Effect of Potato virus Y on the NADP-malic enzyme from Nicotiana tabacum L.: mRNA, expressed protein and activity

Abstract

The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVY(O). The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVY(NTN) strain than in the case of strain PVY(O). The relevance of NADP-malic enzyme in plants under stress conditions was discussed.

Keywords: NADP-malic enzyme; Nicotiana tabacum L.; Potato virus Y strain NTN (PVYNTN); Potato virus Y strain O (PVYO); biotic stress; real time-PCR.

Figure 1.

The relative content of PVY^O (grey columns) and PVY^NTN (black columns) in leaves of Nicotiana tabacum L. in the course of infection. The relative virus content was determined by DAS-ELISA with p-nitrophenylphosphate as substrate for alkaline phosphatase. Absorbance at 405 nm is proportional to the virus content. The absorbance of each sample was measured in triplicate, S.D. are shown.

Figure 2.

Activities of NADP-ME from Nicotiana tabacum L. leaves during PVY^O (grey columns) and PVY^NTN (black columns) infections calculated per fresh mass. The activity of each sample is shown as percentage of the non-infected control in particular day, where 100% NADP-ME is 0.053 ± 0.017 μmol·min⁻¹·g⁻¹. NADP-ME activity in mock-inoculated leaves (not shown) was the same as in non-inoculated controls. The activity was measured in at least three samples, S.D. are shown. Statistical analysis was done using t-test. * denotes significant difference from controls at P<0.05.

Figure 3.

Activities of NADP-ME from Nicotiana tabacum L. stems (a) and roots (b) collected 11^th day after PVY^O inoculation and 13^th day after PVY^NTN inoculation calculated per fresh mass. The activity of each sample is shown as percentage the activity value of the non-infected control, where 100% NADP-ME is in stems 0.040 ± 0.005 μmol·min⁻¹·g⁻¹, in roots 0.076 ± 0.016 μmol·min⁻¹·g⁻¹. The activity was measured in at least six samples, S.D. are shown. Statistical analysis was done using t-test. * denotes significant difference from controls at P<0.05.

Figure 4.

Detection of NADP-ME in 10% polyacrylamide gel after non-denaturating electrophoresis. Control (marked as C), PVY^O and PVY^NTN infected leaves, stems and roots of Nicotiana tabacum L. plants were analysed. Samples were collected at the maximal symptoms occurrence (11^th day after PVY^O inoculation and 13^th day after PVY^NTN inoculation).

Figure 5.

Immunochemical detection of NADP-ME. (a) Test of linearity of Western blot analysis signal using various quantities of purified NADP-ME protein from Nicotiana tabacum L. leaves. Estimated relative molecular mass according to standard protein was 66,000. Plot indicates decrease in relative intensity of signal (expressed in %) evaluated densitomerically by Elfoman 2.0. (b) Detection of NADP-ME protein in control (white columns) and PVY^NTN infected (black columns) leaves collected 6^th–17^th day after inoculation by Western blot analysis. At least four Western blot analyses were performed. 100% corresponds to intensity of protein NADP-ME in control sample in the 6^th day. Statistical analysis was done using t-test. * denotes significant difference from controls at P<0.05.

Figure 6.

Quantification of cytosolic and choroplastic NADP-ME mRNA. (a) Amount of cytosolic and (b) chloroplast NADP-ME mRNA in PVY^NTN infected Nicotiana tabacum L. leaves (black columns) compared to healthy control (white columns) leaves measured by reverse transcription followed by real-time PCR method 6^th – 17^th day of viral infection. Result corresponds to ratio of cytosol NADP-ME transcript (a) or chloroplast NADP-ME transcript (b) and standard gene Actin9. The amount of NADP-ME mRNA was calculated from at least three samples, S.D. are shown. Statistical analysis was done using t-test. * denotes significant difference from controls at P<0.05.

Figure 7.

Concentrations of important cell metabolites. Measurement of concentrations of L-malate, D-glucose and D-glucose-6-phosphate in Nicotiana tabacum L. leaves of healthy plants and those infected by PVY^NTN. Relative concentrations (expressed as % of healthy controls) are presented (L-malate: 100% corresponds to 10.5 mmol·g⁻¹ F.W.; D-glucose: 100% corresponds to 2.8 mmol·g⁻¹ F.W.; D-glucose-6-phosphate: 100% means 0.06 mmol·g⁻¹ F.W.). Statistical analysis was done using t-test. * denotes significant difference from controls at P<0.05.