Analysis of xylem formation in pine by cDNA sequencing

Abstract

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

Figure 1

Northern blot results using cell wall-related cDNA clones identified during this sequencing project. (A) Total RNA samples separated in formaldehyde-agarose gels and stained with ethidium bromide to estimate total loading. (B) Results of hybridization of the immobilized total RNA samples to clones 1C8A and 3N5D, which encode different putative xyloglucan endotransglycosylases (XET). (C) Results of hybridization of total RNA samples to two cDNA clones similar to cell wall proteins. 6N5F is more similar to glycine-rich cell wall proteins, whereas 1CAB3A is more similar to proline-rich cell wall proteins.

Figure 2

Northern blot results using cDNAs similar to regulatory proteins. (A) The same total RNA stained with ethidium bromide, provided to allow lane identification. (B) Results of hybridization with clones similar to calcium-related proteins. 1C10G is similar to calreticulin, 5N7E is similar to calnexin, and 3N4A is similar to calcium-dependent protein kinase. (C) Results of hybridization with clones similar to homeodomain or LIM domain proteins. 5N3D and 5C7A are similar to homeodomain proteins, and 6C12H is similar to LIM domain proteins.