Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and...

A major goal currently in Arabidopsis research is determination of the (biochemical) function of each of its approximately 27,000 genes. To date, however, 12% of its genes actually have known biochemical roles. In this study, we considered it instructive to identify the gene expression patterns of nine (so-called AtCAD1-9) of 17 genes originally annotated by The Arabidopsis Information Resource (TAIR) as cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologues [see Costa, M.A., Collins, R.E., Anterola, A.M., Cochrane, F.C., Davin, L.B., Lewis N.G., 2003. An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof. Phytochemistry 64, 1097-1112.]. In agreement with our biochemical studies in vitro [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C.-H., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci. USA 101, 1455-1460.], and analysis of a double mutant [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin A., 2005. Cinnamyl Alcohol Dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076.], both AtCAD5 (At4g34230) and AtCAD4 (At3g19450) were found to have expression patterns consistent with development/formation of different forms of the lignified vascular apparatus, e.g. lignifying stem tissues, bases of trichomes, hydathodes, abscission zones of siliques, etc. Expression was also observed in various non-lignifying zones (e.g. root caps) indicative of, perhaps, a role in plant defense. In addition, expression patterns of the four CAD-like homologues were investigated, i.e. AtCAD2 (At2g21730), AtCAD3 (At2g21890), AtCAD7 (At4g37980) and AtCAD8 (At4g37990), each of which previously had been demonstrated to have low CAD enzymatic activity in vitro (relative to AtCAD4/5) [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C.-H., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci. USA 101, 1455-1460.]. Neither AtCAD2 nor AtCAD3, however, were expressed in lignifying tissues, with the latter being found mainly in the meristematic region and non-lignifying root tips, i.e. indicative of involvement in biochemical processes unrelated to lignin formation. By contrast, AtCAD7 and AtCAD8 [surprisingly now currently TAIR-annotated as probable mannitol dehydrogenases, but for which there is still no biochemical or other evidence for same] displayed gene expression patterns largely resembling those of AtCAD4/5, i.e. indicative perhaps of a quite minor role in monolignol/lignin formation. Lastly, AtCAD1 (At1g72680), AtCAD6 (At4g37970) and AtCAD9 (At4g39330), which lacked detectable CAD catalytic activities in vitro, were also expressed predominantly in vascular (lignin-forming) tissues. While their actual biochemical roles remain unknown, definition of their expression patterns, nevertheless, now begins to provide useful insights into potential biochemical/physiological functions, as well as the cell types in which they are expressed. These data thus indicate that the CAD metabolic network is composed primarily of AtCAD4/5 and may provisionally, to a lesser extent, involve AtCAD7/8 based on in vitro catalytic properties and (promoter regions selected to obtain) representative gene expression patterns. This analysis has, therefore, enabled us to systematically map out bona fide CAD gene involvement in both the assembly and differential emergence of the various component parts of the lignified vascular apparatus in Arabidopsis, as well as those having other (e.g. putative plant defense) functions. The data obtained also further underscore the ongoing difficulties and challenges as regards current limitations in gene annotations versus actual determination of gene function. This is exemplified by the annotation of AtCAD2, 3 and 6-9 as purported mannitol dehydrogenases, when, for example, no in vitro studies have been carried out to establish such a function biochemically. Such annotations should thus be discontinued in the absence of reliable biochemical and/or other physiological confirmation. In particular, AtCAD2, 3, 6 and 9 should be designated as dehydrogenases of unknown function. Just as importantly, the different patterns of gene expression noted during distinct phases of growth and development in specific cells/tissues gives insight into the study of the roles that these promoters have.