Reduced nitric oxide levels during drought stress promote drought tolerance in barley and is associated with elevated polyamine biosynthesis

Abstract

Nitric oxide (NO) is a key messenger in plant stress responses but its exact role in drought response remains unclear. To investigate the role of NO in drought response we employed transgenic barley plants (UHb) overexpressing the barley non-symbiotic hemoglobin gene HvHb1 that oxidizes NO to NO₃^-. Reduced NO production under drought conditions in UHb plants was associated with increased drought tolerance. Since NO biosynthesis has been related to polyamine metabolism, we investigated whether the observed drought-related NO changes could involve polyamine pathway. UHb plants showed increases in total polyamines and in particular polyamines such as spermidine. These increases correlated with the accumulation of the amino acid precursors of polyamines and with the expression of specific polyamine biosynthesis genes. This suggests a potential interplay between NO and polyamine biosynthesis during drought response. Since ethylene has been linked to NO signaling and it is also related to polyamine metabolism, we explored this connection. In vivo ethylene measurement showed that UHb plants significantly decrease ethylene production and expression of aminocyclopropane-1-carboxylic acid synthase gene, the first committed step in ethylene biosynthesis compared with wild type. These data suggest a NO-ethylene influenced regulatory node in polyamine biosynthesis linked to drought tolerance/susceptibility in barley.

Figure 1

Polyamine metabolism and interaction with other metabolic routes modified from Alcazar et al., 2010 and Imgarberdiev et al., 2011. Red names indicate polyamines, blue names indicate aminoacids, green names indicate the simplified haemoglobin cycle, grey abbreviations correspond to enzymes: NR, nitrate reductase; NIR: nitrite reductase; GlnS, glutamine synthetase; GluS: glutamate synthase; GluDC: glutamate decarboxylase; OAT: ornithine δ-aminotransferase; AR: Arginase; ODC: ornithine decarboxylase; ADC: arginine decarboxylase; MAT: synthetase, AdoMetDC: decarboxylase; SpmS spermine synthase, SpdS spermidine synthase; DAO: diamine oxidase: PAO: polyamine oxidase, ACS: ACC synthase; ACO: ACC oxidase. It has been shown recently that polyamine oxidase is not only involved in the terminal catabolism of polyamines, but also in the back-conversion of spermine to spermidine and spermidine to putrescine.

Figure 2

In vivo NO generation and hemoglobin gene expression. (a) In vivo NO measurement in barley UHb-05 and UHb-06 lines and WT Golden Promise intact plants. (b) Expression of HvHb1 gene. White bar = watered controls; Black bars = plants exposed to drought treatment. Data are mean of four replicates ± standard error. *, **, *** indicate significant differences at P < 0.05, 0.01 and 0.001, respectively with respect to WT control plants.

Figure 3

Drought stress symptoms in UHb and wild type (WT) Golden Promise plants. (a) Drought symptoms were evaluated after withholding water according to a visual scale (Sánchez-Martín et al.) in UHb-05 (triangles), UHb-06 (squares) and WT (circles) plants. Bars in the right panel represent the area under the drought progress curve to assess quantitative stress tolerance, (for details see). Data are mean of five replicates ± standard error. *, ** indicate significant differences at P < 0.05 and 0.01, respectively between WT and UHb plants. (b) Pictures of WT and UHb plants at 20–25% of sRWC showing more drought symptoms in WT plants.

Figure 4

Water related parameters in UHb and wild type (WT) Golden Promise plants. (a) Leaf relative water content. (b) Midday water potential, and (c) Transpiration. Transpiration rate per unit area was measured at different sampling times (left panel) and as accumulated transpiration per unit area (right panel) representing the sum of all transpired water over the assessed period. All parameters were evaluated at 20–25% soil relative water content in UHb-05 (triangles), UHb-06 (squares), and WT (circles) plants. White bars/symbols = control, well-watered plants; Black bars/symbols = plants exposed to water stress. Data are mean of five replicates ± standard error. *, *** indicate significant differences between droughted and well-watered plants at P < 0.05, and 0.001 respectively.

Figure 5

Polyamine content in UHb and wild type (WT) Golden Promise plants. Putrescine, spermidine, spermine, agmatine, their total content (total PA) and the degradation product, 1,3-diamino-propane (DAP) were measured at 20% soil relative water content in UHb and WT plants. White bars = control, well-watered plants; Black bars = plants exposed to drought treatment. Data are mean of five replicates ± standard error. *, **, indicate significant differences between droughted and well-watered plants at P < 0.05, and 0.01 respectively.

Figure 6

Amino acids content in UHb and wild type (WT) Golden Promise plants. Amino acids related with polyamine biosynthesis pathway were measured at 20% soil relative water content in UHb and WT plants. White bars = control, well-watered plants; Black bars = plants exposed to drought treatment. Data are mean of five replicates ± standard error. *, **, *** indicate significant differences between droughted and well-watered plants at P < 0.05, 0.01 and 0.001 respectively.

Figure 7

Scheme of Pearson correlations between total polyamines (PA) and their most direct amino acids precursors (Arginine, ARG; Citrulline, CIT; Methionine, MET; Ornithine, ORN) and derivatives (ϒ-aminobutiric acid, GABA; and 1,3-diaminopropane, DAP) in barley WT and UHb plants. +/ red-colored squares indicated significant positive correlations and −/blue-colored squares indicate significant negative correlations. More details about the correlation coefficients and significance can be accessed in Supplementary Table 1.

Figure 8

Expression of several genes involved in the polyamine pathway in UHb and wild type (WT) Golden Promise plants. Expression of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), methionine adenosyltransferase (MAT), S-Adenosylmethionine decarboxylase (AdoMetDC), and 1-aminocyclopropane-1-carboxylate [ACC] synthase (ACS) were measured at 20% soil relative water content in UHb and WT plants. White bars = control, well-watered plants (W); Black bars = plants exposed to drought treatment (D). Data, expressed as fold change in expression respect to WT well-watered plants, are mean of at least 3 independent biological plus 3 technical replications ± standard error. *, **, *** indicate significant differences with respect WT well-watered plants at P < 0.05, 0.01 and 0.001, respectively.

Figure 9

Ethylene generation and senescence symptoms in UHb and WT Golden Promise plants. (a). In vivo ethylene generation in barley UHb plants and WT Golden Promise. Ethylene was quantified in whole intact UHb and wild type plants at 20–25% soil relative water content. Data are mean of four replicates + standard error. (b). Spad chlorophyll meter readings (SCMR) of barley UHb plants and WT Golden Promise. SCMR were assessed in the first leaf at 20–25% sRWC. Data are based on five replicates per genotype and treatment and three SCMR per leaf. White bar = watered controls (W); Black bars = plants exposed to drought treatment (D). **, *** indicate significant differences at P < 0.01, and 0.001 with respect to control plants.