Research article Changes in cytokinin form and concentration in developing kernels correspond with variation in yield among field-grown barley cultivars Adrian F. Powell a, * , Andrea R. Paleczny a , Henry Olechowski b , R.J. Neil Emery a a Trent University, Peterborough, ON K9J 7B8, Canada b Hyland Seeds e Dow AgroSciences, Ailsa Craig, ON N0M 1A0, Canada article info Article history: Received 8 August 2012 Accepted 4 December 2012 Available online 3 January 2013 Keywords: Cytokinins Barley Yield CKX activity IPT activity Developmental stages abstract The aim of the present study was to determine if relationships between cytokinin (CK) profiles and corresponding enzymatic regulation were consistent with differences in kernel yield among commercial barley (Hordeum vulgare L.) cultivars, differing in parameters relating to productivity and grown under agronomically-relevant field conditions. Quantification of the CKs at six distinct stages of kernel devel- opment by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) revealed a significant increase in CKs, especially trans-zeatin (tZ), during the stage when grain filling occurs, with a greater CK concentration observed in the cultivar with the greatest number of kernels per spike, which was also the highest yielding cultivar. High levels of the cis-zeatin (cZ) CK form were observed at the first developmental stage, indicating a possible role in early embryo development and viability. Cytokinin oxidase (CKX; EC 1.5.99.12) activity was evaluated at each stage through a colori- metric assay, as the enzyme provides a primary mechanism for the irreversible degradation and, thus, regulation of CKs. However, no peaks in CKX activity were observed and no differences were detected at the particular stages examined. Isopentenyl transferase (IPT) gene expression was also examined and suggests that biosynthesis contributes to regulation of CK concentrations in developing barley kernels. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Cytokinins (CKs) have long been connected with plant produc- tivity, development and yield. The earliest research into the func- tions of CKs established their relevance to cell division [1,2]. This gave rise to research examining the specific mechanisms by which CKs affected crop productivity and grain development in species such as maize (Zea mays L.) [3,4], rice (Oryza sativa L.) [5], chickpea (Cicer arietinum L.) [6], and lupins (Lupinus albus L.) [7]. In the context of grain development and plant yield, previous research conducted with barley kernels determined a relationship between CK content and increased kernel mass, showing that barley plants producing kernels of greater average mass, due to either genetic or environmental factors, consistently exhibited higher levels of CK-like activity [8]. In particular, plants producing higher mass kernels appeared to sustain peak CK activity at higher levels and for longer periods of time [8]. More recently, several studies have suggested complex re- lationships between CKs and crop productivity, seed number and seed mass. In agreement with Michael and Seiler-Kelbitsch [8], Zhang et al. [9] observed a relationship whereby CK accumulation corresponded with increased kernel mass in wheat. A study by Ashikari et al. [5] indicated that there was a positive relationship between the number of grains per panicle, branching and CK content in rice. Analysis of genes serving as markers of hormone action in Arabidopsis thaliana (L.) Heynh. revealed that CK signal- ing was important in the early period of endosperm development, when syncytial development was occurring, which is a vital period in the determination of final seed size [10]. Furthermore, a study by Emery et al. [7] in lupines found that a crucial switch occurred from the cis- to trans-forms of CKs, and the timing of this switch was developmentally important in determining whether a seed would be viable or aborted. While these studies confirmed the importance of CKs to seed and grain development, further study is needed to fully elucidate the role that CKs play. In particular, these studies were not conducted under field conditions, which is Abbreviations: CK, cytokinin; tZ, trans-zeatin; cZ, cis-zeatin; HPLC-MS/MS, high- performance liquid chromatography tandem mass spectrometry; CKX, cytokinin oxidase; IPT, isopentenyl transferase; FW, fresh weight. * Corresponding author. Current address: Cornell University, Ithaca, NY 14850, USA. Tel.: þ1 607 255 6330. E-mail addresses: afp43@cornell.edu (A.F. Powell), andreapaleczny@trentu.ca (A.R. Paleczny), holechowski@hylandseeds.com (H. Olechowski), nemery@ trentu.ca (R.J.N. Emery). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.12.010 Plant Physiology and Biochemistry 64 (2013) 33e40