Progress 10/01/02 to 09/30/07
Outputs
Relative quantitative RT-PCR methods for gene expression studies were optimized by addition of a genomic DNA digestion step and primers designed at intron/exon junctions to distinguish PCR products resulting from cDNA and residual genomic DNA. Ipt (isopentyl transferase) gene expression analysis of gene dosage studies indicates that there are no significant transcript level differences across ipt gene dosages suggesting a larger degree of post-translational control in the non-additive dosage effect on cytokinin levels. Cytokinin oxidase (Ckx) activity is increased significantly by any dosage of ipt compared to the non-transgenic control and preliminary expression analysis shows similar results. These results suggest that Ckx may be inducible by substrate levels. We are continuing to investigate Ckx substrate inducibility by studying the reponse to the addition of Zeatin and Zeatin Riboside.
Impacts
Our data continues to support the hypothesis that Ckx activity and/or expression play an important role in the regulation of kernel development in maize. Furthermore, the regulation of Ckx expression or overexpresson of cytokinin synthesis genes in specific kernel component tissues may be a viable molecular strategy to stabilize grain yield of maize against the periodic occurrence of heat stress or long-term global climate change.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
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.
Impacts
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
- 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.
- 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.
- 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.
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Progress 01/01/05 to 12/31/05
Outputs
A study on the effects of high temperature during endosperm cell division on protein accumulation was published in Crop Science 45:2005. In this study, we (Manjardino, Smith and Jones) 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. A subsequent L-[35S] methinonine incorporation study confirmed that heat stress during early stages of endosperm development repressed zein accumulation at the synthesis level, whereas later in development repression occurs mainly by protein degradation. A second manuscript focusing on the mechanisms by which heart stress affects early zein accumulation in maize kernels was prepared for submission for publication. 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. Cytokinin oxidase (Ckx) is the principal enzyme involved in cytokinin catabolism. Research focused on characterization of the developmental regulation and temporal and tissue specific expression of cytokinin level and Ckx during maize kernel development was also continued. Previous studies have shown that the inbred line B73 is more tolerant to heat-stress during endosperm cell division than Mo17 due mainly to maintain higher cytokinin levels and lower Ckx activity after exposure to heat-stress. Recent studies, using reciprocal crosses provided strong evidence that the heat tolerance attributes of B73 are maternally inherited. We continued to use molecular tools in order to manipulate maize kernel endogenous 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.
Impacts
These data 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
- Monjardino, P., A.G. Smith and R.J. Jones. 2005. Heat Stress Effects on Protein Accumulation of Maize Endosperm. Crop Sci. 45:1203-1210.
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Progress 01/01/04 to 12/31/04
Outputs
Studies on the effects of high temperature during endosperm cell division on protein accumulation were completed and a manuscript submitted and accepted for publication in Crop Science. This study (Manjardino, Smith and Jones, 20005) 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. A subsequent L-[35S] methinonine incorporation study confirmed that heat stress during early stages of endosperm development repressed zein accumulation at the synthesis level, whereas later in development repression occurs mainly by protein degradation. Cytokinin oxidase (Ckx) is the principal enzyme involved in cytokinin catabolism. Research focused on characterization of the developmental regulation and temporal and tissue specific
expression of cytokinin level and Ckx during maize kernel development was also continued. Previous studies have shown that the inbred line B73 is more tolerant to heat stress during endosperm cell division than Mo17 due mainly to maintain higher cytokinin levels and lower Ckx activity after exposure to heat stress. Recent studies using reciprocal crosses provided strong evidence that the heat tolerance attributes of B73 are maternal inherited. We continued to use molecular tools in order to manipulate maize kernel endogenous 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.
Impacts
These data 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
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Studies on the effects of high temperature during endosperm cell division on protein accumulation were completed and a manuscript submitted for publication in Crop Science. The study (Manjardino, Smith and Jones) confirmed that a 2 or 4-day heat stress 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. A subsequent L-[35S] methinonine incorporation study confirmed that heat stress during early stages of endosperm development repressed zein accumulation at the synthesis level, whereas later in development repression occurs mainly by protein degradation. Cytokinin oxidase (Ckx) is the principal enzyme involved in cytokinin catabolism. In collaboration with Pioneer Hi-Bred International, a study characterizing cytokinin oxidase gene expression, localization and induction
was published in Plant Physiology. These studies confirmed that Ckx is expressed in the vascular bundle of kernels, coleoptiles and leaves and is developmentally regulated in a manner that is correlated with cytokinin levels and cytokinin oxidase activity. In addition, we confirmed that Ckx is induced by abscisic acid, which may control cytokinin oxidase expression and thus cytokinin levels and subsequent kernel development under abiotic stress. Research focused on characterization of the developmental regulation and temporal and tissue specific expression of cytokinin level and Ckx during maize kernel development, was also continued using molecular tools in order to manipulate maize kernel endogenous 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. In one of the transgenics, the expression of ipt is under the control of a gamma zein
promoter and is thus endosperm specific. The over-expression of ipt in the endosperm begins to occur at approximately 12 days after pollination resulting in more than a 100-fold increase in [9R]Z levels and only a slight increase in CKO activity. The up regulation of endogenous cytokinin levels by ipt is also stable under heat stress. Gene dose experiments also revealed that only one dose of the ipt gene is sufficient to result in the highest level of ipt expression. These data 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.
Impacts
These studies suggest that heat stress during early stages of endosperm development represses zein accumulation at the synthesis level. Moreover, the data 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
- Brugiere, N, S. Jiao, S. Hantke, C. Zinselmeier, J. Roessler, X. Niu, R.J. Jones and J. E. Habben. 2003. Cytokinin oxidase (Ckx1-2) gene expresssion in Zea mays is localized to the vasculature, and is induced by cytokinins, abiotic stress and abscisic acid. Plant Physiol. 132: 1228-1240.
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