutrients is essential for successful pathogenesis. Equally, the host may reallocate its nutrients to defence responses or away from the site of attempted infection. Exogenous application of N fertilizer can, therefore, shift the balance in favour of the host or pathogen. In line with this, increasing N has been reported either to increase or to decrease plant resistance to pathogens, which reflects differences in the infection strategies of discrete pathogens. Beyond considering only N content, the use of Symbol or Symbol fertilizers affects the outcome of plant‐pathogen interactions. Symbol feeding augments hypersensitive response‐ (HR) mediated resistance, while ammonium nutrition can compromise defence. Metabolically, Symbol enhances production of polyamines such as spermine and spermidine, which are established defence signals, with Symbol nutrition leading to increased &ggr;‐aminobutyric acid (GABA) levels which may be a nutrient source for the pathogen. Within the defensive N economy, the roles of nitric oxide must also be considered. This is mostly generated from Symbol by nitrate reductase and is elicited by both pathogen‐associated microbial patterns and gene‐for‐gene‐mediated defences. Nitric oxide (NO) production and associated defences are therefore Symbol dependent and are compromised by Symbol. Conclusion This review demonstrates how N content and form plays an essential role in defensive primary and secondary metabolism and NO‐mediated events.

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant-adaptive response to hypoxia is not known. Here, we investigated whether ethylene-induced aerenchyma requires hypoxia-induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia-induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt) and ethylene biosynthetic inhibitor CoCl2 both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium-dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca-CAMK, inositol 1,4,5-trisphosphate 5-phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.

Interaction of nitric oxide with the components of the plant mitochondrial electron transport chain

Mitochondria are not only major sites for energy production but also participate in several alternative functions, among these generation of nitric oxide (NO), and its different impacts on this organelle, is receiving increasing attention. The inner mitochondrial membrane contains the chain of protein complexes, and electron transfer via oxidation of various organic acids and reducing equivalents leads to generation of a proton gradient that results in energy production. Recent evidence suggests that these complexes are sources and targets for NO. Complex I and rotenone-insensitive NAD(P)H dehydrogenases regulate hypoxic NO production, while complex I also participates in the formation of a supercomplex with complex III under hypoxia. Complex II is a target for NO which, by inhibiting Fe-S centres, regulates reactive oxygen species (ROS) generation. Complex III is one of the major sites for NO production, and the produced NO participates in the phytoglobin-NO cycle that leads to the maintenance of the redox level and limited energy production under hypoxia. Expression of the alternative oxidase (AOX) is induced by NO under various stress conditions, and evidence exists that AOX can regulate mitochondrial NO production. Complex IV is another major site for NO production, which can also be linked to ATP generation via the phytoglobin-NO cycle. Inhibition of complex IV by NO can prevent oxygen depletion at the frontier of anoxia. The NO production and action on various complexes play a major role in NO signalling and energy metabolism.

VisiSens Technique to Measure Internal Oxygen and Respiration in Barley Roots

Respiration is vital for production of energy in plants where oxygen is essential for conducting respiration. Internal oxygen levels in plants depend on respiratory rates. Tissues such as underground roots experience hypoxia due to their isolation from atmospheric oxygen and their internal oxygen depends on tissue size and local oxygen in soil and rhizosphere. Here, we used the ViSisens imaging technique, in which an optical sensor foil (i.e., the planar optode) is attached surface of root. This sensor measures oxygen values from fluorescence. Visisens microscope captures an entire area root surface and gives profile of internal oxygen. Interestingly, this system is also able to measure respiratory rate by measuring oxygen levels in a vial that contains root tissue.

Chemiluminescence Detection of Nitric Oxide from Roots, Leaves, and Root Mitochondria

NO is a free radical with short half-life and high reactivity; due to its physiochemical properties it is very difficult to detect the concentrations precisely. Chemiluminescence is one of the robust methods to quantify NO. Detection of NO by this method is based on reaction of nitric oxide with ozone which leads to emission of light and amount of light is proportional to NO. By this method NO can be measured in the range of pico moles to nano moles range. Using direct chemiluminescence method, NO emitted into the gas stream can be detected whereas using indirect chemiluminescence oxidized forms of NO can also be detected. We detected NO using purified nitrate reductase, mitochondria, cell suspensions, and roots; detail measurement method is described here.

A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production

&NA; Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport and can prevent reactive oxygen species (ROS) and nitric oxide (NO) production under non‐stressed, normoxic conditions. Here we assessed the roles of AOX by imposing stress under normoxia in comparison to hypoxic conditions using AOX over expressing (AOX OE) and anti‐sense (AOX AS) transgenic Arabidopsis seedlings and roots. Under normoxic conditions stress was induced with the defence elicitor flagellin (flg22). AOX OE reduced NO production whilst this was increased in AOX AS. Moreover AOX AS also exhibited an increase in superoxide and therefore peroxynitrite, tyrosine nitration suggesting that scavenging of NO by AOX can prevent toxic peroxynitrite formation under normoxia. In contrast, during hypoxia interestingly we found that AOX is a generator of NO. Thus, the NO produced during hypoxia, was enhanced in AOX OE and suppressed in AOX AS. Additionally, treatment of WT or AOX OE with the AOX inhibitor SHAM inhibited hypoxic NO production. The enhanced levels of NO correlated with expression of non‐symbiotic haemoglobin, increased NR activity and ATP production. The ATP generation was suppressed in nia1,2 mutant and non symbiotic haemoglobin antisense line treated with SHAM. Taken together these results suggest that hypoxic NO generation mediated by AOX has a discrete role by feeding into the haemoglobin‐NO cycle to drive energy efficiency under conditions of low oxygen tension. Graphical abstract Figure. No caption available. HighlightsUnder normoxic conditions AOX acts as scavenger of NO when treated with flg22.AOX over expressing line displayed less NO under normoxia in response to flg22.Scavenging of NO by AOX can prevent peroxynitrite formation under normoxia.In contrast to normoxia, during hypoxia AOX is a generator of NO.The enhanced levels of NO under hypoxia correlated with ATP production.Hypoxic NO generation, mediated by AOX, drives the Hb‐NO cycle under hypoxia.

Measurement of Respiration and Internal Oxygen in Germinating Cicer arietinum L. Seeds Using Optic Microsensor

Internal oxygen concentrations vary in different tissues depending on tissue size, developmental stage, and their location. Respiratory rate of tissue also determines internal oxygen levels. For studying various signaling pathways it is essential to establish a correlation between respiration and internal oxygen. Seed germination is associated with increase in respiration which can dictate the internal oxygen and subsequent production of reactive oxygen species. Using optic oxygen microsensor we made an attempt to measure respiratory rate and internal oxygen. We found that microsensor is able to sense internal oxygen and it is also possible to measure oxygen levels in a close vial that contains seeds. Step-by-step protocol is described here along with illustration.

Isolation of Physiologically Active and Intact Mitochondria from Chickpea

Chickpea is an important leguminous crop that belongs to Fabaceae family, highly valued for its nutritious seeds. Seeds contain reserve food for the developing embryos. Mitochondria are crucial organelle for generation of chemical energy in the form of ATP which is required for achieving metabolically active state; therefore, investigating mitochondrial function and respiration rate is crucial for exploring various metabolic and physio-biochemical changes that occur during seed germination. Here we describe a method for isolation of mitochondria from germinating seeds of two chickpea varieties, i.e., Desi and Kabuli. Structure of Mitotracker-stained isolated mitochondria was observed by confocal microscopy and respiration rate was measured using an oxygen microsensor.

Nitric Oxide Measurement from Purified Enzymes and Estimation of Scavenging Activity by Gas Phase Chemiluminescence Method.

In plants, nitrate reductase (NR) is a key enzyme that produces nitric oxide (NO) using nitrite as a substrate. Lower plants such as algae are shown to have nitric oxide synthase enzyme and higher plants contain NOS activity but enzyme responsible for NO production in higher plants is subjected to debate. In plant nitric oxide research, it is very important to measure NO very precisely in order to determine its functional role. A significant amount of NO is being scavenged by various cell components. The net NO production depends in production minus scavenging. Here, we describe methods to measure NO from purified NR and inducible nitric oxide synthase from mouse (iNOS), we also describe a method of measure NO scavenging by tobacco cell suspensions and mitochondria from roots.