Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies.

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Brassinolide promotes interaction between chloroplasts and mitochondria during the optimization of photosynthesis by the mitochondrial electron transport chain in mesophyll cell protoplasts of Arabidopsis thaliana.

The current experimental data unveils the role of brassinolide (BL), a phytohormone of class brassinosteroids (BRs), in augmenting the cross-talk between the mitochondrial electron transport chain (mETC) and chloroplasts to strengthen the efficiency of the Calvin-Benson cycle (CBC) for higher assimilation of carbon dioxide in the mesophyll cell protoplasts (MCP) of Arabidopsis thaliana. The outcome of total respiration (TR) and photosynthetic carbon assimilation (PCA) was monitored as O2 uptake under dark and NaHCO3-dependent O2 evolution under light, respectively, after pre-incubation of MCP at a broad spectrum of BL concentration from 0.05 pM to 5 pM at 25 °C and optimum light intensity of 1000 µmol m-2 s-1. The addition of optimal concentration (0.5 pM) of BL to MCP stimulated the (i) TR, (ii) PCA, and (iii) para-benzoquinone-dependent O2 evolution (PSII activity). Further, in response to BL, the enzyme activity or transcript levels of redox-regulated CBC enzymes and glucose-6-phosphate raised considerably. Also, the addition of BL to MCP remarkably accelerated the capacity of the cytochrome oxidase (COX) and alternative oxidase (AOX) pathways concurrently with an increase in total cellular pyruvate and reactive oxygen species (ROS) levels. Besides, malate valve components (Malate, Chl-MDH, M-MDH) increased in response to BL. At the same time, the cellular redox ratios of pyridine nucleotides (NADPH and NADH) were kept low in the presence of BL. However, BL could not keep up the CBC activity of photosynthesis along with its associated light-activated enzymes/transcripts when mETC through COX or AOX pathway is restricted by antimycin A (AA) or salicylhydroxamic acid (SHAM), respectively. In contrast, adding BL to MCP under restricted mETC showed aggravation in total cellular ROS, pyruvate, malate, and redox ratio of pyridine nucleotides with a concomitant increase in transcripts associated with malate valve and antioxidant systems. These results suggest that BL enhances the PCA by coordinating in cross-talk of chloroplasts and mitochondria to regulate the cellular redox ratio or ROS through the involvement of COX and AOX pathways along with the malate valve and antioxidant systems.

Development of a rapid process for purification of Bowman-Birk and Kunitz inhibitors from legume seeds, and evaluation of their biophysical, insecticidal, and antimicrobial properties.

Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.

Structural and Biophysical Characterization of Purified Recombinant Arabidopsis thaliana's Alternative Oxidase 1A (rAtAOX1A): Interaction With Inhibitor(s) and Activator.

In higher plants, alternative oxidase (AOX) participates in a cyanide resistant and non-proton motive electron transport pathway of mitochondria, diverging from the ubiquinone pool. The physiological significance of AOX in biotic/abiotic stress tolerance is well-documented. However, its structural and biophysical properties are poorly understood as its crystal structure is not yet revealed in plants. Also, most of the AOX purification processes resulted in a low yield/inactive/unstable form of native AOX protein. The present study aims to characterize the purified rAtAOX1A protein and its interaction with inhibitors, such as salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG), as well as pyruvate (activator), using biophysical/in silico studies. The rAtAOX1A expressed in E. coli BL21(DE3) cells was functionally characterized by monitoring the respiratory and growth sensitivity of E. coli/pAtAOX1A and E. coli/pET28a to classical mitochondrial electron transport chain (mETC) inhibitors. The rAtAOX1A, which is purified through affinity chromatography and confirmed by western blotting and MALDI-TOF-TOF studies, showed an oxygen uptake activity of 3.86 µmol min-1 mg-1 protein, which is acceptable in non-thermogenic plants. Circular dichroism (CD) studies of purified rAtAOX1A revealed that >50% of the protein content was α-helical and retained its helical absorbance signal (ellipticity) at a wide range of temperature and pH conditions. Further, interaction with SHAM, n-PG, or pyruvate caused significant changes in its secondary structural elements while retaining its ellipticity. Surface plasmon resonance (SPR) studies revealed that both SHAM and n-PG bind reversibly to rAtAOX1A, while docking studies revealed that they bind to the same hydrophobic groove (Met191, Val192, Met195, Leu196, Phe251, and Phe255), to which Duroquinone (DQ) bind in the AtAOX1A. In contrast, pyruvate binds to a pocket consisting of Cys II (Arg174, Tyr175, Gly176, Cys177, Val232, Ala233, Asn294, and Leu313). Further, the mutational docking studies suggest that (i) the Met195 and Phe255 of AtAOX1A are the potential candidates to bind the inhibitor. Hence, this binding pocket could be a 'potential gateway' for the oxidation-reduction process in AtAOX1A, and (ii) Arg174, Gly176, and Cys177 play an important role in binding to the organic acids like pyruvate.

Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure.

The midgut of lepidopteran larvae is a multifunctional tissue that performs roles in digestion, absorption, immunity, transmission of pathogens and interaction with ingested various molecules. The proteins localized at the inner apical brush border membrane are primarily digestive proteases, but some of them, like aminopeptidase N, alkaline phosphatase, cadherins, ABC transporter C2, etc., interact with Crystal (Cry) toxins produced by Bacillus thuringiensis (Bt). In the present study, aminopeptidase N (APN) was characterized as Cry-toxin-interacting protein in the larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which have less than 40% sequence similarity but show the presence of characteristic 'GAMENEG' and zinc-binding motifs. Feeding a sublethal dose of Cry toxin caused differential expression of various APN isoform. Further, 6thgeneration Cry-toxin-exposed larvae showed reduced expression of APN2. This report suggests that A. janata larvae exploit altered expression of APNs to overcome the deleterious effects of Cry toxicity, which might facilitate toxin tolerance in the long run.

Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI.

Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel ß-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by 'red shift' in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea.

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ±â€¯4.5 nM) and chymotrypsin (Ki = 807.8 ±â€¯23.7 nM) while RsKI (Ki = 172.0 ±â€¯9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > ß-sheets/ß-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata.

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest - Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ±â€¯11 nM) and chymotrypsin (Ki of 446 ±â€¯9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 µg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera.

Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus.

Leucaena leucocephala (Lam.) de Wit., "subabul" stem lignin: Isolation, structural characterization and thermal properties.

Lignin is the second most abundant renewable biopolymer on earth after cellulose. It is being used in many industrial applications due to its abundance. In the present study, lignin was isolated from the stems of Leucaena leucocephala (Lam.) de Wit., a high biomass yielding plant using acidic dioxane under N2 atmosphere. Structural characterization of isolated dioxane lignin (DL) was performed by analytical techniques: UV, FT-IR, ¹H NMR and ¹³C NMR. Their monolignol content was determined by nitrobenzene oxidation followed by HPLC-MS/MS analysis. The data was compared with commercial alkali lignin (AL). The results showed that DL is of hardwood guaiacyl-syringyl (GS) type, whereas AL is softwood type with more guaiacyl units and trace amounts of p-hydroxyphenyl units (H). Thermogravimetric analysis (TGA) of DL showed two stage thermal degradation profile similar to AL. The DTGmax for DL and AL were found in the second major loss event of second stage of TGA at 424°C and 404°C, respectively. Differential scanning calorimetry (DSC) study exhibited the glass transition temperatures (Tg) at 132°C and 122°C for DL and AL, respectively. The results from thermal stability studies suggest that dioxane lignin isolated from the "miracle tree" (subabul) can be exploited in various thermoplastic industrial applications.

Exploitation of subabul stem lignin as a matrix in controlled release agrochemical nanoformulations: a case study with herbicide diuron.

The utilization of nanomaterials in the domain of agriculture is at an inception, especially in the development of controlled release agrochemical nanoformulations. The present study demonstrated the potential of subabul stem lignin as a matrix material in agrochemical formulations using nanotechnology. In this study, "nanoprecipitation" method was employed and "optimized" to fabricate a stable herbicide, "diuron nanoformulation" (DNF). "Optimized DNF" (ODNF) has 5.17 ± 0.49 % diuron loading efficiency (DLE) and 74.3 ± 4 % encapsulation efficiency (EE). The size of nanoparticles in ODNF was 166 ± 68 nm as revealed by FESEM/TEM studies. Physicochemical characterization of ODNF by UV, FT-IR, and DSC studies revealed the successful loading of diuron within the lignin matrix. The ODNF exhibited nonlinear biphasic release profile for diuron. Further, the bioefficacy of diuron released from ODNF was tested using canola (Brassica rapa). B. rapa seedlings grown in the soil supplemented with ODNF showed early signs of leaf chlorosis and mortality when compared with seedlings grown in the presence of commercial diuron formulation (CDF) or bulk diuron (BD), respectively. This study not only revealed the exploitation of subabul stem lignin as a "matrix" in the controlled release nanoformulation of diuron but also opened up new avenues for utilizing it as matrix for several other agrochemicals associated with the growth and development of the plant.

Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis.

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)-sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD (+)/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems.

The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 µmoles m(-2) s(-1) at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated the importance of AOX pathway in optimizing photosynthesis under both hyper-osmotic stress and sub-optimal temperatures. Regulation of ROS through redox couples related to malate valve and antioxidant system by AOX pathway to optimize photosynthesis under these stresses are discussed.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.

BACKGROUND AND AIMS: The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. METHODS: Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. KEY RESULTS: In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. CONCLUSIONS: These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III.

Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata.

Physiological role of AOX1a in photosynthesis and maintenance of cellular redox homeostasis under high light in Arabidopsis thaliana.

As plants are sessile, they often face high light (HL) stress that causes damage of the photosynthetic machinery leading to decreased photosynthesis. The importance of alternative oxidase (AOX) in optimizing photosynthesis is well documented. In the present study, the role of AOX in sustaining photosynthesis under HL was studied using AOX1a knockout mutants (aox1a) of Arabidopsis thaliana. Under growth light (GL; 50 µmol photons m(-2) s(-1)) conditions, aox1a plants did not show any changes in photosynthetic parameters, NAD(P)/H redox ratios, or respiratory O2 uptake when compared to wild-type (WT). Upon exposure to HL (700 µmol photons m(-2) s(-1)), respiratory rates did not vary between WT and aox1a. But, photosynthetic parameters related to photosystem II (PSII) and NaHCO3 dependent O2 evolution decreased, while the P700 reduction state increased in aox1a compared to WT. Further, under HL, the redox state of cellular NAD(P)/H pools increased with concomitant rise in reactive oxygen species (ROS) and malondialdehyde (MDA) content in aox1a compared to WT. In presence of HL, the transcript levels of several genes related to antioxidant, malate-oxaloacetate (malate-OAA) shuttle, photorespiratory and respiratory enzymes was higher in aox1a compared to WT. Taken together, these results demonstrate that under HL, in spite of significant increase in transcript levels of several genes mentioned above to maintain cellular redox homeostasis and minimize ROS production, Arabidopsis plants deficient in AOX1a were unable to sustain photosynthesis as is the case in WT plants.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae.

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX.

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O(2) evolution and O(2) uptake in mesophyll protoplasts of pea pre-incubated under different light intensities. Under HL (3000 micromol m(-2) s(-1)), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO(3)-dependent O(2) evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O(2) uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO(3)-dependent O(2) evolution or p-benzoquinone (BQ)-dependent O(2) evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation.

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO2. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components--ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate-glutathione (Asc-GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.