Purification and cloning of an arabinogalactan-protein from xylem of loblolly pine.

An arabinogalactan-protein (AGP) was purified from differentiating xylem of loblolly pine (Pinus taeda L.) and the N-terminal sequence used to identify a cDNA clone. The protein, PtaAGP3, was not coded for by any previously identified AGP-like genes. Moreover, PtaAGP3 was abundantly and preferentially expressed in differentiating xylem. The encoded protein contains four domains, a signal peptide, a cleaved hydrophilic region, a region rich in serine, alanine, and proline/hydroxyproline, and a hydrophobic C-terminus. It is postulated to contain a GPI (glycosylphosphatidylinositol) anchor site. If the protein is cleaved at the putative GPI anchor site, as has been observed in other classical AGPs, all but the Ser-Ala-Pro/Hyp-rich domain may be missing from the mature protein. Xylem-specific AGPs are hypothesized to be involved in xylem development.

Sequences upstream and downstream of two xylem-specific pine genes influence their expression.

The identification of regulatory elements conferring high levels of expression in differentiating pine xylem will be valuable for genetic engineering of wood properties and will contribute to our understanding of gene regulation in this important group of forest trees. We examined the roles of both upstream and downstream elements in regulating the expression of two genes with preferential expression in developing xylem of loblolly pine. Gene constructs containing a PtX3H6, PtX14A9, or CaMV 35S promoter, the uidA gene encoding beta-glucuronidase, and a PtX3H6, PtX14A9, or NOS terminator were used to transform tobacco and hybrid poplar. When combined with the NOS terminator, neither pine promoter conferred xylem-specific expression in tobacco. When combined with the PtX3H6 promoter, an element at the 3' end of PtX3H6 reduced GUS expression resulting in preferential expression in vascular tissues. This silencing effect was not observed when the pine terminator was tested in conjunction with the CaMV 35S promoter. The PtX14A9 terminator did not increase tissue specificity. In leaves of transgenic poplar, both pine promoters conferred preferential GUS expression in veins when combined with the NOS terminator. The PtX3H6 terminator greatly decreased expression in leaves and stems when combined with the PtX3H6 promoter but only slightly altered expression when combined with the CaMV 35S promoter. An element at the 3' end of PtX14A9 increased GUS expression in veins when used in conjunction with either the PtX14A9 or CaMV35S promoter.

Two pine endo-beta-1,4-glucanases are associated with rapidly growing reproductive structures.

Two cDNA clones encoding endo-beta-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (inus adiata cellulase ) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved 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 cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.

Xylem-specific gene expression in loblolly pine.

Two genes preferentially expressed in differentiating xylem of loblolly pine (Pinus taeda L.) were cloned from cDNA and genomic libraries and designated PtX3H6 and PtX14A9. Transcripts of PtX3H6 and PtX14A9 are very abundant in differentiating xylem, less abundant in needles, and very low or non-detectable in embryos and megagametophytes. PtX3H6 contains a putative signal peptide, a threonine-rich region, a proline-rich region, and a hydrophobic tail. Repeats of Pro-Pro-Pro-Val-X-X are similar to repeats found in proline-rich cell wall proteins. The amino acid compositions of PtX3H6 and PtX14A9 are similar to those of arabinogalactan proteins (AGPs). PtX14A9 contains an 8 amino acid sequence similar to amino terminal sequences of ryegrass, carrot and rose AGPs. Upstream sequences have been determined from genomic clones encoding PtX3H6 and PtX14A9. A 7 bp sequence found in the 5' flanking regions of both genes has previously been shown to be involved in the vascular-specific expression of GRP 1.8, a glycine-rich protein found in bean. The sequence is also present upstream of another glycine-rich protein from bean, GRP 1.0, and may be partially responsible for the xylem-specific expression of pTx3H6 and PtX14A9.

Agrobacterium-mediated DNA transfer in sugar pine.

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.