Ronald J. Newton

Expression analysis of a gene family in loblolly pine (Pinus taeda L.) induced by water deficit stress

A cDNA clone (lp3) from loblolly pine induced by water deficit stress (WDS) has been isolated. It is preferentially induced in roots with a constitutive basal level of expression also observed in stems and needles. Northern blot analysis with well irrigated ABA-treated seedlings indicated that the overall accumulation of lp3 transcripts in the roots was lower than that of water deficit-stressed seedlings. However, within roots, lp3 was induced by ABA indicating that the expression of lp3 in roots under WDS conditions was partly mediated by ABA. The lp3 clone is similar to a group of genes called asr (ABA stress and ripening) genes identified in several species. A genomic clone (lp3-1) was identified and its putative protein has the hydrophylicity profile similar to that of lp3 except for two deletions in the 5′ region. The genomic Southern and RT-PCR (reverse transcriptase-polymerase chain reaction) analyses indicate that the lp3 gene belongs to a small multigene family of at least four members with a distinct pattern of expression during WDS.

Growth enhancement of loblolly pine (Pinus taeda L.) seedlings by silicon

Summary The effect of silicon on growth and water status of Pinus taeda L. seedlings was studied under normal growth conditions and during induced water-stress. Seedlings were grown either on a sand or a fritted clay medium supplemented with nutrient solutions containing 0, 60 and 100 mg l −1 K 2 SiO 3 . Silicon-treated seedling fascicles accumulated 3 times more silicon than did controls. Fresh and dry weights of seedlings were enhanced by silicon; the percentage increase in growth compared with the controls was greater under water-stress conditions. The enhanced growth was associated with higher water and osmotic potentials, a greater symplastic water volume, increased tissue elasticity, and less turgor. These data suggest that silicon enhances pine seedling growth by increasing cell expansion.

Expression of an abscisic acid responsive promoter in Picea abies (L.) Karst. following bombardment from an electric discharge particle accelerator

The 1.5 kilobase promoter sequence upstream of Dc8, a late embryo abundant gene of Daucus, fused to the reporter β-glucuronidase gene was introduced into several tissues of Picea abies via a custom-made electric-discharge particle accelerator. Transient expression was measured histochemically as spot number 2 d after bombardment. Embryogenic suspensions gave higher levels of expression depending upon cell line than embryogenic callus or zygotic embryos. Expression was enhanced when cultures were treated with abscisic acid for 3 d before bombardment. A mean and maximum of 17 and 34 spots/disk, respectively, were observed with the best cell line, which was comparable with the level of expression driven by an enhanced 35S promoter.

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants.

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10−7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10−7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.

Cloning of a cDNA for a chitinase homologue which lacks chitin-binding sites and is down-regulated by water stress and wounding.

A cDNA clone (pLP6) of a gene which is repressed under water deficit was isolated from a loblolly pine (Pinus taeda L.) cDNA library and characterized. The predicted polypeptide encoded by pLP6 bears strong resemblance to a number of Class I chitinases. Howerver, LP6 lacks most of the amino-terminal and, consequently the signal peptide, cysteine-rich chitin-binding domain and glycine/proline-rich ‘hinge’ region, diagnostic of Class I chitinases, are absent. Although the cDNA is similar in size to its mRNA, the long open reading frame encoding the LP6 protein commences halfway through the mRNA, implying a 5′-untranslated region of over 700 nucleotides. Subfragments from the 5′ end of pLP6 hybridize to the same mRNA as do probes consisting of the entire cDNA. Reverse transcription (RT)-PCR experiments confirm that the cDNA derives from a single mRNA molecule. Analysis of the 5′-UTR revealed six upstream open reading frames and four inverted repeat structures. Expression of the pLP6 gene is repressed by water deficit stress and wounding. Possible functions and origin of this gene are discussed.

A Simple Method for Identifying Plant/T-DNA Junction Sequences Resulting from Agrobacterium-mediated DNA Transformation

Genomic integration of transferred T-DNA is traditionally analyzed by Southern hybridization; however, these analyses often do not provide sufficient information pertaining to the transformation event. Analysis of the junction sequences spanning the region between the T-DNA borders and plant genomic DNA, give a clear demonstration of genomic integration. The procedures available for border junction analysis can be problematic, therefore a simplified method was developed for plants transformed by Agrobacterium tumefaciens harboring the binary vector with pBI121 backbone.

Water stress-induced changes in protein synthesis of slash pine hypocotyls

Summary Hypocotyl segments from growing regions of four-week-old slash pine ( Pinus elliottii Engelm) seedlings exhibited reduced protein synthesis when subjected to water stress imposed directly by incubation in solutions of mannitol. Hypocotyl segments recovered full protein synthesis upon return to fully-hydrated conditions following stress up to −0.8 MPa, while slow recovery was observed under greater stresses (−1.3 MPa to −2.5 MPa). Using one-dimensional electrophoresis and fluorography of de novo synthesised proteins, it was possible to follow changes in the pattern of protein synthesis in slash pine hypocotyls. Of the newly-synthesised proteins visualized by fluorography three new proteins (71 kD, 70 kD, 58 kD) showed strong enhancement in hypocotyls exposed to −2.5 MPa and a 46 kD protein was induced in hypocotyls exposed to −1.8 MPa for 24 h.

Growth response of loblolly pine (Pinus taeda L.) seedlings to ozone fumigation

Twenty-two week-old Pinus taeda L. (loblolly pine) seedlings of 30 open-pollinated and five full-sib families, representing a wide range in geographic origin, were grown in charcoal-filtered (CF) air or CF-air supplemented with 160 or 320 nl liter(-1) ozone for 8 h day(-1), 4 days week(-1), for 9 weeks. Visible foliar injury (banded chlorosis, tip burn and premature senescence) was apparent in many families after 3 weeks in 320 nl liter(-1) and 6 weeks in 160 nl liter(-1) ozone. Decreases in relative height and root collar diameter growth rates, total dry weight, root dry weight, shoot dry weight, and root/shoot ratios were evident after 9 weeks of treatment with both 160 and 320 nl liter(-1) ozone. For relative height growth rates, family differences in response to ozone were observed. By the studys end, net photosynthesis rates were 15% less for the 320 nl liter(-1) ozone treatment as compared to the CF-air treatment. Total soluble sugar and total starch content of roots were not changed after 9 weeks of ozone exposure.

Solute contributions to osmotic potential in loblolly pine (Pinus taeda L.) callus

Summary Solute contributions to the callus Ψ s were assessed in loblolly pine ( Pinus taeda L.) callus while growing on medium at 2 Ψ w s over a 5-week period. Murashige and Skoog medium with 30 g 1- 1 sucrose was used to produce a high Ψ w of −0.4 MPa (H) and the same medium was used to create a moderate Ψ w of −0.7 MPa (M) by adding 10% (w/v) polyethylene glycol (PEG, MW = 8000). Callus growth, Ψ w , Ψ s , and 4 solute classes (potassium, malate, soluble carbohydrates and amino acids) were measured and Ψ p (Ψ w -Ψ s ) was calculated. Callus Ψ p was maintained over time on both H and M media. H-callus Ψ w approached equilibrium with that of the external H medium after 5 weeks, whereas M-callus Ψ w was lower than that of the M medium. After 5 weeks on either medium, no increase was observed in the level of total solutes, when expressed per unit of either tissue DW or FW. At 5 weeks, the 4 solute classes were calculated to account for 87% of the H-callus Ψ s and 42% of the M-callus Ψ s . Apoplastic PEG accounted for an additional 14% (0.21 MPa) of theM-callus Ψ s . H-callus Ψ s increased with time because of increased water uptake, whereas M-callus Ψ s decreased because of PEG accumulation and probable PEG blockage of Mcallus hydration. Osmotic adjustment was not observed in this callus system.

Analysis And Localization of the Water-Deficit Stress-Induced Gene (lp3)

LP3 is a water-deficit-induced protein, which is highly homologous to ASR (ABA, stress and ripening) proteins. Homology was found in the C-terminal region of the putative LP3 protein while lower homologies were found in the N-terminal region. The goal of this study was to investigate the function of the LP3 protein and the mechanism of the lp3 promoter in response to water-deficit stress (WDS) and other stresses. In regenerated transgenic tobacco (T0), expression of β-glucuronidase (GUS) from the lp3 promoter-GUS construct was observed in polyethylene glycol (PEG), abscisic acid (ABA), methyl-jasmonate (MeJa), and fluridone (Flu) treatments. GUS expression was not observed following gibberellin (GA3), 2-methyl-4-dichlorophenoxy acetic acid (2,4-D), silver nitrate, or ethephon (ethylene releasing agent) treatments. Germinated T1 seedlings containing the lp3 promoter-GUS construct exhibited GUS activity up to 40 days postgermination. Expression could be restored when 5-azacytidine was included in the culture media, indicative of a developmentally regulated silencing mechanism involving methylation. In transgenic tobacco, the LP3 protein localized in the cell nucleus was induced by WDS and appeared to be developmentally regulated.