Physiologia Plantarum 2012 Copyright © Physiologia Plantarum 2012, ISSN 0031-9317 Seed development, seed germination and seedling growth in the R50 (sym16) pea mutant are not directly linked to altered cytokinin homeostasis Chengli Long a , Mark Held a,† , Allison Hayward b , Jaroslav Nisler c , Lukas Sp´ ıchal c,d , R. J. Neil Emery b , Barbara A. Moffatt e and Fr ´ ed ´ erique C. Guinel a,∗ a Department of Biology, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada b Department of Biology, Trent University, Peterborough, Ontario K9J 7B8, Canada c Laboratory of Growth Regulators, Institute of Experimental Botany, ASCR & Palack ´ y University, 78371 Olomouc, Czech Republic d Centre of the Region Han ´ a for Biotechnological and Agricultural Research, Faculty of Science, Palack ´ y University, 783 71 Olomouc, Czech Republic e Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada Correspondence *Corresponding author, e-mail: fguinel@wlu.ca Received 30 July 2011; revised 13 November 2011 doi:10.1111/j.1399-3054.2012.01594.x R50 (sym16) is a pea nodulation mutant that accumulates cytokinin (CK) in its vegetative organs. Total CK content increases as the plant ages because of the low activity of the enzyme cytokinin oxidase/dehydrogenase (CKX) responsible for CK degradation. R50 exhibits a large seed with high relative water content, and its seedling establishes itself slowly. Whether these two traits are linked to abnormal CK levels was considered here. R50 was found to have a similar germination rate but a much slower epicotyl emergence than Sparkle, its wild-type (WT). At the onset of emergence, the starch grains in R50 cotyledons were larger than those of WT; furthermore, they did not degrade as fast as in WT because of low amylase activity. No differences between the pea lines were observed in the CK forms identified during seed embryogenesis. However, while CK content compared to that of WT was reduced early in R50 embryogenesis, it was elevated later on in its dry seeds where CKX activity was low, although CKX transcript abundance remained high. Transcripts of the two known PsCKX isoforms exhibited tissue- and development-specific profiles with no detectable PsCKX2 expression in cotyledons. There were more of both transcripts in R50 roots than in WT roots, but less of PsCKX2 than PsCKX1 in R50 shoots compared to WT shoots. Thus, although there is a definite CKX post-transcriptional defect in R50 dry seeds, an abnormal CK homeostasis is not the basis of the delay in R50 seedling establishment, which we linked to abnormal amylase activity early in development. Abbreviations – AGP, ADP-glucose pyrophosphorylase; BA, N 6 -benzyladenine; BAP, 6-benzylaminopurine; CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase; DAB, days after blooming; DAP, days after planting; DAI, days after imbibition; DMSO, dimethyl sulfoxide; DW, dry weight; FW, fresh weight; HK, histidine kinase; iP, N 6 -(2-isopentenyl)adenine; iPNT, phosphorylated isopentenyl; LR, lateral root; PL, plumule length; RL, radicle length; RT, radicle thickness; RW, radicle width; RWC, relative water content; WT, wild-type. † Present address: Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, 140 Gortner Laboratory, Saint Paul, MN 55108, USA Physiol. Plant. 2012