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In phyllosphere microbiology, the distribution of resources available to bacterial colonizers of leaf surfaces is generally understood to be very heterogeneous. However, there is little quantitative understanding of the mechanisms that underlie this heterogeneity. Here, we tested the hypothesis that different parts of the cuticle vary in the degree to which they allow diffusion of the leaf sugar fructose to the surface. To this end, individual, isolated cuticles of poplar leaves were each analyzed for two properties: (1) the permeability for fructose, which involved measurement of diffused fructose by gas chromatography and flame ionization detection (GC-FID), and (2) the number and size of fructose-permeable sites on the cuticle, which was achieved using a green-fluorescent protein (GFP)-based bacterial bioreporter for fructose. Bulk flux measurements revealed an average permeance P of 3.39 × 10(-9) ms(-1), while the bioreporter showed that most of the leaching fructose was clustered to sites around the base of shed trichomes, which accounted for only 0.37% of the surface of the cuticles under study. Combined, the GC-FID and GFP measurements allowed us to calculate an apparent rate of fructose diffusion at these preferential leaching sites of 9.15 × 10(-7) ms(-1). To the best of our knowledge, this study represents the first successful attempt to quantify cuticle permeability at a resolution that is most relevant to bacterial colonizers of plant leaves. The estimates for P at different spatial scales will be useful for future models that aim to explain and predict temporal and spatial patterns of bacterial colonization of plant foliage based on lateral heterogeneity in sugar permeability of the leaf cuticle.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino- terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a Km for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 µM, a Vmax of 38.3 ± 4.5 nmol mg-1 min-1, and a catalytic efficiency/Km of 1,873 M-1 S-1. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis. [ABSTRACT FROM AUTHOR]