Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions

Abstract

Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.

Fig. 1

Summary of oilseed rape culture conditions and experimental workflow designed to apply contrasted nitrogen and/or water treatments. N+W+, control plants received 8 mM nitrate and were watered throughout; N−W+, N-deprived plants received 0.4 mM nitrate for 5 weeks and were watered throughout; N+W−, water-stressed plants received 8 mM nitrate but were not watered for 2 weeks from day 0; N−W−, N and water-deprived plants received 0.4 mM nitrate when watered but were not watered for 2 weeks from day 0

Fig. 2

Phenological response of oilseed rape plants subjected to nitrogen depletion and/or water shortage as estimated by the number of leaves per plant during treatments. Treatments are referred to as described in Fig. 1. The oldest leaves were ranked #1 and were taken into account when their petiole was still attached to the stem. Cotyledons were not considered. The youngest leaves were counted when their petiole became visible. The total leaf number was obtained by difference between the youngest and the oldest leaf ranks. Values are expressed as means of five independent plants ± standard error

Fig. 3

Changes in chlorophyll content (a, c) and maximum photosynthetic efficiency (F _v/F _m; b, d) in leaves of oilseed rape plants with high or low N input with or without water shortage (N+W+, line with black filled square; N−W+, line with grey filled square; N+W−, line with open square; N−W−, line with grey open square; treatment codes as in Fig. 1). a, b Results shown are for leaf ranks #6, #9, #12 and #15 after 14 days of treatment (day 14). c, d Results for leaf rank #9 during the 22 days of the experiment. Values are expressed as means of five independent replicates ± standard error. Different letters indicate significant differences in the Tukey HSD test (P ≤ 0.05) between leaf ranks (a, b) or treatments after 14 and 22 days of treatment (c, d). na Leaf not apparent

Fig. 4

Changes in stomatal conductance (a, f), relative water content (RWC, b, g), water content (c, h), water potential (d, i) and osmotic potential (e, j) in leaves of oilseed rape plants receiving high or low N input with or without water shortage (N+W+, line with black filled square; N−W+, line with grey filled square; N+W−, line with open square; N−W−, line with grey open square; treatment codes as in Fig. 1). a–e Results for leaf ranks #6, #9, #12 and #15 after 14 days of treatment (day 14). f Results for leaf rank #12. g–j Results for leaf rank #9 during the 22 days of the experiment. Values are expressed as means of five independent replicates ± standard error. Different letters indicate significant differences in the Tukey HSD test (P ≤ 0.05) between leaf ranks (a–e) or treatments after 14 and 22 days of treatment (f–j). f Leaf fallen, na leaf not apparent, nm not measured as not possible to collect leaf juice

Fig. 5

Free proline content of leaves of oilseed rape plants with high or low N input (N+W+, black filled square; N−W+, grey filled square, a) followed or not by water shortage (N+W−, open square; N−W−, grey open square, b) after 14 days of treatment. Values are expressed as means of five independent replicates ± standard error

Fig. 6

Proline as a percentage of total free amino acids (TFAA) in laminae (a) and phloem sap (b) of leaf ranks of oilseed rape plants with high or low N input (N+W+, black filled square; N−W+, grey filled square) followed or not by water shortage (N+W−, open square; N−W−, grey open square) after 14 days of treatment. Values are expressed as means of five independent replicates ± standard error

Fig. 7

Linear relationships between free proline content and relative water content in leaves of N+W− (a, c) and N−W− (d) oilseed rape plants subjected to water shortage during 14 days (day 0–14), and followed by rehydration of 8 days (day 14–22) for N+W− plants (b). Values are expressed as means of five independent replicates

Fig. 8

Apparent proline biosynthesis capacity of explants from distinct leaf ranks of oilseed rape plants receiving high or low N input for 10 days followed or not by water shortage for 7 days. Treatment codes are as for Fig. 1. Proline content was measured at time of sample collection (T0) and after 16 h of hyper-osmotic stress at −2.5 MPa (PEG-induced). a Results were obtained for leaf ranks #3, #6, #9 and #12 for control plants (N+W+). b Results for leaf ranks #2, #4, #6 and #8 for N-deprived plants (N−W+). c Results for leaf ranks #4, #7, #9 and #11 for water-stressed plants (N+W−). d Results for leaf ranks #2, #4, #6 and #8 for N− and water-deprived plants (N−W−). Values are expressed as means of four independent replicates ± standard error. Different letters indicate significant differences using the Tukey HSD test (P ≤ 0.05) between leaf ranks after PEG incubation

Fig. 9

Apparent proline consumption capacity of explants from leaf ranks #3 and #8 of oilseed rape plants receiving high or low N input for 10 days (N+W+ black filled square/N−W+ grey filled square) followed or not by water shortage for 7 days (N+W− open square/N−W− grey open square). The percentage of proline consumed after 4 hours in −0.04 MPa medium was estimated relative to the proline content measured after 6 hours of enrichment with proline (40 mM). Values are expressed as means of five independent replicates ± standard error. Asterisks indicate significant differences between leaf ranks for each treatment in the Student’s t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001)

Fig. 10

Relative expression levels measured by qRT-PCR of BnP5CS1 (a), BnP5CS2 (b) and BnPDH1 (c) genes and free proline contents (d) in leaf ranks #7, #8, #10, #11 and #13 of oilseed rape plants grown with high or low N input for 14 days (N+W+ black filled square/N−W+ grey filled square) followed or not by water shortage for 10 days (N+W− open square/N−W− grey open square). Values are expressed as means of three independent replicates ± standard error. Letters indicate significant differences in the Tukey HSD test (P ≤ 0.05) between leaf ranks for each treatment