2 PINE ENDO-BETA-1,4-GLUCANASES ARE ASSOCIATED WITH RAPIDLY GROWING REPRODUCTIVE STRUCTURES

Two cDNA clones encoding endo-beta-1,4-glucanases (EGases) were isolat ed from a radiata pine (Pines radiata) cDNA library prepared from imma ture female strobili. The cDNAs PrCel1 (Pines radiata cellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectivel y, including putative signal peptides. Both proteins contain domains c onserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase clone d from tomato (Lycopersicon escelentum) pistils, than to any other rep orted EGases. Northern-blot analyses indicated that both genes display ed a similar pattern of expression. The only significant difference wa s in the level of expression. In situ hybridizations were used to demo nstrate that, within differentiating pine reproductive structures, PrC el1 expression was greatest in microsporangia in pollen strobili and n ear the developing ovule in the seed strobiii. Expression was also fou nd in vegetative tissues, especially in regions experiencing cell elon gation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot a nalysis indicated the presence of a family of EGase genes in the radia ta pine genome, and that PrCel1 and PrCel2 are transcribed from distin ct one-copy genes.