Summary• Expression of 4600 poplar expressed sequence tags (ESTs) was studied over the [2001][2002] growing seasons using trees of the moderately ozone (O 3 )-tolerant trembling aspen ( Populus tremuloides ) clone 216 exposed to elevated CO 2 and /or O 3 for their entire 5-yr life history.• Based on replication of the experiment in years 2001 and 2002, 238 genes showed qualitatively similar expression in at least one treatment and were retained for analysis. Of these 238 genes, 185 were significantly regulated (1.5-fold) from one year to the other in at least one treatment studied. Less than 1% of the genes were regulated 2-fold or more.• In the elevated CO 2 treatment, relatively small numbers of genes were upregulated, whereas in the O 3 treatment, higher expression of many signaling and defense-related genes and lower expression of several photosynthesis and energyrelated genes were observed. Senescence-associated genes (SAGs) and genes involved in the flavanoid pathway were also up-regulated under O 3 , with or without CO 2 treatment. Interestingly, the combined treatment of CO 2 plus O 3 resulted in the differential expression of genes that were not up-regulated with individual gas treatments.• This study represents the first investigation into gene expression following longterm exposure of trees to the interacting effects of elevated CO 2 and O 3 under field conditions. Patterns of gene-specific regulation described in this study correlated with previously published physiological responses of aspen clone 216.

Summary• This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO 2 ] (e[CO 2 ]) at the Aspen free-air CO 2 enrichment site near Rhinelander, WI, USA.• The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e [CO 2 ], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO 2 ], the transcriptional activity of leaf e[CO 2 ]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons.• An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO 2 -responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO 2 -unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening.• This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO 2 ]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO 2 responsiveness.

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola-specific phage (phi 11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola phi 11P, 19H and P. aeruginosa phi PLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux+ bacteria were inoculated onto French bean leaf (Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux+ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.

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