Session 10: Biochemical Genetics
Chair: Chris Somerville
email: rll3@cornell.edu
Summary: Rob Last, Boyce Thompson Institute for Plant Research at Cornell University, USA.
Although the Biochemical Genetics session covered many biochemical processes, the theme of sensing and responding to environmental and internal stimuli ran through many of the talks. This is an interesting area of plant biology given that plants grow under and adapt to a wide variety of climatic conditions.Rob Last, of the Boyce Thompson Institute for Plant Research at Cornell began the session by describing his group's genetic dissection of oxidative stress. This is an important topic because plants experience oxidative stress caused by normal metabolic processes and during growth under extreme environmental conditions, and plant pathogenesis. Last and coworkers are using the air pollutant ozone as a convenient way to increase reactive oxygen species in Arabidopsis. In published work, they and Keith Davis' lab at Ohio State University have shown that Arabidopsis induces rapid changes in gene expression following ozone fumigation. More recently, Patricia Conklin in Last's group has identified ozone sensitive mutants that are deficient in L-ascorbic acid (Vitamin C). These plants, which still have 1 millimolar concentrations of ascorbate, are sensitive to a variety of abiotic stress conditions including sulfur dioxide and UV-B. They also show altered responses to pathogens. Future studies with these mutants should reveal the ascorbate biosynthetic pathway in plants (which is still not known despite its importance in human nutrition), and will tell us more about the role of this abundant antioxidant in physiological processes and cell division.
As photosynthetic creatures, sugar metabolism and regulation of genetic and biochemical processes in response to carbohydrate status are near and dear to the hearts of plants. Several talks described approaches to the dissection of sugar sensing mechanisms using genetics and reverse genetics. By using reverse genetics, Jen Sheen's group at Massachussets General Hospital has built upon published work from her lab and others to obtain strong evidence that the enzyme hexokinase acts as a sugar sensor in higher plants. Jyan-Chyun Jang presented data showing results with transgenic plants that overexpress or underexpress hexokinase. Analysis of seedling phenotype and expression of genes whose regulation is known to be influenced by sugar showed that overexpresser plants are sugar hypersensitive and antisense transgenic plants are relatively insensitive. Because the regulatory activity of yeast hexokinase is thought to require its catalytic activity, Jang tested the effects of the fungal enzyme in Arabidopsis: overexpressing yeast hexokinase confered elevated HXK activity but did not affect sugar sensitivity in transgenic plants, implying the catalytic but not the regulatory functions of HXK are shared between plants and yeast. Based on these results, Sheen's group propose that Arabidopsis hexokinase has both catalytic and regulatory functions that are not completely overlapping.
Two groups reported using sugar sensitivity of seedlings as a way to identify mutants that may be altered in carbohydrate sensing and response. To complement and extend their work with transgenic plant, Jen Sheen' s lab has obtained several classes of glucose insensitive (gin) mutants. These mutants show altered responses to glucose at both biochemical and molecular levels. Ian Graham's group in Glasgow has taken advantage of the observation that seedlings accumulate high levels of anthocyanins and low levels of chlorophyll in their hypocotyl and cotyledons on agar medium containing high sucrose (100 mM) and depleted nitrate. They have used these growth conditions to obtain a number of cai (carbohydrate insensitive) mutants that do not accumulate high levels of anthocyanins yet still have significant levels of chlorophyll. The characterization of cai and gin mutants should help to define upstream sensing mechanisms and downstream pathways in sugar signal transduction.
Large-scale genomic DNA sequencing promises to revolutionize all aspects of plant biology, and biochemical genetics is no exception. One glimpse of this brave new world was provided by Alain Lecharny from CNRS, Universite Paris-Sud. Free arabinose and galactose, possibly released during the degradation or turn over of polysaccharides, can be metabolised along a salvage pathway starting with a sugar kinase to form a sugar-1-phosphate and then by a second enzyme to produce the UDP-sugar. As part of the ESSA (European Scientists Sequencing Arabidopsis) project, Lecharny and
coworkers discovered a galactokinase family member near the FCA locus. They identified two ESTs with homolgy to this putative sugar sensing protein. The smaller one (496 aa) maps to chromosome 3 and complements the E. coli galK galactokinase structural gene mutation, suggesting that this may be a galactokinase cDNA. In contrast, the gene on chromosome 4, which encodes a protein of twice the size and with less similarity to the galK cDNA, maps very near to the arabinose hypersensitivity mutation ara1. This mutation was discovered by Chris Cobbett's group at the University of Melbourne, where it was shown to cause an arabinose kinase deficiency. The working hypothesis of the Lecharny/Cobbett collaborators is that the galK homologue discovered by genomic sequencing encodes the arabinose kinase affected in ara1.Although the most of the inorganic nutrients plants require are available in abundance, not all are easily accessible. Mary Lou Guerinot (Dartmouth University) described molecular and genetic approaches to dissecting the processes that plants use to acquire iron from the soil. When iron is limiting, Arabidopsis roots show three responses that make iron more available to the plant: increased Fe(III) chelate reductase activity, increased Fe(II) transport and increased acidification of the rhizosphere. Mutants that have altered Fe(III) chelate reductase activities have been isolated and characterized and the mutations have been mapped in anticipation of walking to the genes. To approach Fe(II) transport, they took advantage of what is known about iron transport in yeast. The IRT1 (iron-regulated transporter) gene, encoding a probable Fe(II) transporter, was cloned by functional expression of an Arabidopsis cDNA in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. In Arabidopsis, IRT1 is expressed in roots and is induced within 24 hours after transfer of plants to iron deficient growth conditions. Database comparisons and genomic Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Interestingly, related sequences are also found in the genomes of rice, yeast, nematodes, and humans. In yeast, two of theIRT-related genes, ZRT1 and ZRT2, encode high affinity and low affinity zinc transporters, respectively. Using a zrt1;zrt2 yeast strain defective in zinc uptake, they have isolated several Arabidopsis genes that functional studies in yeast suggest may encode zinc transporters. Understanding the amino acid residues that determine ion specificity may allow creation of designer metal transport systems in plants.
Mark Knight of Oxford University described an approach to studying a process that may be common to the various signaling processes described above. Intracellular calcium acts as a second messenger in Arabidopsis transducing many signals including changes in temperature, mechanical stimulation, oxidative stress and circadian rhythms. Knight and his colleagues measure intracellular calcium non-invasively in living plants by expressing the calcium activated photoprotein, aequorin, in transgenic Arabidopsis. This allows the measurement of cytosolic free calcium in real time by measuring luminescence. A recent advancement is that they have targeted aequorin to various organelles and membranes to measure calcium fluxes at specific locations of the cell. This approach is being bolstered by identifying mutants showing altered calcium dynamics.
Participants were reminded of the interesting results being obtained in plant cellular biochemisty during a talk from Alex Conceicao of Natasha Raikhel's group at Michigan State University. Syntaxin and its homologues are membrane proteins involved in docking and/or fusion of transport vesicles. Recently the aPEP12 gene, encoding a syntaxin homologue, was cloned from Arabidopsis plants (Bassham et al., 1995, PNAS 92, 7262-7266). This plant gene product is proposed to play an important role in protein targeting because the yeast protein is involved in protein trafficking to yeast vacuoles, and may be associated with a prevacuolar organelle (Becherer et al., 1996, Mol. Biol. Cell 7, 579-594). Conceicao showed that the Arabidopsis PEP12 protein (aPEP12p) is present either as one or two major isoforms depending on the tissue. Several subcellular fractionation techniques indicate that both aPEP12p isoforms are localized to the same organelle. Furthermore, immunocytochemical studies of seeds showed that aPEP12p is present on the membranes of smooth-vesicles; they are grouped to form multivesicular bodies which could correspond to endocytic or prevacuolar organelles. Prevacuoles have not been biochemically characterized in plants and aPEP12p could be a first membrane marker of this organelle type.
I thank those speakers who helped to write this summary, and apologize and take responsibility for any mistakes included.