OBJECTIVES: Although recent evidence clearly shows that cytokinin levels and cytokinin oxidase (ckx) activity are tightly regulated, little work has been done to characterize this regulation in a plant system where cytokinin action may play a major role in development or an agronomically important crop like maize. How maize kernel cytokinin levels and ckx activity are regulated, and when and in what tissues ckx is expressed, is not well understood. The recent cloning of ckx and the development of other cellular and molecular techniques makes it possible to further elucidate its role in regulation of levels of active cytokinins in developing maize kernels under optimal environmental conditions and in response to environmental perturbation. Therefore, one of our primary goals is to characterize this enzyme (or possible isozyme forms) at the protein and molecular level in developing maize kernels. Our ultimate goal is to understand how cytokinin levels and ckx are controlled at the organ, tissue, protein, mRNA and gene levels and their role in the modulation of other genes that regulate kernel development under both stress and non-stress conditions. Specifically, our objectives are: 1. To determine the role and function of cytokinins and their metabolism by ckx in maize kernel development; a) Characterize the developmental regulation, temporal expression and localization of cytokinins and ckx in maize kernels and kernel constituent tissues, b) determine if the transient accumulation of cytokinins in kernels and kernel component tissues is associated with local de novo biosynthesis, c) determine the influence of elevated cytokinin and auxin levels on ckx activity and gene expression. 2. Determine the role of sucrose metabolism and elevated cytokinin levels in the amelioration of detrimental effects of high temperature during endosperm cell division. 3. Contribute to graduate student and postdoctoral training in crop physiology and the applied plant sciences.

APPROACH: Objective 1: To determine the role and function of cytokinins and cytokinin oxidase in maize kernel development. Generally, maize plants used in these studies will be grown under field, greenhouse, or growth chamber conditions. Samples will generally be collected at daily intervals from 5- 14 days after pollination (DAP) and at 3-day intervals until the kernels reach physiological maturity. Objective 1a: Developmental regulation, temporal expression and localization of ckx in maize kernels and kernel constituent tissues. The aim of this line of research is a more rigorous identification of the full spectrum of cytokinins in both whole maize grains and component tissues (i.e. endosperm, embryo and pedicle) during their transient accumulation following pollination. Moreover, the levels of major cytokinins will be determined as well as ckx activity and expression. Objective 1b. Determine if the transient accumulation in kernel and kernel constituents tissues is associated with local de novo biosynthesis. Ultimately, we will attempt to isolate the ckx promoter and develop a reporter gene system to address this question. In the interim, cytokinin biosynthesis in developing grains will be tested by in vivo incorporation of potential precursor(s) into cytokinins. In addition, an attempt will be made to test the in vitro activity of the cytokinin biosynthetic enzyme dimethylallyl diphosphate:ATP/ADP isopentenyl transferase activity using in vitro assay. Objective 1c:Determine the influence of elevated cytokinins and auxin levels on ckx and other gene expression. In general, our approach will be to increase endogenous levels in developing maize kernels by: 1) the application of exogenous cytokinins (via in vitro culture of isolate kernels or stem-infusion of whole ears attached to field-grown plants) and 2) the use of a transgenic lines of maize that over-produce cytokinins in either the endosperm or embryo. Profiles of cytokinin levels and ckx activity during the grain development will be compared and induction of the enzyme by elevated cytokinin or auxin levels will be determined. Objective 2. Determine the role of sucrose metabolism and high cytokinin levels in the amelioration of detrimental effects of high temperature during endosperm cell division. Our general approach will be to use temperature shift experiments concurrent with the application of hexoses and cytokinins by stem infusion to ears of plants or by application to the culture media of kernels grown in vitro. The focus will be on studying the effects of 0, 4 and 6 days of high temperature beginning 3-4 days after pollination (DAP) and the application of hexoses (8% fructose + 8% glucose) or cytokinins (BA, 4PU-30 and/or thidiazuron). Hexose and cytokinin treatments will be imposed from 0 - 10 DAP. Kernel samples will be taken daily during the treatment period. Our approach will be to measure carbohydrate levels, the activity of soluble and insoluble invertase activity, and the steady state mRNA level of Ivr l and Ivrl I. In vitro hybridization experiments will also be conducted to localize acid soluble invertase transcripts and to determine effects of hexose and cytokinin supply.

KEYWORDS: cytokinins; heat stress; high temperature; gene expression; metabolic regulation; thidiazuron; in situ hybridization; kernels; seed development; oxidases; enzyme activity; abortion; plant embryos; temporal distribution; localization; biosynthesis; auxins; transgenic plants; transferases; corn; stress tolerance; plant physiology; molecular biology; seed physiology; heat

PROGRESS: 2006/01 TO 2006/12
A second study on the effects of high temperature during endosperm cell division on protein accumulation was published in Crop Science xx:2006. In the first study, we (Manjardino, Smith and Jones, Crop Science 45: 2005) confirmed that a 2- or 4-day heat stress (35 degrees C) caused a 20 to 48% reduction in total protein content. Specifically, zein content was reduced by an average of 53%, but zein composition was only mildly affected where the concentration of glutelins and albumins plus globulins were negatively affected. The second study focused on the mechanisms by which heart stress affect early zein accumulation. This study showed that both the 27 kD and cluster 1 zeins of the sub-family 4 (ZSF4C1) zein mRNA steady-state levels are significantly delayed by 4 days of high temperature and the transcription rate of both zein proteins were reduced. Cytokinin oxidase (Ckx) is the principal enzyme involved in cytokinin catabolism. The core of research continues to focus on characterization of the developmental regulation and temporal and tissue specific expression of cytokinin level and Ckx during maize kernel development. We continue to use molecular tools to characterize and to manipulate cytokinin levels in planta. Transgenic plants over expressing the bacterial form of the cytokinin synthesis gene (isopentyl transferase-ipt) have been developed and moved into a homogenous background. Gene dose experiments continue to support the finding that only one dose of the ipt gene is sufficient to result in the highest level of ipt expression compared to non-transgenic control and that increased gene dose has no additive affect suggesting control at the transcriptional and/or translational levels. The ipt gene expression experiments were complete, but results are confounded by genomic DNA. A genomic DNA digestion step has been added and primers designed at intron/exon junctions to distinguish PCR products resulting from cDNA and residual genomic dna in order to optimize gene expression studies.

IMPACT: 2006/01 TO 2006/12
Our data continues to suggest that regulation of Ckx expression or overexpression of cytokinin synthesis genes in specific kernel component tissues may be a viable molecular approach to increasing endogenous cytokinin levels during kernel development and thus to stabilizing grain yield of maize against the periodic occurrence of heat stress or more long-term global climate change.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Monjardino, P., Smith A.G., and Jones, R.J. 2006. Zein transcription and endoreduplication in maize endosperm are differentially affected by heat stress. Crop Sci. 46:2581-2589.
2. Liu, A., Malzer, G.L., Rehm, G.W., and Jones, R.J. 2006. Degradation of 14C-Zinc ammonium acetate in soils as influenced by soil type, soil sterilization and carriers. J. Plant Nutr. 29:1003-1019.
3. Liu, A., Malzer, G.L., Rehm, G.W., and Jones, R.J. 2006. Fate of ammonium acetate in soils and its uptake by corn. J. Plant Nutr. 29:797-708.

PROJECT CONTACT:
Name: Jones, R. J.
Phone: 612-625-1276
Fax: 612-625-1268
Email: jones012@umn.edu