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Nitric oxide production by tobacco plants and cell cultures under normal conditions and under stress
(2004)
Nitric oxide (NO) is a gaseous free radical involved in the regulation of diverse biochemical and physiological processes in animals. During the last decade, evidence has accumulated that NO might also play an important role as a second messenger in plants. Of special interest were observations that NO was involved in a signal chain leading to the hypersensitive response (HR) in incompatible plant-pathogen interactions. In contrast to animals, plants have probably several enzymes that may produce NO. Potential candidates are: Cytosolic nitrate reductase (NR; EC 1.6.6.1), plasma-membrane (PM)-nitrite: NO reductase (Ni:NOR), nitric oxide synthase (NOS; EC 1.14.13.39) and Xanthine dehydrogenase (XDH; EC 1.1.1.204). The major goal of this work was to quantify NO production by plants, and to identify the enzymes responsible for NO production. As a major method, NO production by tobacco leaves or cell suspensions was followed under normal, non-stress conditions, and under biotic stress, through on-line measurement of NO emission into the gas phase (chemiluminescence). Plants used were tobacco wild-type (N. tabacum cv Xanthi or cv Gatersleben), NR-free mutants grown on ammonium in order to prevent NR induction, plants grown on tungstate to inhibit synthesis of functional MoCoenzymes, and a NO-overproducing nitrite reductase (NiR)-deficient transformant. Induction of HR in tobacco leaves and in cell suspensions was achieved using the fungal peptide elicitor cryptogein. Non-elicited leaves from nitrate-grown plants showed a typical NO-emission pattern where NO-emission was low in dark, higher in the light and very high under dark-anaerobic conditions. Even at maximum rates, NO production in vivo was only a few percent of total NR activity (NRA). Consistent with that, with a solution of purified NR as a simple, “low quenching” system, NO-emission was also about 1 % of NRA. Thus, NO scavenging by leaves and stirred cell suspensions appeared small and NO-emission into purified air should give a reliable estimate of NO production. NO-emission was always high in a NiR-deficient transformant which accumulated nitrite, and NO-emission was completely absent in plants or cell suspensions which did not contain NR. Thus, in healthy plants or cell suspensions, NO-emission was exclusively due to the reduction of nitrite to NO, mainly by cytosolic NR. In addition to nitrite, cytosolic NADH appears as an important factor limiting NO production. Unexpectedly, plants (in absence of NR) were able to reduce nitrite to NO under anaerobic conditions through an unknown enzyme system that was not a MoCo-enzyme and was cyanide-sensitive. When infiltrated into leaves at nanomolar concentrations, the fungal elicitor cryptogein provoked cell death in tobacco leaves and cell suspensions. The HR could be prevented by the NO-scavengers PTIO or c-PTIO, suggesting that NO production was indeed required for the HR. However, the product of the reaction of c-PTIO with NO, c-PTI, also prevented cell death without quenching NO emission. Thus, prevention of cell death by c- PTIO is no proof for an involvement of NO. No differences were found in the HR induction between NR-free plants and/or cell suspensions and WT plants. Thus, NR appears not necessary for the HR. Further, and in contrast to literature suggestions, a continuously high NO-overproduction by a NiR-free mutant did not interfere with the development of the HR. Most surprisingly, no additional NO-emission from tobacco leaves was induced by cryptogein at any phase of the HR. In contrast, some NO-emission, paralleled by nitrite accumulation, was detected 3-6 h after cryptogein addition with nitrate grown cell suspensions, but not with NR free, ammonium- grown cells. Thus, induction of NO-emission by cryptogein appeared somehow correlated with NR and nitrite, at least in cell suspensions. But since cryptogein induced the HR even in NR-free cell suspensions, this nitrite-related NO- emission was not required for cell death. NOS inhibitors neither prevented cell death nor did they affect nitrite-dependent NO-emission. Thus, in total these data question the often proposed role of NO as a signal in the HR, and of NOS as source for NO.
Normoxic and anoxic metabolism of Nicotiana tabacum transformants lacking root nitrate reductase
(2002)
The aim of this work was to find out whether and how nitrate reduction in roots would facilitate survival of hypoxic and anoxic (flooding)-phases. For that purpose, we compared the response of roots of hydroponically grown tobacco wildtype (Nicotiana tabacum cv. Gatersleben) and of a transformant (LNR-H) with no nitrate reductase (NR) in the roots but almost normal NR in leaves (based on a nia2-double mutant). As an additional control we used occasionally a 35S-transformant of the same nia2-double mutant, which on the same genetic background constitutively expressed NR in all organs. In some cases, we also compared the response of roots from WT plants, which had been grown on tungstate for some time in order to completely suppress NR activity. The following root parameters were examined: 1) Growth and morphology 2) Root respiration rates and leaf transpiration 3) Metabolite contents in roots (ATP, hexosemonophosphates, free sugars, starch, amino acids, total protein) 4) Inorganic cation and anion contents 5) Lactate and ethanol production 6) Extractable LDH-and ADH-activities 7) Cytosolic pH values (by 31P-NMR) 8) NO Cation and anion contents of roots from WT and LNR-H were only slightly different, confirming that these plants would be better suited for our purposes than the widely used comparison of nitrate-versus ammonium-grown plants, which usually show up with dramatic differences in their ion contents. Normoxia: LNR-H-plants had shorter and thicker roots than WT with a lower roots surface area per leaf FW. This was probably the major cause for the significantly lower specific leaf transpiration of LNR-H. WT-roots had lower respiration rates, lower ATP-and HMP-contents, slightly lower sugar- and starch contents and somewhat lower amino acid contents than LNR-H roots. However, total protein/FW was almost identical. Obviously the LNR-H transformants did not suffer from N-defciency, and their energy status appeared even better than that of WT-roots. Data from the 35S-transformant were similar to those of WT. This indicates that the observed differences between WT and LNR-H were not due to unknown factors of the genetic nia2-background, but that they could be really traced back to the presence resp. absence of nitrate reduction. Anoxia: Under short-term anoxia (2h) LNR-H plants, but not WT-plants exhibited clear symptoms of wilting, although leaf transpiration was lower with LNR-H. Reasons are not known yet. LNR-H roots produced much more ethanol (which was excreted) and lactate compared to WT, but extractable ADH and LDH activities, were not induced by anoxia. However, the LDH activity background was twice as high as that of the WT troughout the time period studied. Tungstate-treated WT-roots also gave higher fermentation rates than normal WT roots. Sugar- and HMP-contents remained higher in LNR-H roots than in WT. NR in WT roots was activated under anoxia and roots accumulated nitrite, which was also released to the medium. 31P-NMR spectroscopy showed that LNR-H- roots, in spite of their better energy status, acidified their cytosol more than WT roots. Conclusions: Obviously nitrate reduction affects - by as yet unknown mechanisms - root growth and morphology. The much lower anoxic fermentation rates of WT-roots compared to LNR-H roots could not be traced back to an alternative NADH consumption by nitrate reduction, since NR activity was too low for that. An overall estimation of H+-production by glycolysis, fermentation and nitrate reduction (without nitrite reduction, which was absent under anoxia) indicated that the stronger cytosolic acidification of anoxic LNR-H roots was based on their higher fermentation rates. Thus, nitrate reduction under anoxia appears advantageous because of lower fermentation rates and concomitantly lower cytosolic acidification. However, it remained unclear why fermentation rates were so different. Perspective: Preliminary experiments had indicated that WT-roots produced more nitric oxide (NO) under anoxia than LNR-H-roots. Accordingly, we suggest that nitrate reduction, beyond a merely increased NADH-consumption, would lead to advantageous changes in metabolism, eventually via NO-production, which is increasingly recognized as an important signaling compound regulating many plant functions.
The presented work shows the analysis of the correlation between the spatial and temporal expression pattern of NtAQP1 and its function in water relation in planta. In situ immunological studies indicated NtAQP1-protein accumulation in the root exodermis and endodermis, in the cortex, close to vascular bundles, in the xylem parenchyma and in cells of the stomatal cavities. The aquaporin was also found to be abundant in longitudinal cell-rows in the petioles. Expression studies with generated transgenic plants (Ntaqp1-promoter::gus or luc) confirmed the Ntaqp1 accumulation in the root, stem and petioles but also revealed further localization in pollen grains, adventitious roots and leaf glandular hairs. Ntaqp1-expression was induced during growth processes, like stem bending after gravistimulation or photostimulation, seed germination and hypocotyl elongation as well as during the comparatively fast circadian leaf movement. The expression was further stimulated by phytohormones, especially gibberellic acid (GA) and osmotic stress. Further analysis displayed a diurnal and even circadian expression of Ntaqp1 in roots and petioles. The functional analysis of the aquaporin was accomplished by reverse genetics and biophysical studies. The antisense technique was used to reduce NtAQP1-expression in tobacco plants. The antisense (AS) plants exhibited a severe reduction of Ntaqp1-mRNA, less reduction of the highly homologous NtPIP1a RNA and no effect on expression of other aquaporin family genes (PIP2, TIP). The function of NtAQP1 at the cellular level was investigated by a newly developed experimental setup to record the osmotically induced increase in protoplast volume. The reduction of NtAQP1 by the antisense expression decreased the overall cellular waterpermeability Pos for more than 50 %. Function of NtAQP1 at the whole plant level was e.g. measured by the “high-pressure flow meter method”. Those measurements revealed that the root hydraulic conductivity per unit root surface area (KRA) of roots from the AS-lines was reduced by more than 50 %. KRA displayed a strong diurnal and circadian variation with a maximum in the middle of the light period, similar to the expression pattern of Ntaqp1 in roots. Gas exchange-, stem (Ystem) and leaf (Yleaf) water potential measurement gave dissimilar values in AS and control plants under well-watered conditions. Under a water-limiting environment the Y of AS-plants remained at more negative water values, even though a further decrease in transpiration of AS-plants was detected. Quantitative analysis displayed a much stronger wilting reaction in the AS than in the control plants. Quantitative studies of the leaf movement in AS compared to control plants exhibited a dramatic reduction in velocity and also in the extent of the process. The following conclusions can be drawn. NtAQP1 was expressed at sites of anticipated high water fluxes from and to the apoplast or symplast. Additionally, the specific distribution pattern and temporal expression of NtAQP1 in petioles and the bending stem strongly indicate a role in transcellular movement of water. The reduction of NtAQP1 by the antisense expression decreased the overall cellular Pos. Conclusively, NtAQP1-function increases membrane water permeability of tobacco root protoplasts. The decrease of the specific root hydraulic conductivity (KRA) was in the same order of magnitude as the mean cellular water permeability reduction, indicating that aquaporin expression is essential in maintaining a natural root hydraulic conductance. Reduction of KRA in AS plants might be the first definitive proof that the pathway of water uptake from the root surface to the xylem involves passage across membranes. The absence of NtAQP1 resulted in a water stress signal, causing a certain stomatal closure. NtAQP1 seems to contribute to water stress avoidance in tobacco. NtAQP1 plays an essential role in fast plant movements and transcellular water shift.