Isolation and Functional Analysis of Drought-Induced Genes in Pinus

Abstract

Drought is a common factor limiting timber production in the U.S. and Israel. Loblolly (Pinus taeda) and alleppo pine (Pinus halepensis) seedling survival is reduced when out planted, and growth and reproduction are often hindered by periodic droughts during later stages of tree development. Molecular and gene responses to drought stress have not been characterized. The objectives were to characterize drought-induced gene clones from these pines, to determine the effects of a growth regulator on drought tolerance, ABA levels, and drought-induced gene expression in alleppo pine, and to develop procedures for loblolly pine transformation. Nearly 20 cDNA clones influenced by gradual, prolonged drought stress have been isolated. Many of these have been shown to be induced by drought stress, whereas several others are down-regulated. These are the first drought-induced genes isolated from a pine species. Two genomic clones (lp5-1 and lp3-1) have been sequenced and characterized, and each has been found to be associated with a gene family. Clone lp5 appears to code for a cell wall protein, and clone lp3 codes for a nuclear protein. The former may be associated with changing the elastic properties of the cell wall, while the latter may be involved in signal transduction and/or protection from desiccation in the nucleus. Clone lp3 is similar to a drought-induced gene from tomato and is regulated by ABA. Several DNA sequences that are specific to induction during growth-retardation in alleppo pine by uniconazole have been identified. The active DNA species is now being identified. Promoters from genomic clones, lp3 and lp5, have been sequenced. Both are functional when fused with the gus reporter gene and transferred to other plant tissues as well as responding to a simulated drought stress. Through exodeletion analysis, it has been established that the promoter ABRE element of lp3 responds to ABA and that drought-induction of lp3 expression may also involve ABA. Stable tobacco transformants carrying either the lp5 or the lp3 promoter fused to a reporter gus gene have been obtained. The lp5lgus fusion was expressed at several stages of tobacco development and differentiation including the reproductive stage. There was no difference in phenotype between the transformants and the wild type. Embryogenesis procedures were developed for slash pine, but attempts to couple this process with gene transfer and plantlet transformation were not successful. Transformation of pine using Agrobacterium appears tractable, but molecular data supporting stable integration of the Agrobacterium-transferred gene are still inconclusive.