A mutant of Arabidopsis thaliana deficient in the accumulation of inorganic phosphate has been isolated by screening directly for plants with altered quantities of total leaf phosphate. The mutant plants accumulate approximately 5% as much inorganic phosphate, and 24 to 44% as much total phosphate, as wild-type plants in aerial portions of the plant. Growth of the mutant is reduced, relative to wild type, and it exhibits other symptoms normally associated with phosphate deficiency. The phosphate deficiency is caused by a single nuclear recessive mutation at a locus designated phol. The rate of phosphate uptake into the roots was similar between mutant and wild-type plants over a wide range of external phosphate concentrations. In contrast, when plants were grown in media containing 200 micromolar phosphate or less, phosphate transfer to the shoots of the mutant was reduced to 3 to 10% of the wild-type levels. The defect in phosphate transfer to the shoots could be overcome by providing higher levels of phosphate. Transfer of sulfate to the shoots was essentially normal in the mutant, indicating that the phol lesion was not a general defect in anion transport. Movement of phosphate through the xylem of the shoots was not impaired. The results suggest that the mutant is deficient in activity of a protein required to load phosphate into the xylem.in uptake kinetics (3,7,11). Under conditions of limited phosphate availability, the capacity to transfer the absorbed phosphate to the shoots is also increased, suggesting an increase in phosphate release into the xylem (3, 7, 11). Compartmentalization of Pi appears to be important at the cellular level, with the vacuole acting as a storage site for excess Pi that can be released under conditions ofcytosolic Pi deficiency (3, 12). Thus, understanding the mechanisms that regulate Pi acquisition is complicated by both the existence of several potential sites of control and the presence of adaptive responses.As one approach to understanding how Pi levels are regulated, a genetic study was initiated to define loci that play a role in this process. The goal was to identify plants from a mutagenized population of Arabidopsis thaliana that display quantitative differences in leaf total phosphate content when compared with wild-type plants. We describe here the properties of a novel mutant that displayed a leaf Pi content that is only approximately 5% of the wild-type level. Measurements of the rate of root Pi uptake and transfer to the shoots, under various external phosphate concentrations and physiological conditions, indicate that the mutant is most likely deficient in activity of a protein required to load phosphate into the xylem.The mechanisms that regulate fluxes of phosphate between various tissues of higher plants are not well characterized. Potential points of control are the level of root Pi uptake, transfer of the absorbed Pi to the shoot, and compartmentalization into different subcellular organelles. There appear to be two major points of regulation for ion...

Nonspecific lipid transfer proteins (LTPs) from plants are characterized by their ability to stimulate phospholipid transfer between membranes in vitro. However, because these proteins are generally located outside of the plasma membrane, it is unlikely that they have a similar role in vivo. As a step toward identifying the function of these proteins, one of severa1 LTP genes from Arabidopsis has been cloned and the expression pattern of the gene has been examined by analysis of the tissue specificity of 8-glucuronidase (CUS) activity in transgenic plants containing LTP promoter-CUS fusions and by in situ mRNA localization. l h e LTPl promoter was active early in development in protoderm cells of embryos, vascular tissues, lignified tips of cotyledons, shoot meristem, and stipules. In adult plants, the gene was expressed in epidermal cells of young leaves and the stem. In flowers, expression was observed in the epidermis of all developing inflorescence and flower organ primordia, the epidermis of the siliques and the outer ovule wall, the stigma, peta1 tips, and floral nectaries of mature flowers, and the petal/sepal abscission zone of mature siliques. l h e presence of CUS activity in guard cells, lateral roots, pollen grains, leaf vascular tissue, and interna1 cells of stipules and nectaries was not confirmed by in situ hybridizations, supporting previous observations that suggest that the reporter gene is subject to artifactual expression. These results are consistent with a role for the LTPl gene product in some aspect of secretion or deposition of lipophilic substances in the cell walls of expanding epidermal cells and certain secretory tissues. l h e LTPl promoter region contained sequences homologous to putative regulatory elements of genes in the phenylpropanoid biosynthetic pathway, suggesting that the expression of the LTPl gene may be regulated by the same or similar mechanisms as genes in the phenylpropanoid pathway.LTPs are small, soluble, basic proteins that are characterized by an ability to catalyze the transfer or exchange of lipids between membranes in vitro (reviewed in Arondel and Kader, 1990). The plant proteins of this class have a broad specificity for the type of lipid they will transfer in vitro, and

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNAblotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.Ensiling crops is a popular method of preserving forage for animal feed, especially in the humid northern regions of the United States. During harvesting and early stages of ensiling, plant membranes are ruptured, releasing proteolytic enzymes that rapidly degrade available substrates. Once fermentation by lactobacteria has progressed sufficiently to lower silage pH to less than about 5, proteolytic activity slows significantly. The wider the window between harvest and reaching a low pH, the more extensive is the proteolytic degradation. Unfortunately, excessive proteolysis in ensiled forages results in economic losses to farmers (Rotz et al., 1993) amounting to approximately $100 million annually. This economic loss is due to purchasing additional protein to supplement diets because of poor utilization of the nonprotein nitrogen products of proteolysis (ammonia, amino acids, and small peptides) by ruminant animals. Ruminants excrete much of this nonprotein nitrogen as urea, resulting in increasing nitrogen burdens upon the environment.For some ensiled forages, such as alfalfa (Medicago sativa), proteolytic losses are especially high with degradation of 44% to 87% of the forage protein (Papadopoulos and McKersie, 1983;Muck, 1987). By contrast, red clover (Trifolium pratense), a forage with protein content similar to alfalfa, has up to 90% less proteolysis than alfalfa during ens...