David E. Harry

Molecular genetics and physiology of alcohol dehydrogenase in woody plants.

Abstract Alcohol dehydrogenase (ADH) is an enzyme that plays a central role in the anaerobic metabolism of plants. In herbaceous and woody plants, ADH activity occurs in root apices and in germinating seeds. In trees, ADH activity also occurs in leaves and in stems, and it is particularly high in the vascular cambium, a site of ethanol synthesis. We are investigating the biochemical properties and physiological functions of ADH in trees, as well as the environmental and developmental circumstances in which ADH is expressed. ADH genes from a number of herbaceous angiosperms have been isolated and characterized, and specific DNA sequences have been implicated in regulating ADH gene expression. This information provides a good foundation from which we are beginning to characterize the regulation of ADH genes in hardwoods and conifers. The ability to manipulate ADH genes using molecular technology will facilitate detailed explorations of the unusual physiology and biochemistry of ADH expression in woody plants. Such experiments are especially important in trees because of the apparently widespread occurrence of hypoxic conditions in their tissues.

Identification of a locus modifying the electrophoretic mobility of malate dehydrogenase isozymes in incense-cedar (Calocedrus decurrens), and its implications for population studies

Using megagametophyte (maternal haploid) and embryo (diploid) tissues of incense-cedar seeds, the expression of one of three malate dehydrogenase (MDH) loci was found to be influenced by a second, unlinked, modifier locus. Whereas alleles of the affected structural locus are codominant, the modifier alleles show dominance. The action of the modifier, limited to 1 of 28 structural loci examined, results in a shift of electrophoretic mobility detectable in conventional starch gels. Both the structural and the modifier MDH loci are polymorphic in all populations surveyed. Studies of genetic variation in natural populations made without rigorous genetic analysis may not detect such modification. By misinterpreting the genetic basis of enzyme phenotypes, such undetected modification can result in overestimates of genetic diversity in natural populations and can cause an apparent excess of homozygotes relative to expectations. These effects on allele and genotype frequency estimates are dependent on the levels of polymorphism at both the structural and the modifier loci. Using procedures common to many surveys of electrophoretic variation, the frequency of a recessive modifier allele could be as high as 0.3 before being detected.

GENE DISCOVERY IN LOBLOLLY PINE THROUGH cDNA SEQUENCING

We have initiated efforts to identify functions of loblolly pine genes based upon partial sequences of random copy DNAs. These efforts are coordinated with the construction of genetic maps (Devey et al., 1991; Neale and co-workers, unpublished data). In addition to providing possible biochemical functions encoded by individual random cDNAs, our work has allowed for the identification of classes of genes actively transcribed in tissues from actively growing seedlings or developing phloem and cambium. Also, we have gained insight into the nature of complex gene families within pine genomes.

Transcriptomic profile of leaf tissue from the leguminous tree, Millettia pinnata

Millettia pinnata (formerly Pongamia pinnata) is a fast-growing leguminous tree indigenous to the Indian subcontinent, Southeast Asia, and Australia. This species has been introduced to subtropical and arid regions of Africa, India, the Philippines, Malaysia, Australia, and the USA for commercial growth. Exhibiting saline and drought tolerance, as well as nitrogen-fixing properties, M. pinnata has been used extensively for traditional medicine and agriculture and, more recently, for the production of a biofuel feedstock. The large size, high oil content, and fatty acid profile of the seeds are well suited for biofuel production. In this study, we characterized the leaf transcriptome that was assembled de novo from 72 seedlings pooled into eight libraries. Deep paired-end short-read sequencing was performed on individual libraries using the Illumina HiSeq 2000 platform. The Trinity-assembled transcriptome of 25,146 unique genes was annotated with a combination of open-source tools. Functional annotation was facilitated through sequence homology searches, Gene Ontology term assignment, and protein domain identification. A total of 11,873 genes were classified as full-length, and 22,603 sequences were functionally annotated. Predominate Gene Ontology biological process categories included phosphorylation, metabolic processes, and oxidation-reduction processes. Orthologous gene family analysis identified 19,640 families among the 11 sequenced plant species compared. A total of 4280 were conserved across all species, and 103 were unique to the M. pinnata leaf transcriptome. The unique M. pinnata gene families included transcripts with an array of functions including ubiquitin-like modifier proteins and BED zinc finger proteins with membership in pathways related to salt tolerance and disease resistance.

Recent Advances in the Molecular Genetics of Tree Species

O n 20-23 May 1994 a group of 70 scientists gathered at Prouts I Neck, Maine, USA, to discuss progress toward understanding and manipulating the molecular genetics of a diverse group of ecologically and economically important species, forest trees. Like the organisms themselves, the presentations were diverse, spanning genes, genomes, and populations of both angiosperms and gymnosperms. The meeting was organized by Michael Greenwood and Keith Hutchison of the University of Maine (USA). Compared to previous meetings of this group, significant progress has been made in several areas. Research teams continue to isolate and characterize new genes, but transgenic plants are increasingly being used to study gene function in vivo. Genome mapping has also matured. In earlier meetings presentations described the construction of genetic linkage maps, but at this meeting maps were being used as tools to identify and dissect quantitative trait loci (QTL). The biological distinctiveness of forest trees provides both challenges and opportunities for forest biologists. Transformation systems remain recalcitrant, especially for conifers, but progress is apparent even here. On the other hand, advantages of the haploid genetics offered by conifer gametophytes continues to be exploited. Recent advances in model organisms such as Arabidopsis continue to influence studies in forest

Molecular forest genetics comes of age

Over80participants from 9countriesand 21 statesattended the fourth meeting of the Molecular Genetics Working Party, International Union of Forestry Research Organizations (I UFRO). The conference was organized by David Neale, U.S. Forest Service, Institute of Forest Genetics, Berkeley, California, USA, with corporate sponsorship provided by Westvaco and Weyerhaeuser Corporations. Held at the Stanford Sierra Camp, Lake Tahoe, California, USA, 30 September to 4 October 1990, the meeting provided an ample dose of good science in an atmosphere embellished by grandiose Sierra Nevada mountain scenery, sunny and crisp atttumn days, a wealth of recreational activities, and fine accommoda tions. While the previous three meetings of the group were dominated by ideas, techniques, and progress reports, this meeting was dominated by detailed molect,lar studies of physiological processes, gene structure, and genetic variation. The field has matured rapidly and is now asking sophisticated qttestions using much of the same repertoire of molecular tools available for studying maize and tobacco. The meeting included eight technical sessions, two poster sessions, and three concurrent discussion sessions or workshops. Many of the talks dealt with conifers, reflecting their worldwide importance to forestry and their d~stinct evohttionary position relative to the more commonly studied flowering plants. Among angiosperm tree species, members of the genus Populus (poplars) received the most scrutiny.