The process of leaf senescence is biochemically characterized by the transition from nutrient assimilation to nutrient remobilization. The nutrient drain by developing vegetative and reproductive structures has been implicated in senescence induction. The steady-state levels of amino acids in senescing leaves are dependent on the rate of their release during protein degradation and on the rate of efflux into growing structures. To determine the possible regulatory role of amino acid content in leaf senescence, an in planta non-destructive, semi-quantitative method for the analysis of endogenous levels of free amino acids has been developed. The method is based on in vivo bioluminescence of amino acid-requiring strains of recombinant Escherichia coli carrying the lux gene. The luminescence signal was found to be proportional to the levels of added exogenous tryptophan and to the free amino acid levels in the plant tissues analysed. During the senescence of tobacco flowers and of detached leaves of oats and Arabidopsis, a progressive increase in the levels of free amino acids was monitored. By contrast to the detached leaves, the attached oat leaves displayed a decrease in the levels of free amino acids during senescence. In Arabidopsis, both the attached and detached leaves exhibited a similar pattern of gradual increase in amino acid content during senescence. The differences between the sink-source balance of the two species and the possible relationships between amino acid content and leaf senescence are discussed.

Cytokinin replaces light in several aspects of the photomorphogenesis of dicot seedlings. Arabidopsis thaliana seedlings grown under red light have been shown to become disoriented, losing the negative hypocotyl gravitropism that has been observed in seedlings grown in darkness or white light. We report here that cytokinin at micromolar concentrations restores gravitropism to seedlings grown under red light. Cytokinin cancels the effect of red light on the gravity-sensing system and at the same time replaces light in the inhibition of hypocotyl elongation. Furthermore, application of the ethylene precursor 1 -aminocyclopropane-I -carboxylic acid acts similarly to cytokinin. Cytokinin cannot restore gravitropism under red light to an ethylene-insensitive mutant that is defective at the NN2 locus. Stimulation of ethylene production, therefore, can explain the action of cytokinin in restoring negative gravitropism to the hypocotyls of Arabidopsis seedlings grown under continuous red light.

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development.

The release of the ribonucleic acid (RNA)-containing phage MS2 from Escherichia coli is accompanied by cellular lysis at 37 C, whereas at 30 C phage are released from intact cells. Chloramphenicol or rifampin prevents the release of progeny phage particles at both temperatures. Neither drug causes an immediate cessation of phage release and after inhibition of protein synthesis by chloramphenicol phage release proceeds for about 17 min at 37 C and about 35 min at 30 C. Rifampin does not inhibit phage release from mutant cells possessing a rifampin-resistant deoxyribonucleic acid-dependent RNA polymerase. The results indicate that a short-lived host-controlled protein(s) is essential for the release of RNA phage particles at both temperatures.

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serumfree medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, >90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.In embryonic development, secondary stem cells of mesenchymal origin give rise to a number of differentiated cell types including adipocytes, myoblasts, myotubes, chondrocytes, osteocytes, and adult fibroblasts. This process has been mimicked in cell culture by treating fibroblastic cells of embryonic origin with 5-azacytidine for as short a time as a single cell cycle (1-3). Indeed, the response of embryonic fibroblastic cell lines to this drug has provided the basis for their identification as secondary mesenchymal stem cells. Since 5-azacytidine decreases the extent of cytosine methylation in DNA, its effect on stem cells has suggested that decreased DNA methylation is involved in the differentiation process. The results reported here are part of an ongoing investigation of molecular mechanisms underlying specific steps in development and the distribution of stem cells into alternative pathways of terminal differentiation.In a previous report, we described the ability of 5-azacytidine to induce Chinese hamster embryonic fibroblastic (CHEF/18) ~ stem cells to become preadipocytes, myoblasts, or chondrocytes, and showed further that the same clonal CHEF/18 cell populations could be directed exclusively into the adipocyte pathway if the cells were grown in the presence J Abbreviations used in this paper: CHEF cells, Chinese hamster embryonic fibroblastic cells; FBS, fetal bovine serum; GPDH, snglycerol-3-phosphate dehydrogenase.

Rifampin, in addition to interfering with intracellular growth of the ribonucleic acid-containing phage MS2, also inhibits the release of mature phage particles from Escherichia coli cells.