|Home | About | Journals | Submit | Contact Us | Français|
Nitric oxide (NO) plays a key role in plant diseases resistance. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated NO and reactive nitrogen species production during early infection stage not only in infected leaf but also in root and shoot. Production of NO was further confirmed by oxyhemoglobin assay. Furthermore, we used Phenyl alanine ammonia lyase as marker of pathogenesis related enzyme. In addition evidence for protein tyrosine nitration during the early stage of viral infection clearly showed the involvement of nitrosative stress.
A growing body of evidences suggests that nitric oxide (NO) plays a key role in plant disease resistance.1–6 Apart from that NO plays a diverse physiological role in plants.7 Most of the experimental data available on NO detection during plant-pathogen interaction come from studies of infections by biotrophic pathogens.8 Rapid accumulation of NO in response to avirulant bacteria has been observed in Soybean and Arabidopsis suspension cultured cells1,9 as well as in Arabidopsis plants.10 However, except in tobacco plants, there is a very little information available on the generation of NO with respect to plant-virus interaction.
Kenaf (Hibiscus cannabinus L.) and roselle (Hibiscus subdariffa L.), together known as mesta (family Malvaceae), are cultivated primarily for the production of bast fibres in different states of India. Besides their use as traditional packaging material in the fibre industry, the cultivated species are also used as a leafy vegetable. Their seeds are rich in oil which has a low proportion of unsaturated fatty acids and they have potential medicinal value.11,12 Mesta, particularly H. cannabinus, is receiving increasing global attention as an alternative source of good quality paper pulp due to an increasing shortage of hard wood sources. A yellow vein mosaic disease, characterized by yellowing of leaf veins followed by complete chlorosis of the leaves, is spreading rapidly in many parts of eastern Northern and Southern India13–15 and thus has become a serious problem in the cultivation of these crops. The height of diseased plants is reduced significantly and thus adversely affects the bast fibre yield.16 In eastern India, the disease was found to be associated with a recently described begomovirus, Mesta yellow vein mosaic virus (MeYVMV), and an isolate of Cotton leaf curl Multan betasatellite (CLCuMB).17,18 Symptomatic samples obtained from Northern India showed the association of another recently described species of begomovirus, Mesta yellow vein mosaic Bahraich virus (MeYVMBV) and an isolate of Ludwigia leaf distortion betasatellite (LuLDB).19,20 Interestingly, in Southern India the begomovirus complex consisted of MeYVMV and LuLDB.15 These begomovirus complexes have been shown to be transmitted efficiently by a whitefly (Bemisia tabaci) and they have a very narrow host range.16,19
With the aim of understanding the plant-pathogen interaction associated with yellow vein mosaic disease of H. cannabinus, biochemical approach was attempted to study the cellular nitric oxide production under diseased condition. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated nitric oxide production not only in infected leaf but also in root and shoot. Furthermore, we used Phenyl alanine ammonia lyase (PAL) as marker of pathogenesis related enzyme. In addition we have provided evidences of NOS like enzymes measured by oxyhemoglobin assay. In this work, using H. cannabinus plants, evidence is presented showing that under viral infection, NO and other NO-derived products are overproduced, which leads to tyrosine nitration of proteins indicating that in plant cells, nitrosative stress could participate, as a significant component.
Disease development was observed sequentially. Initially, 8–10 days after whitefly inoculation, mild chlorotic flecks appeared along the veins on one half of the lamina. As the disease progressed, entire veins of the lamina turned yellow and formed a yellow vein network. The interveinal chlorosis started as mild greenish yellow discolouration which progressed until the entire lamina turned whitish yellow. The impact of Mesta yellow vein mosaic disease, on growth and yield, was greatest when the plants were inoculated at the earliest stage of growth. However, there were no significant differences in disease incidence among the groups of plants that were inoculated either at the early or later stages of growth.15 So, we studied in detail the production of nitric oxide in early stages of infection in different ages plant. At the early stage of infection, inoculated plants exhibited yellow fleck type of symptom in the emerging leaves. Gradually the flecks converted into a typical ‘yellow vein network’ phenotype characterized by yellowing of veins and veinlets followed by complete chlorosis and defoliation of leaves at an advanced stage of infection (Fig. 1A–E). The height of diseased plants are usually reduced significantly and thus adversely affected the bast fibre yield (Fig. 1F).
The specific primers used for amplification successfully amplified the complete genome (c. 2.7 kb) of the begomovirus and betasatellite (c. 1.3 kb) (Fig. 2A and B) and thus confirmed the association of such begomovirus complex in the infected plants. Such infected plants containing the begomovirus and betasatellite were used further for all the biochemical studies.
NO has been shown to play a key role during plant-pathogen interactions by triggering resistance associated cell death and inducing defense-related genes. In order to gain a precise view of NO production during early infection in different ages (45, 90 and 120 days) of H. cannabinus plant, the presence of NO in leaf tissues of the control plant and the yellow vein mosaic virus infected plant were analyzed, using the cell permeable NO specific probe 4,5-diaminofluorescein diacetate (DAF-2DA),21 which is converted to its fluorescent triazole derivative DAF-2T upon reaction with NO (Figs. 3 and and44). All the infected leaf sections of different age's plant appeared strongly fluorescent in relation to NO production in these tissues particularly in the vascular bundle region than the control plants. Leaf sections incubated with a well known scavenger of NO such as cPTIO did not show any NO specific fluorescence (Fig. 5) which indicated that our experimental procedure is suitable for NO detection in H. cannabinus leaves. To further show the specificity of DAF-2DA, control leaf sections incubated with 250 µM SNP as NO donor confirmed the presence of NO (Fig. 6). Infected leaf section showed high fluorescence in xylem, phloem, epidermis and in spongy and palisade mesophyll also. This signal did not correspond to autofluorescence, as it was not observed in the absence of DAF-2DA. In addition, the fluorescence presented a localized pattern which ruled out the possibility of a general stress response induced by wounding. To determine whether infected H. cannabinus stem and root can produce NO or not, we monitored NO specific fluorescence in infected stem and infected root. Interestingly, there was a significant difference in NO specific fluorescence in infected stem and root compared to the control plant. There was negligible NO specific fluorescence in control stem and root but infected stem and root showed significant fluorescence in xylem, phloem and in epidermis layer (Fig. 7). Not only that, NO specific fluorescence was observed in conjunctive tissue of infected root.
Dihydrorhodamine 123 has also been reported to react with reactive oxygen species as well as reactive nitrogen species.22 As shown in Figures 8 and and99, a significant fluorescent signal was observed in all the sections of infected root, leaf and stem indicating the presence of either reactive oxygen or reactive nitrogen species. All the sections were further tested with DHE and DCF-DA, well known fluorescent probes for the detection of superoxide generation, however, no superoxide specific fluorescence was observed in all the sections (data not shown). As we observed significant NO specific fluorescence in all the infected plants, then it could be assumed that reactive nitrogen species would have generated in the infected plants. So fluorescent signal using Dihydrorhodamine 123 might be due to the production of reactive nitrogen species. In addition, high fluorescence is presented in all the portions of infected stem particularly in the collenchyma layer including the vascular bundle and cambium regions.
NOS like activities have been detected in several plant tissues and purified organelles, including mitochondria, the nucleus and peroxisomes.2,23–27 To determine whether any NOS like activity present in infected leaf of H. cannabinus or not, we measured NO synthase activity using oxyhemoglobin assay. Interestingly, Ca2+/CaM independent NOS activity was found in infected leaf only. We did not observe any NOS like activity in control leaf. NOS like activity was completely inhibited using L-NAME, a well known NOS inhibitor indicating the presence of NOS like enzyme in plant system. This is further supported by NADPH dependent Griess assay (data not shown). Although NOS like activity was very low (4.1 n mole min−1mg−1 protein) in the infected plant, still the presence of NOS like activity in viral infected plant is significant. As the NOS like activity was very low in the crude leaf extract, it was not possible to further characterize the enzyme. An attempt was made to assess the activity profile of nitrate reductase, however, no enzymatic activity was observed under our experimental conditions.
In plants, nitrations of tyrosine residues in target proteins have drawn the least attention to date as a mechanism of NO signaling. Several groups pointed to the involvement of protein tyrosine nitration in plants using antibodies raised against 3-NO2-Tyr residues. The tyrosine nitration of proteins is considered as an indicator of the peroxynitrite action.28 A similar immunological based strategy was adopted to detect protein tyrosine nitration and the immunoblot analysis of the proteins that could be affected by this process (Fig. 10) showed that yellow vein mosaic virus infection caused an increase in the intensity and number of tyrosine nitrated proteins in the molecular mass range between 44 and 60 kDa. Nitrated BSA was used as control. Immuno positive bands appeared both in control leaf extracts also indicated in vivo production of reactive nitrogen species, but there was a marked increase inimmunopositive bands in infected leaves collected from infected plant.
The involvement of cGMP dependent components in NO dependent defense gene activation is suggested by accumulation of PAL and PR-1 transcript in tobacco cell suspensions.2 So, to determine PAL level in control and infected plant, we monitored the PAL activity at different time point of viral infection. PAL activity in early infected plant showed a 2.48 fold increase with respect to the control plant (Fig. 11A). PAL activity remained at high level in early infection of different ages plants. However, the folds increase of PAL activity at mid and late infection compared to the control was less than the initial stage of infection. To confirm whether PAL induction is mediated by NO or not, control leaf was incubated with different concentrations of Sodium nitroprusside (SNP) as NO donor. PAL activity level was increased in a dose dependent manner (Fig. 11B). Similar result was obtained using DETA NONOate as NO donor (data not shown).
The main objective of this study was to elucidate the status of NO production in early Begomovirus infection of different age's susceptible plant Hibiscus cannabinus. Previously it was shown that the activity of iNOS was strongly induced in resistant tobacco leaves with tobacco mosaic virus (TMV) but not in susceptible variety.2 In fact Durner et al. showed that upon shifting the infected tobacco plants from 32 to 22°C, the NOS activity was induced 4.5 times within 2–3 hrs in resistant plants but not in susceptible plants. However, our study demonstrates that the production of nitric oxide in early stage of infection (45, 90 and 120 days old plant) in Hibiscus cannabinus after Begomovirus infection in susceptible cultivars which is evident from our fluorescent microscopy data. In addition we have provided evidences for NOS like enzyme activity. Not only that NO specific expression of PAL was induced in early infection of different ages plants.
The finding of NO production in infected leaf, stem and root of H. cannabinus was very much surprising to us because the presence of NO was mainly shown in plant defense mechanism in resistant plant. Interestingly, the main production of NO took place in the xylem; phloem in control leaves indicating NO might have other signaling action in healthy plant. In the infected leaves, NO specific fluorescence was distributed not only in vascular bundle region but also in spongy and palisade parenchyma. In addition distribution of NO and RNS were observed in collenchyma, cambium region of the infected stem and in epidermis, conjunctive tissue, vascular bundle region of the infected root. It has been shown that in cell wall lignification of xylem elements, an oxidative burst is involved and a NO burst also participates in the programmed cell death associated to the differentiating vessels.29 The obvious question came in our mind that what could be the role of this high amount of NO production in infected leaves. It is clearly evident that high amount of NO generation could not impart any protective role in this plant because yellow vein was very much prominent at this stage. It is possible that the amount of NO is not sufficient enough to induce the defense gene expression. At this stage it is not clear what role is played by NO in infected plant.
In previous studies PAL expression was studied in resistant plant and it was controlled by NO.1,2 Here we first time showed that susceptible cultivars also produce PAL and administration of NO donors also induced it. Apart from the synthesis of flavonoids and other phenolic derivatives, the product of PAL enzyme reaction, trans-cinnamic acid, provides phenylpropanoid skeletons which serve as building subunits for lignin biosynthesis.
In plants, there are very few reports on the specific activity of L-arginine-dependent nitric oxide synthases (NOSs) perhaps due to the difficulties in measuring this activity. In our work, the NOS activity in infected H. cannabinus leaves was measured by L-Arginine dependent oxyhemoglobin assay set up for plant tissues, which has been demonstrated to be very sensitive.24 The enzyme activity was independent of calcium and calmodulin similar like mammalian iNOS. In olive leaves the NOS activity determined was 0.280 nmol NO mg−1 protein min-1 by an ozone chemiluminiscence method, set up for plant tissues, it was dependent on L-Arginine and required NADPH, calcium and different cofactors (FAD, FMN and BH4). The NOS activity of infected leaves was much higher than that previously reported in pea leaf extracts (0.120 nmol NO mg−1 protein min−1).24 and in olive leaf extract (ca. 0.300 nmol NO mg−1 protein min−1). However, it must be mentioned that to our knowledge there are no reports on the presence of BH4 in plants. This cofactor promotes and/or stabilizes the active dimeric form of the three mammalian NOS isoforms.30 In plants, this function perhaps could be carried out by tetrahydrofolate (FH4) whose biosynthesis and distribution is well known in higher plants.31 However, further research is necessary to demonstrate this function of FH4 as a NOS cofactor in plants.
In plants, apart from the NOS-like activity, other documented sources of NO are two nitrate reductases, different plant organelles like peroxisomes,21 mitochondria and chloroplasts,32,33 and also non-enzymatic systems.34,35 The AtNOS1 protein which until very recently was considered as the characteristic plant NOS36 has just been demonstrated that does not produce NO and, therefore, is not a real L-Arginine-dependent NOS enzyme.37,38 The NOS activity values reported in this work cannot be easily compared with the values of NO emitted by certain plants,32,39 because the NOS activity determined must be just a part of the whole endogenous production of NO.
In plants, to our knowledge, there is no information on the effect of viral infection on the tyrosine nitration of proteins. Using an antibody against nitro tyrosine, the results obtained in this work indicated that in H. cannabinus leaves viral infection produced an increase in the number and intensity of proteins that experimented tyrosine nitration (Fig. 10). Similar observation was found in olive leaves under salt stress.40 In nitrite reductase antisense tobacco leaves the induction of several tyrosine-nitrated polypeptides with molecular masses between 10 kDa and 50 kDa was described.41 Moreover, in tobacco BY-2 suspension cells treated with a fungal elicitin, the induction of tyrosine nitration in proteins with molecular masses in the range 20–50 kDa was demonstrated.42 Conversely, in tobacco transgenic plants with genetically increased cytokinin levels the content of tyrosine nitrated proteins decreased.43
In conclusion, the results presented in this work indicate that in leaves from H. cannabinus plants infected with yellow vein mosaic virus produce an increase in the NO, RNS and as a result of this; there is a rise in tyrosine-nitrated proteins, which are considered biomarkers of nitrosative stress. These data indicate that in H. cannabinus leaves viral infection induces nitrosative stress. The results described in this work apart from providing new insights into the physiological response of susceptible H. cannabinus plants to yellow vein mosaic virus, also evidence that vascular tissues could play in plants an important function in the redistribution of RNS (NO and/or RSNOs) during normal and stress conditions. These considerations perhaps could be extended to other plant species under different viral infected situations.
All the reagents are of highest purity and purchased from Sigma Chemical Co., (St. Louis, MO, USA) unless otherwise stated.
Diseased plant samples, showing typical yellow vein symptoms, were collected from the cultivated fields of Bongaon region of West Bengal state of India these plants were used as sources of virus inocula. The associated begomovirus complex were maintained in susceptible kenaf plants cv HC-583, grown under insect-proof glasshouse of Central Research Institute for Jute and Allied Fibres (CRIJAF), through successive whitefly (B. tabaci) transmission. For whitefly transmission, the tobacco whiteflies (B. tabaci) were collected from kenaf crop grown at the CRIJAF research farm and reared on tobacco plants (Nicotiana tabacum) in an insect-proof wood-framed cage for few generations to make the whitefly culture virus-free. The adult whiteflies, which emerged from nymphs present on tobacco, were used for transmission studies. Five virus-free whiteflies were separately fed on the diseased samples collected from the farmers' field and after 12 h of acquisition access period the viruliferous whiteflies were released on the healthy plants (10 seedlings per pot) grown under glasshouse. After 12 h of inoculation access period, inoculated plants were sprayed with 0.2% Dimethoate (Rogor) and kept in cages until symptom development. Successive back-inoculation to new sets of healthy kenaf plants was carried out to avoid mixed infection.
For detection of the associated begomovirus complex, total plant DNA was isolated from the diseased plants maintained under glasshouse by a modified CTAB method recently developed at CRIJAF.44 The modification included the use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 (M) NaCl, which markedly reduced the viscosity of the mucilage and thus the final purification step yielded more quantity of mucilage-free DNA suitable for subsequent PCR based detection of begomoviruses. To amplify the begomovirus(es) associated with MeYVMD, one primer pair (FL[H]F: 5′-AAG CTT AAA TAA ATY TCC YGC YTA T-3′ and FL[H]R: 5′AAG CTT TGA GCG CGT CAT ATG ATT G-3′) was designed from the AV2 gene of the previously characterized sequence of MeYVMV (accession no. EF373060) and another primer pair [NIYVM(SP)-FLF (5′-CAG AAG TCC GGA TGT TCC AAG-3′)/NI-YVM(SP)-FLR(5′-TAC ATC CGA TAC ATT CTG GGC-3′)], was designed from the AV1 gene of the MeYVMBV (accession no. EU360303). For the amplification of betasatellite genomes, PCR was carried out using the universal betasatellite primers.15
All operations were performed at 0–4°C. Leaves were ground to powder in a mortar with liquid nitrogen, and were suspended in 10 ml of 100 mM Tris-HCl buffer (pH 8.8), containing 1 mM EDTA, 7% (W/V) PVPP, 15 mM DTT, 15 mM PMSF and centrifuged at 10,000 r.p.m for 10 min to remove the cell debris. Then, the supernatants were used for enzyme assay and western blot.
NO was detected in kenaf leaf cross sections that were incubated for 1 h at 25°C, in darkness, with 10 µM 4,5-diaminoflorescein diacetate (DAF-2 DA, Calbiochem) prepared in 10 mM Tris-HCl (pH 7.4).21 Background staining, routinely negligible, was controlled with unstained sections. As control, sections were pre-incubated for 30 min at 25°C with 200 µM cPTIO, an ·NO scavenger. Moreover, control leaf sections were incubated with 250 µM SNP as NO donor which further confirmed the presence of NO as well as infiltration of DAF-2DA (Fig. 6). For Reactive Nitrogen Species, the samples were incubated with 10 µM DHR 123. After incubation, samples were washed twice in the same buffer for 15 min each. Then the sections were examined by Olympus BX51 fluorescence microscope attached with Olympus CoolSNAP cf color/OL camera using appropriate filter. Light intensity and exposure times were kept constant for a given set of experiment and collection modalities for DAF-2 DA green fluorescence (excitation 495 nm; emission 515 nm), DHR 123 green fluorescence (excitation 488 and emission 525–550 nm) and chlorophyll autofluorescence (chlorophyll a and b, excitation 429 and 450 nm, respectively; emission 650 and 670 nm, respectively) as orange.
The initial rate of NO synthesis was measured at 25°C using oxyhemoglobin assay for NO.45 The leaf extract was added to the cuvette containing 40 mM Tris, pH-7.6, containing 0.3 mM DTT, 1 mM L-Arginine, 4 µM FAD, 4 µM FMN, 10 µM H4B, 100 U/ml catalase, 10 µM oxyhemoglobin to give a final volume 0.5 ml. The reaction was started by adding NADPH to give 0.2 mM. The NO mediated conversion of oxyhemoglobin to methhemoglobin was monitored over time as an absorbance increase at 401 nm and measured using the coefficient of 38 mM−1cm−1.
15 µg protein was separated in 10% SDS-polyacrylamide gel electrophoresis. Protein was transferred (250 mA, 90 min) to PVDF membrane according to standard protocol using Bio-Rad wet transfer apparatus. Blot was then blocked in Tris-buffered saline (TBS; 20 mM Tris.Cl, 137 mM NaCl, pH 7.6) containing 3% (w/v) non-fat dry milk and 0.1% (v/v) Tween-20 at 4°C overnight. The blot was incubated with primary antibody (Monoclonal anti nitrotyrosine antibody Upstate, USA, Catalogue No.CA92590), diluted in TBS containing 0.1% (v/v) Tween-20 (TBST) for 1 hour at room temperature. After washing with TBST, the blot was incubated with HRP conjugated rabbit antimouse IgG (secondary antibody, 1:10,000 diluted in TBST) for 30 min. It was then washed extensively with TBS and then the immunopositive spots were visualized by using chemilluminiscent reagent (Perkin Elmer, Boston, MA) as directed by the manufacturer.
The PAL assay46 was performed at 37°C for 1 h in an assay mixture (500 µl) containing 225 µl of 50 mM Tris-HCl (pH 8.8), 250 µl of enzyme extract and 25 µl of 100 mM L-phenylalanine. The reaction was terminated by the addition of 100 µl of 5 M HCl and the reaction mixture was centrifuged at 11,700 g for 10 min to remove the precipitate that might have influenced the absorbance readings. PAL activity was determined spectrophotometrically by measuring the amount of trans-cinnamic acid formed at 290 nm.
Specific activity of PAL was determined in leaf extracts following 0, 150 and 250 µM Sodium Nitroprusside (SNP) treatments. Single leaf was incubated in a sterile Petri plate in 50 mM Potassium Phosphate buffer, pH-7.0 containing different SNP concentrations at 25°C and 16/8 hr photoperiod at 200 µmol m−2s−1 in plant growth chamber for 18 hours. Control leaf experiment was performed under similar conditions.
The authors would like to acknowledge the help of all the research scholars of the Division of Crop Protection and would like to thank the Director of CRIJAF for providing the infrastructural support. They would also like to acknowledge DST-FIST, UGCCAS program, Govt. of India for equipment and infrastructural support.
Previously published online: www.landesbioscience.com/journals/psb/article/11282
This work was supported by Indian Council of Agricultural Research under Technology Mission on Jute (TMJ) MM 1.6. Research fellowship of Tuhin Subhra Sarkar was supported by University Grants Commission (UGC), Govt. of India.