Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA

Abstract

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3- decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Figure 1

Biomass accumulation in well-irrigated control plants (stippled bars), in plants deprived of water for 5 d (black bars), and in plants deprived of water for 3 d and subsequently rehydrated for 2 d (gray bars). Effects on shoot biomass (A), shoot dry weight (B), and shoot dry weight to fresh weight ratio (C) were measured in untransformed N. plumbaginifolia (WT) and 35S-NR transformants (C1).

Figure 2

The effect of water deprivation (•, ▴) and rehydration (shaded symbols) compared with well-watered conditions (○, ▵) on ambient photosynthesis in untransformed (circles and dotted lines) N. plumbaginifolia and 35S-NR transformants (triangles and bold lines).

Figure 3

The effect of water deprivation on the carbohydrate contents of leaves of untransformed N. plumbaginifolia (stippled bars) and of 35S-NR transformants (shaded bars). Foliar Suc (A), Fru (B), Glc (C), and starch (D) were measured in plants deprived of water for 4 d. The effect of rehydration on d 3 untransformed N. plumbaginifolia (white bars) and 35S-NR transformants (black bars) is also shown. Chl, Chlorophyll.

Figure 4

The effect of water deprivation on the foliar NO₃⁻ content (A) and on foliar amino acid accumulation (B) in untransformed N. plumbaginifolia (stippled bars) and in 35S-NR transformants (shaded bars). The effect of rehydration after 3 d of water stress in untransformed N. plumbaginifolia (white bars) and in 35S-NR transformants (black bars) is also shown. Chl, Chlorophyll.

Figure 5

The effect of water deprivation on maximal extractable NR activity (A), on NR activity extracted and assayed in the presence of Mg²⁺ (B), and on the NR activation state (C). A and B, Leaves from untransformed N. plumbaginifolia (stippled bars) and in 35S-NR transformants (shaded bars) were compared. The effects of rehydration after 3 d of water stress on untransformed N. plumbaginifolia (white bars) and in 35S-NR transformants (black bars) were measured on d 4. C, Untransformed controls (□) and 35S-NR transformants (⋄) were subjected to water stress for 3 d and then water was restored for a further 2 d (▪,♦). Chl, Chlorophyll.

Figure 6

The effect of water deprivation on NR mRNA accumulation in leaves of untransformed N. plumbaginifolia (WT) and 35S-NR transformants (C1) expressed as a percentage of βATPase mRNA. Plants were deprived of water immediately after the first measurement on day t₀. mRNA abundance was then measured at the same point in the photoperiod on consecutive days of water stress (t₁, t₂, t₃, and t₄) and after 1 d of rehydration following 3 d of water deprivation (t₄R).

Figure 7

The relationship between maximal extractable NR activity and ambient photosynthesis in leaves of untransformed (▪) N. plumbaginifolia and of 35S-NR (□) transformants during water stress. Chl, Chlorophyll.