Vegetative storage protein expression during terminal bud formation in poplar 1 Susan D. Lawrence, Janice E.K. Cooke, John S. Greenwood, Theresa E. Korhnak, and John M. Davis Abstract: Trees recycle nitrogen (N) to conserve this valuable nutrient. The processes that regulate N recycling within trees are poorly understood at the molecular level. Because vegetative storage proteins (VSPs) are thought to play im- portant roles in within-plant N cycling, we are investigating the expression of VSP genes to gain insights into how sea- sonally controlled N cycling is regulated in trees. We compared steady-state mRNA levels of three different VSP homologs during short day induced terminal bud formation in hybrid poplar (Populus trichocarpa Torr. & Gray × Populus deltoides Bartr. ex Marsh.) – WIN4 (wound-inducible protein 4), BSP (bark storage protein), and pni288 (pop- lar nitrogen-regulated cDNA 288, a newly identified sequence). We determined that win4 and pni288 transcripts de- crease, while bsp transcripts increase, as the terminal bud is formed. Immunolocalization analysis indicated that, during apical bud formation, BSP accumulates in the ground meristem and in parenchyma cells adjacent to xylem and proxi- mal to the apical dome. Based on messenger RNA and protein expression analysis, we conclude that different VSPs play distinct roles in the poplar shoot apex, with BSP accumulating as a reserve near the shoot apex during terminal bud formation. Résumé : Les arbres recyclent l’azote afin de conserver ce précieux nutriment. Les processus qui contrôlent le recy- clage de l’azote dans les arbres à l’échelle moléculaire sont peu connus. Puisque les protéines végétatives d’entreposage (VSP) jouent vraisemblablement un rôle majeur dans le cycle de l’azote à l’intérieur des plantes, les au- teurs ont étudié l’expression des gènes VSP afin de mieux comprendre comment le cycle saisonnier de l’azote est con- trôlé chez les arbres. Les niveaux d’ARNm stable de trois homologues différents de VSP ont été comparés pendant la formation du bourgeon terminal induite sous courte photopériode chez le peuplier hybride (Populus trichocarpa Torr. & Gray × Populus deltoides Bartr. ex Marsh.), à savoir : WIN4 (la protéine 4 induite par une blessure), BSP (la protéine d’entreposage dans l’écorce) et pni288 (l’ADNc 288 contrôlé par l’azote chez le peuplier, une nouvelle séquence ve- nant d’être identifiée). Au fur et à mesure de la formation du bourgeon terminal, les niveaux de transcrits de win4 et pni288 diminuent alors que les transcrits de bsp augmentent. L’analyse d’immunolocalisation a indiqué que pendant la formation du bourgeon apical, BSP s’accumule dans le méristème périphérique et les cellules du parenchyme adjacen- tes au xylème et à proximité du dôme apical. À partir de l’analyse de l’expression des protéines et de l’ARNm, les au- teurs concluent que les différentes VSP jouent des rôles distincts au sein de l’apex de la pousse terminale chez le peuplier, dont BSP qui s’accumule sous forme de réserve proche de l’apex de la pousse durant la formation du bour- geon terminal. [Traduit par la Rédaction] Notes 1103 Introduction Temperate zone hardwoods cycle nitrogen (N) and carbon (C) seasonally in association with the establishment of dor- mancy in the autumn and the breaking of dormancy in the spring (Millard 1993). As daylengths shorten, terminal buds are formed, and there is a net shift in N and C allocation from leaves to perennating tissues such as phloem paren- chyma in bark and xylem parenchyma in wood. This shift in resource allocation is marked by the accumulation of pro- tein, starch, and lipid reserves in terminal buds, stems, and roots. Synthesis of starch and vegetative storage proteins (VSPs) allows for the sequestration of N and C resources into an osmotically inactive form (Staswick 1994; Martin and Smith 1995). Breakdown of these reserves provides sub- strates for respiration and other maintenance metabolic pro- cesses that occur during the winter, as well as to support bud burst and the early stages of shoot growth during the follow- ing spring until the transpiration stream is re-established (Hansen 1971; Dickson 1991; Rowland and Arora 1997). Can. J. For. Res. 31: 1098–1103 (2001) © 2001 NRC Canada 1098 DOI: 10.1139/cjfr-31-6-1098 Received November 12, 2000. Accepted January 17, 2001. Published on the NRC Research Press Web site on June 9, 2001. S.D. Lawrence, 2 J.E.K. Cooke, T.E. Korhnak, and J.M. Davis. 3 School of Forest Resources and Conservation and Program in Plant Molecular and Cellular Biology, P.O. Box 110410, University of Florida, Gainesville, FL 32611, U.S.A. J.S. Greenwood. Department of Botany, University of Guelph, Guelph, ON NIG2W1, Canada. 1 Journal Series No. R-08168 of the Florida Agricultural Experimental Station. 2 Present address: U.S. Department of Agriculture, Agricultural Research Service, Insect Biocontrol Laboratory, Building 011A, Room 214, Beltsville, MD 20705, U.S.A. 3 Corresponding author (e-mail: jmdavis@ufl.edu).