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Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H⁺-ATPase activity and abundance.

Nikolic M, Cesco S, Monte R, Tomasi N, Gottardi S, Zamboni A, Pinton R, Varanini Z - BMC Plant Biol. (2012)

Bottom Line: Chinese long) plants was studied and compared with that of the root.However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species.Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves.

View Article: PubMed Central - HTML - PubMed

Affiliation: IMSI, University of Belgrade, Kneza Viselslava, 111030 Belgrade, Serbia.

ABSTRACT

Background: The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level.

Results: The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process.

Conclusions: Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.

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Hydrolytic-activity and western-blot analyses of PM H+-ATPase from cucumber roots (A) or leaves (B). The plants were treated as described in the legend of Figure 4. Data are means ± SD of three independent experiments with three replicates. Immunodetection of the enzyme isolated from the three independent experiments was performed using antibodies raised against the C-terminal part of the Arabidopsis AHA3 PM H+-ATPase. Blots of a representative experiment are shown.
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Figure 7: Hydrolytic-activity and western-blot analyses of PM H+-ATPase from cucumber roots (A) or leaves (B). The plants were treated as described in the legend of Figure 4. Data are means ± SD of three independent experiments with three replicates. Immunodetection of the enzyme isolated from the three independent experiments was performed using antibodies raised against the C-terminal part of the Arabidopsis AHA3 PM H+-ATPase. Blots of a representative experiment are shown.

Mentions: To verify whether the modification of PM H+-ATPase occurred concomitantly with the nitrate-uptake induction, PM -enriched vesicles were isolated from roots and leaves of N-deprived or nitrate-induced (roots after 4 h and leaves after 12 h of exposure to 4 mM nitrate, respectively) of intact cucumber plants. Based on the effects of selective inhibitors on PM H+-ATPase activity, membrane preparations appeared enriched in plasma membrane vesicles. Vanadate inhibited PM H+-ATPase activity by 85% and 89% in leaf and root respectively. Moreover, the nitrate treatment did not significantly modify the composition of the isolated membrane-vesicles preparations. The hydrolytic activities and the amounts of PM H+-ATPase measured in the vesicle preparations are presented in Figure 7. When plants were placed in contact with nitrate for 4 h the specific activity of root PM H+-ATPase increased (+35%) in relation to that measured in membrane vesicles isolated from roots of N-deprived plants (Figure 7A). Western blot analysis indicated a relative increase (+31%) of the enzyme-steady-state level within plasma-membrane proteins from nitrate-induced roots. For the leaves, following treatment with nitrate for 12 h, the hydrolytic activity and the relative amount of PM H+-ATPase were respectively (+50%) and (+42%) higher than the comparative values detected in vesicle preparations isolated from leaves of N-deprived plants (Figure 7B).


Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H⁺-ATPase activity and abundance.

Nikolic M, Cesco S, Monte R, Tomasi N, Gottardi S, Zamboni A, Pinton R, Varanini Z - BMC Plant Biol. (2012)

Hydrolytic-activity and western-blot analyses of PM H+-ATPase from cucumber roots (A) or leaves (B). The plants were treated as described in the legend of Figure 4. Data are means ± SD of three independent experiments with three replicates. Immunodetection of the enzyme isolated from the three independent experiments was performed using antibodies raised against the C-terminal part of the Arabidopsis AHA3 PM H+-ATPase. Blots of a representative experiment are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403936&req=5

Figure 7: Hydrolytic-activity and western-blot analyses of PM H+-ATPase from cucumber roots (A) or leaves (B). The plants were treated as described in the legend of Figure 4. Data are means ± SD of three independent experiments with three replicates. Immunodetection of the enzyme isolated from the three independent experiments was performed using antibodies raised against the C-terminal part of the Arabidopsis AHA3 PM H+-ATPase. Blots of a representative experiment are shown.
Mentions: To verify whether the modification of PM H+-ATPase occurred concomitantly with the nitrate-uptake induction, PM -enriched vesicles were isolated from roots and leaves of N-deprived or nitrate-induced (roots after 4 h and leaves after 12 h of exposure to 4 mM nitrate, respectively) of intact cucumber plants. Based on the effects of selective inhibitors on PM H+-ATPase activity, membrane preparations appeared enriched in plasma membrane vesicles. Vanadate inhibited PM H+-ATPase activity by 85% and 89% in leaf and root respectively. Moreover, the nitrate treatment did not significantly modify the composition of the isolated membrane-vesicles preparations. The hydrolytic activities and the amounts of PM H+-ATPase measured in the vesicle preparations are presented in Figure 7. When plants were placed in contact with nitrate for 4 h the specific activity of root PM H+-ATPase increased (+35%) in relation to that measured in membrane vesicles isolated from roots of N-deprived plants (Figure 7A). Western blot analysis indicated a relative increase (+31%) of the enzyme-steady-state level within plasma-membrane proteins from nitrate-induced roots. For the leaves, following treatment with nitrate for 12 h, the hydrolytic activity and the relative amount of PM H+-ATPase were respectively (+50%) and (+42%) higher than the comparative values detected in vesicle preparations isolated from leaves of N-deprived plants (Figure 7B).

Bottom Line: Chinese long) plants was studied and compared with that of the root.However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species.Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves.

View Article: PubMed Central - HTML - PubMed

Affiliation: IMSI, University of Belgrade, Kneza Viselslava, 111030 Belgrade, Serbia.

ABSTRACT

Background: The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level.

Results: The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process.

Conclusions: Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.

Show MeSH
Related in: MedlinePlus