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Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositolspecific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca 2+ modulator which induces distinctive oscillating bursts of cytosolic Ca 2+ , resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stressrelated genes such as glutathione S-transferase, hormoneregulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

The cloning and identification of full-length cDNA fragments coding for the Brassica napus phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) is described. In addition, two complementary fragments (120 nucleotides long) corresponding to Arabidopsis PtdIns 4-kinase (PtdIns 4-K) and PtdIns-4-phosphate 5-kinase (PtdIns4P 5-K) sequences were chemically synthesized. These, as well as the cDNA clones, were used as probes to study the corresponding steady state mRNA levels in different tissues and developmental stages of B. napus, as well as in response to different environmental conditions. Transcripts corresponding to BnPLC2, BnPtdIns S1, BnVPS34 and PtdIns 4-K were found constitutively expressed at different levels in most tissues, with young leaves, siliques, and developing seeds showing the lowest levels. No detectable PtdIns4P 5-K transcripts were found in buds or flowers. Up-regulation of BnPLC2 was seen in response to low temperature stress, which was notably accompanied by a parallel down-regulation of BnPtdIns S1, while BnVPS34 and PtdIns 4-K remained at control levels. A moderate increase in PtdIns4P 5-K levels was noted. In high salinity conditions BnPtdIns S1, BnVPS34 and BnPLC2 transcripts had similar responses but at different levels, with no major changes detected for PtdIns 4-K or PtdIns4P 5-K. Significantly, all five transcripts increased under drought stress conditions and all stressed plants clearly showed relatively higher levels of total inositol trisphosphate.

The response of living cells to change in cell environment depends on the action of second messenger molecules. The two second messenger molecules cAMP and Ca 2+ regulate a large number of eukaryotic cellular events. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interaction between cAMP and Ca 2+ second messenger systems. Some PDE1 isozymes have similar kinetic and immunological properties but are differentially regulated by Ca 2+ and calmodulin. Accumulating evidence suggests that the activity of PDE1 is selectively regulated by cross-talk between Ca 2+ and cAMP signalling pathways. These isozymes are also further distinguished by various pharmacological agents. We have demonstrated a potentially novel regulation of PDE1 by calpain. This study suggests that limited proteolysis by calpain could be an alternative mechanism for the activation of PDE1. We have also shown PDE1 activity, expression and effect of calpain in the rat model in vitro of cardiac ischemiareperfusion. Contents 1. Introducton 2. Kinetic properties of various PDE1 isozymes 3. Differential regulation of PDE1 by CaM and Ca 2+ 4. Coupling between Ca 2+ and cAMP second messengers in the regulation of PDE1 5. Role of autophosphorylation of PDE1B1 by CaMdependent protein kinase II 6. Differential inhibition of PDE1 isozymes and its therapeutic applications 7. Role of proteolysis in regulating PDE1A2 8. Role of PDE1A1 in ischemic-reperfused heart 9. Conclusion

A mutant phenotype isolated from alfalfa (Medicago sativa L) cv. Excalibur appeared to have dramatic changes in source‐sink relations and the partitioning of carbohydrates. Leaves emerged normally, but starch accumulatedin the chloroplasts of the palisade mesophyll cells with maturity. Subsequently the cells of the palisade layer lost chlorophyll, exhibited ultrastructural symptoms of senescence, and necrotic spotting appeared on the adaxiai surface of fully expanded leaves. Segregation analysis of this phenotype, designated as hls (high‐leaf starch), with F1, BC1, and F2 progenies revealed that two independent dominant genes condition the trait. The hls phenotype had five‐fold more starch in mature leaves and less in the taproot than in the normal leaf phenotypes, suggesting a blockage in transport of carbohydrate from the leaf to the root. In vitro activity assays and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that most enzymes involved in carbohydrate metabolism were not altered in the hls phenotype. However, invertase activity of expanding leaves was significantly higher in plants with the hls phenotype than in those with a normal phenotype. The visual appearance of the mutation, high leaf starch content, and high invertase activity co‐segregated in the progeny. Native PAGE revealed that a fast‐moving invertase isozyme (F) was developmentally inactivated in normal phenotypes during leaf expansion but remained active in the hls phenotype. A biochemical model for the hls phenotype is proposed in which high invertase activity blocks phloem loading and reduces the availability of cytosolic inorganic orthophosphate for exchange with triose‐phosphate across the chloroplast envelope, thereby promoting starch accumulation.

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S. 1992. Ultrastructural and genetic characterization of a mutant exhibiting starch accumulation and premature leaf senescence in Medicago sativa. Can. J . Bot. 70: 2245-2253. A mutant was isolated from irradiated seed of alfalfa, Medicago sativa L. cv. Excalibur. The mutant plant, Ex-139, displayed symptoms of premature senescence in the leaf palisade mesophyll. The leaves emerged as a normal phenotype, but light microscopy revealed that they rapidly began to accumulate starch in plastids of some cells in the palisade mesophyll. This accumulation of starch was followed by general cellular autolysis leading to the formation of necrotic regions in the palisade mesophyll. The adjacent epidermal and spongy mesophyll cells were not structurally affected. The mutant otherwise exhibited normal growth and development and was fertile. Inheritance studies indicated that the trait was transmitted to the progeny, preferentially but not exclusively, through the female, which suggests that either there is differential selection among male and female gametes or the trait is controlled by extranuclear DNA. This mutant should be useful in the study of the link between senescence and carbohydrate metabolism and in alfalfa genetics. Key words: starch metabolism, plastid, chloroplast genome, biparental inheritance. MCKERSIE, B. D., PETERSON, R. L., BOWLEY, S. R., et DAS, S. 1992. Ultrastructural and genetic characterization of a mutant exhibiting starch accumulation and premature leaf senescence in Medicago sativa. Can. J . Bot. 70 : 2245 -2253. Un mutant a Ctt is016 a partir de graines irradiCes de la luzerne, Medicago sativa L. cv. Excalibur. La plante mutante, Ex-139, montre des symptdmes de sCnescence prCmaturCe dans le mCsophylle palissadique foliaire. Les feuilles Cmergent comme celles d'un ophCnotype normal, mais la microscopie photonique rCvtle qu'elles commencent rapidement a accumuler de l'amidon dans les plastes de certaines cellules du mCsophylle palissadique. Cette accumulation d'amidon est suivie d'une autolyse cellulaire gCneralisCe conduisant a la formation de rCgions nCcrotiques dans le mCsophylle palissadique. Les structures des cellules adjacentes de 1'Cpiderme et du mCsophylle lacuneux ne sont pas affectCes. Par ailleurs, le mutant montre une croissance et un dCveloppement normaux et garde sa fertilite. Les Ctudes d'hCrCditC indiquent que le caractere se transmet aux descendants, prCfCrentiellement mais non exclusivement, par le cdtC femelle, ce qui suggere qu'il existe soit une sClection diffkrentielle entre les gamktes miles et femelles, ou soit que le caractkre est contrdle par de 1'ADN extranuclkaire. Ce mutant devrait s'avCrer utile pour Ctudier le lien qui existe entrela sCnescence et le mCtabolisme des glucides ainsi que la gCnCtique de la luzerne de faqon plus gCnCrale. Mots c l b : mCtabolisme de I'amidon, plastes, gCnome chloroplastique, hCrCditC bi-parentale. [Traduit par la rCdaction]

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