TITLE In vitro tobacco cultures provide a convenient system for investigating nitrate reductase regulation during shoot morphogenesis
AUTHOR M.A. Roberts
School of Biological and Medical Sciences, University of St Andrews, St Andrews, KY 16 9th FIFE, UK
M.P. Watt
Biology Department, University of Natal, Private Bag X10, Dalbridge 4014, South Africa
B.I. Huckett
Biotechnology Department, SA Sugar Association Experiment Station, Private Bag X02, Mount Edgecombe 4300, South Africa
FULL TEXT [in HTML format] [in PDF format]
ABSTRACT Nicotiana tabacum callus culture was assessed in terms of its usefulness as a system for investigating the regulation of nitrate reductase (NR; EC 1.6.6.1) during shoot morphogenesis. Culture conditions for organogenesis and appropriate methodologies for extraction and assay of NR during callus growth and development were established. The most suitable medium comprised Murashige and Skoog salts and vitamins, sucrose, indoleacetic acid and kinetin. Provided that NO3_-N was present, callus growth and shoot morphogenesis occurred in the same culture step. Optimised in vivo, in situ, and in vitro NR assays yielded similar values and patterns during culture development, and the in vivo assay was selected for subsequent studies on the basis of ease and efficiency of use. With quantitative alteration in nitrogen supply (60 vs. 120 mM; 1:2 NH4+-N : NO3_-N) and variations in light regime (16 /8 h light/dark vs. continuous darkness), callus growth parameters were comparable in terms of nitrate uptake, increase in fresh mass and protein accumulation. However, a marked rise in the level of in vivo NR activity (NRA) was observed just prior to shoot primordia emergence in light/dark-grown but not in dark-grown calli. In protein blot analyses using a polyclonal antibody raised against spinach NR, major protein bands representing holo-NR cleavage products were identified at 71 and 55 kDa. Putative nitrate reductase protein (NRP) levels were compared with in vivo NRA during the final stages of callus differentiation under both light/dark and continuous dark conditions. NRP variations appeared to be much less marked than those of NRA, suggesting the possibility that post-translational modification is a significant feature of NR regulation. The potential value of the system for further studies of NR regulation at the molecular level is discussed.
KEYWORD Callus; Differentiation; Morphogenesis; Nitrate reductase; Nitrate reductase assays; Tobacco;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 37 Number 1 January 1996, page 51-59, 9 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China