Differential response of gray poplar leaves and roots underpins stress adaptation during hypoxia

Abstract

The molecular and physiological responses of gray poplar (Populus x canescens) following root hypoxia were studied in roots and leaves using transcript and metabolite profiling. The results indicate that there were changes in metabolite levels in both organs, but changes in transcript abundance were restricted to the roots. In roots, starch and sucrose degradation were altered under hypoxia, and concurrently, the availability of carbohydrates was enhanced, concomitant with depletion of sucrose from leaves and elevation of sucrose in the phloem. Consistent with the above, glycolytic flux and ethanolic fermentation were stimulated in roots but not in leaves. Various messenger RNAs encoding components of biosynthetic pathways such as secondary cell wall formation (i.e. cellulose and lignin biosynthesis) and other energy-demanding processes such as transport of nutrients were significantly down-regulated in roots but not in leaves. The reduction of biosynthesis was unexpected, as shoot growth was not affected by root hypoxia, suggesting that the up-regulation of glycolysis yields sufficient energy to maintain growth. Besides carbon metabolism, nitrogen metabolism was severely affected in roots, as seen from numerous changes in the transcriptome and the metabolome related to nitrogen uptake, nitrogen assimilation, and amino acid metabolism. The coordinated physiological and molecular responses in leaves and roots, coupled with the transport of metabolites, reveal important stress adaptations to ensure survival during long periods of root hypoxia.

Figure 1.

Effects of hypoxia on expression and activity of fermentative enzymes (A) and general transcript abundance (B) in poplar roots and leaves. A, Transcript levels and activities of PDC (left) and ADH (right) in leaves (top row) and roots (bottom row) of gray poplar. Transcript levels of normoxic controls (black squares) and flooded plants (red circles) are normalized to β-tubulin mRNA levels. Means ± se of the enzyme activities (bars) from four to six independent experiments are shown. Statistically significant differences at P < 0.05 were calculated by Student's t test and are indicated by asterisks. B, Number of differentially expressed genes (≥2-fold changes; q < 0.05) following root hypoxia as determined by microarray analysis. Data represent averages of three biological replicates for each time point/tissue. Applying the criteria given in “Materials and Methods” for leaves, no differentially expressed genes were observed.

Figure 2.

Effects of hypoxia on metabolite abundance in leaves and roots of poplar. Normoxic controls and hypoxically treated trees were harvested and metabolite concentrations were determined by GC-MS (roots) at 5, 24, and 168 h and by HPLC (leaves) at 168 h. Ethanol concentrations were determined enzymatically. Metabolites listed in red showed significantly lower concentrations in treated trees than in controls. Blue and green metabolites indicate higher and unchanged concentrations, respectively. Diagrams behind metabolite names indicate fold changes (log₂ converted) of results obtained by GC-MS.

Figure 3.

Carbohydrate (A), ethanol (B), and amino acid (C) concentrations in leaves and roots of poplar affected by root hypoxia. Normoxic and hypoxically grown trees were harvested and metabolite concentrations were determined. Results shown are means ± se from at least four trees per time point and treatment. Statistically significant differences between treatments per carbohydrate species at P < 0.05 were calculated with the Tukey test under ANOVA (A) or Student's t test (B and C) and are indicated by different letters/asterisks above the bars. Cit, Citrulline; f.wt., fresh weight; 3-m-his, 3-methyl-His.

Figure 4.

Hierarchical clustering of changes in transcript abundances following flooding. Following microarray analysis, fold changes were determined from three biological replicates for each time point/tissue type. A, Hierarchical clustering was performed using the TMeV software package from log₂ signal ratio data. To distinguish between clusters, violet triangles are drawn to the left of clusters and cluster numbers are given. B, Color scale indicating signal log₂ ratios. The criteria given in “Materials and Methods” were applied, and only genes with greater than 2-fold changed expression (q < 0.05) are displayed.

Figure 5.

Expression changes of genes involved in primary carbon metabolism. After 5 h (left squares), 24 h (middle squares), and 168 h (right squares) of hypoxia, roots were harvested and transcript levels determined by microarray analysis. The log₂ values of fold changes of genes involved in carbon metabolism are displayed using the color code indicated. Protein identifiers are indicated. This figure was generated using MapMan (Thimm et al., 2004). Green arrows indicate gluconeogenesis, and orange arrows indicate the glyoxylate cycle. Details on gene expression are given in Supplemental Table S2.

Figure 6.

Effects of root hypoxia on shoot growth (A) and gas exchange (B) of gray poplar. A, Two weeks prior to and after starting the hypoxic treatment, shoot lengths were determined. Means ± se of five to 10 trees per treatment are given. B, Fully mature leaves were chosen and rates of net CO₂ assimilation (A), transpiration (E), and stomatal conductance for water vapor [g(H₂O)] were determined. Means ± se of eight leaves per plant and four plants per treatment are given. Statistically significant differences between treatments and controls at P < 0.05 are indicated by asterisks above the bars. White symbols and bars, Normoxic controls; gray symbols and bars, hypoxically treated plants.