Source: UNIVERSITY OF NEW HAMPSHIRE submitted to
GENETIC MANIPULATION OF FOREST TREES TO ALTER STRESS RESPONSE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0137194
Grant No.
(N/A)
Project No.
NH00024-M
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Minocha, S. C.
Recipient Organization
UNIVERSITY OF NEW HAMPSHIRE
(N/A)
DURHAM,NH 03824
Performing Department
PLANT BIOLOGY
Non Technical Summary
It is known that a number of metabolic pathways involving multiple sets of genes are turned on or off in response to stress; one such pathway that is turned on is the polyamine biosynthetic pathway. The main goal of the proposed research is to genetically manipulate the metabolism of polyamines (putrescine, spermidine and spermine) in poplar cells in order to test if this alteration causes a positive physiological responses of the cells to osmotic, salinity and aluminum stress.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012420104025%
2032420104025%
2062420104050%
Goals / Objectives
1) To produce transgenic cells of poplar (Populus nigra x maximowiczii) using genes for polyamine biosynthetic enzymes and to analyze polyamine and ethylene metabolism in these cells. 2) To analyze physiological responses of the transgenic and the nontransgenic cells of poplar to water stress, salinity and aluminum stress. 3) To characterize the expression patterns of genes for key regulatory enzymes involved in polyamine biosynthesis in response to water stress, salinity and aluminum stress in poplar.
Project Methods
Plasmid vectors containing a poplar arginine decarboxylase, a carrot S-adenosylmethionine decarboxylase, and a Datura spermidine synthase cDNA under the control of 2x35S promoter will be used to transform poplar cell cultures by biolistic bombardment. After confirming the presence and expression of the foreign gene, selected cell lines will be used for further analysis of enzyme activities, polyamine levels and metabolic studies. In addition to studying the cellular titers of polyamines, experiments will be conducted in which 14C-labeld ornithine, putrescine, spermidine and spermine will be used to follow the metabolic flux of these compounds in the polyamine and ethylene pathways. While the polyamines will be analyzed by HPLC, ethylene will be studied by gas chromatography. The transgenic cells showing increased polyamine production will be used for experiments on their responses to water stress, salt treatment, and aluminum chloride treatment. The incorporation of labeled leucine and uridine into proteins and RNA at different times (4, 8 and 24 h) after the application of stress, (b) the metabolic activity of mitochondria, (c) determination of cell viability (cytotoxicity) using Evans blue staining, and (d) the leakage of electrolytes from the cells will be used as means of measuring stress response. Among the different approaches used for the study of gene expression, promoter-reporter gene fusion technique provides a powerful tool for analyzing the expression patterns of a specific gene of interest. The activities of arginine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase will be measured at the same time as the RNA analysis to determine correlations between transcript levels and the actual enzyme activities.

Progress 10/01/03 to 09/30/06

Outputs
Transgenic cell lines of poplar (expressing a transgene for ornithine decarboxylase) with elevated levels of putrescine were analyzed for changes in various metabolites and expression of several genes involved in polyamine metabolism. It was shown that: (1) poplar cells make most of their putrescine by the arginine decarboxylase pathway; (2) this enzyme is not feed-back regulated by high putrescine; (3) the half life of putrescine turnover is around 5-6 h in contrast to that of spermidine and spermine, whose half-lives are in the range of 30-40 h; (4) we propose that increased utilization of ornithine in the transgenic cells is accompanied by concomitant increase in nitrate and ammonium assimilation into glutamate, and the stimulation of key steps in the ornithine/arginine biosynthesis. Quantitative PCR analysis showed that the expression of several genes in polyamine biosynthesis pathway is highly coordinated. Microarray analysis of the two cell lines shows that several hundred genes are up regulated and a similar number is down-regulated in response to increased accumulation of putrescine. The putrescine overproducing cells show altered levels of protein accumulation, mitochondrial activity, and response to treatment with altered levels of calcium and aluminum in the medium.

Impacts
Understanding of the regulation of polyamine metabolism in plants by itself will potentially have profound and far-reaching implications, from manipulating abiotic stress tolerance in plants to those in the field of human oncology and parasitology, where foods with reduced polyamine contents have proven desirable to retard tumor growth and combat parasite infections.

Publications

  • Minocha, S., P. Bhatnagar, R. Minocha, and S. Long. 2003. Genetic manipulation of polyamine metabolism in poplar. In : Espinel S, Barredo Y, Ritter, E (Eds). Sustainable Forestry, Wood Products, and Biotechnology. DFA-AFA Press, Victoria-Gasteiz, Spain, pp 179-194.
  • Minocha, R., S. Minocha, and S. Long. 2003. Monitoring environmental stress in forest trees using biochemical and physiological markers. In : Espinel S, Barredo Y, Ritter, E (Eds). Sustainable Forestry, Wood Products, and Biotechnology. DFA-AFA Press, Victoria-Gasteiz, Spain, pp 109-116.
  • Minocha, R. and S.C. Minocha. 2005. Effects of soil pH and aluminum on plant respiration. In: Lambers, H. and M. Ribas-Carbo (eds.) Plant Respiration: From Cell to Ecosystem. Pp. 159-176; Springer, Dordrecht, The Netherlands.
  • Michelle Serapiglia, 2005. Nitrogen metabolism and its relation to forest decline, MS Thesis, University of New Hampshire, Durham. 119 pp.


Progress 10/01/04 to 09/30/05

Outputs
In attempting to achieve genetic manipulation of cellular metabolism, the impact of altering a single reaction on the regulation of an entire pathway, and also on other related pathways, must be critically examined, particularly if the target metabolite is not the terminal product. Data presented here show that manipulation of a single step in the polyamine (PA) metabolic pathway has far-reaching consequences. The expression of a mouse ornithine decarboxylase transgene in poplar (Populus nigra x maximowiczii) cells has provided evidence for the co-regulation of expression of several genes in the PA biosynthetic pathway. An examination of the transcript levels and enzyme activities of the key regulatory enzymes as well as the metabolites revealed a complex homeostatic mechanism at work involving several parts of the pathway operating in a coordinated manner. Use of Quantitative Reverse-Transcriptase PCR has enabled greater precision in examining expression levels of the various orthologues of S-adenosylmethionine decarboxylase and spermidine synthase, along with those of the transgene (mouse ornithine decarboxylase) and the native gene for putrescine biosynthesis; i.e. arginine decarboxylase. The results show that different orthologues of S-adenosylmethionine decarboxylase and spermidine synthase were regulated differently by altering the cellular putrescine content, and there was a strong correlation between transcript levels, enzyme activities and PA levels in the cells. Furthermore, there was no feedback inhibition of the native arginine decarboxylase gene or its activity by putrescine.

Impacts
The results of research will lead to a better understanding of the regulation of metabolic pathways in plant (metabolic engineering) that will allow the use of transgenic approach to manipulate plant metabolism. In the long run, this will help us produce trees that have the desirable qualities of wood, fiber and other useful products, as well as being tolerant of various types of abiotic stress.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
The consequences of overproduction of putrescine in transgenic cells of poplar (Populus nigra x maximoviczii) expressing an ornithine decarboxylase transgene were studied in relation to the effects of different concentrations of nitrogen in the medium on several physiological parameters. Cells grown in the presence of a broad range of supplemental (to the normal levels of nitrogen sources in the growth medium) and reduced (below those present in the growth medium) amounts of ammonium nitrate and potassium nitrate were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, and growth rates as well as changes in cellular polyamine content. The results show that the amount of nitrogen in the growth medium is not a limiting factor for continued production of higher amounts of putrescine in the transgenic cells. However, continued supply of both ammonium and nitrate is required in order to maintain the homeostatic levels of putrescine in both cell lines. The presence of higher amounts of putrescine in the transgenic cells had significant effects on the physiological parameters that were studied. The transgenic cells had greater plasma membrane permeability as compared to the non-transgenic cells, they were less tolerant to ammonium nitrate and more tolerant to potassium nitrate, and they accumulated higher amounts of soluble protein than the non-transgenic cells. It can be concluded that: (1) upon increased utilization of Orn by the transgenic ODC, there is a coordinated increase in multiple steps in the metabolic pathway for Orn biosynthesis, and (2) excess amounts of either ammonium or nitrate do not lead to sequestration of the excess N in the form of polyamines. This, however, does not exclude the possibility that polyamines play an important role in sequestration of extra N in a whole plant, where, in contrast to the cells in suspension that are rapidly dividing, the sequestration would occur in the leaves and other tissues of the plant which are capable of storing metabolites such as polyamines. Data on the accumulation of polyamines in forest trees in response to N supplementation in soil indeed supports this argument. Number of graduate students = 2

Impacts
The results of research will lead to the modeling of the regulation of metabolic pathways in plant (metabolic engineering) that will allow the use of polyamine pathway as an early indicator of stress in plants. The availability of an early indicator of stress is useful in devising strategies at mitigation of stress and management of forest trees.

Publications

  • Page, A.F., Minocha, S.C. 2004. Analysis of gene expression in transgenic plants. In. Methods in Molecular Biology, Vol. 286: Transgenic Plants. L. Pena (ed.), Humana Press. pp. 291-311.
  • Minocha, R., J.S. Lee, S. Long, P. Bhatnagar, S.C. Minocha. 2004. Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium. Tree Physiol. 24: 551-560.


Progress 10/01/02 to 09/30/03

Outputs
A cDNA library has been created using mRNA isolated from non-transgenic poplar (Populus nigra x maximoviczii) cells. Using degenerate primers, partial gene sequences for arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMD-C), aminocyclopropane carboxylic acid (ACC) synthase (two genes), glutamate decarboxylase, diamine oxidase, and spermidine synthase have been cloned and sequenced. The sequence homology has been used to confirm the gene sequences. Based upon cloned gene sequences, specific primers have been designed for quantitative Reverse-transcriptase Polymerase Chain Reaction to quantify the amounts of each transcript in the non-transgenic and mouse odc (ornithine decarboxylase)- transgenic cells. Using transient expression assays with promoters of two Arabidopsis thaliana genes (adc2 and samdc1) fused with a bacterial Beta-glucuronidase (GUS) gene, it has been determined that the expression of the two genes responds positively to salt and water stress treatments. The results also show that the heterologous Arabidopsis promoters work quite well in poplar. The effects of variation in the nitrogen content of the medium on cellular polyamine content in the transgenic and non-transgenic cells, further show that the amount of nitrogen in the MS medium is not a limiting factor for continued production of higher amounts of putrescine in the transgenic cells. However, continued supply of both ammonium and nitrate is required in order to maintain the homeostatic levels of putrescine in both cell lines. Graduate students - 2

Impacts
The results of research will lead to the modeling of the regulation of ametabolic pathway that will allow the use of polyamine pathway as an early indicator of stress in plants. The availability of an early indicator of stress is useful in devising strategies at mitigation of stress and management of forest trees.

Publications

  • Minocha, S.C., Bhatnagar, P. and Minocha, R. 2003. Genetic manipulation of polyamine metabolism in poplar. Abs. BIOFOR 02 - Sustainable Forestry, Wood Products & Biotechnology; Nov. 11-14, 2002; Vitoria-Gastaiz, Spain.
  • Minocha, S.C. 2003. Functional genomics of a metabolic pathway. Abst. 2nd International Congress of Plant Physiology, Jan 8-12, New Delhi, India.
  • Minocha, S.C., Bhatnagar, P., Quan, Y. and Minocha, R. 2003. The regulation of the polyamine metabolic pathway in plants. Abst. South Dakota Plant Physiology/Plant Biochemistry Symposium; Brookings, SD, Aug. 6-8, 2003.


Progress 10/01/01 to 09/30/02

Outputs
The effect of putrescine overproduction on spermidine was investigated in a nontransgenic (NT) and a transgenic cell line of poplar (Populus nigra x maximowiczii) expressing a transgenic ornithine decarboxylase cDNA. The transgenic cells produce three to four fold higher amounts of putrescine than the NT cells. The rate of spermidine loss and the half-life of cellular spermidine were determined by feeding the cells with radioactive spermidine as a precursor, and following the loss of radioactive spermidine in the cells at various times after transfer to label-free medium. The data show that: (a) the uptake of spermidine by poplar cells is affected by the cellular content of total polyamines, (b) these cells are capable of converting spermidine into putrescine, albeit at a slow rates, (c) the rate of spermidine catabolism is five-to-six fold slower than that of putrescine, (d) the overproduction of putrescine does not affect the overall rates of turnover (including excretion) of spermidine, nor does it affect the rates of conversion of spermidine into spermine, and (e) there probably is a single pool of spermidine in the cells. Also, the overproduction of putrescine has no impact on the biosynthesis of ethylene in these cells. Number of graduate students = 3.

Impacts
A better understanding of plant metabolism through genetic engineering approach will lead to the production of improved varieties of forest tress that are tolerant to various forms of abiotic stress, e.g. salt, drought and aluminum.

Publications

  • 1. Bhatnagar P, Minocha R, Minocha SC (2002) Genetic manipulation of the metabolism of polyamines in poplar cells: the regulation of putrescine catabolism. Plant Physiol 128: 1455-1469.
  • 2. Quan Y, Minocha R, Minocha SC (2002) Genetic manipulation of polyamine metabolism does not alter ethylene biosynthesis. Plant Physiol. Biochem. 40: 929-937.


Progress 10/01/00 to 09/30/01

Outputs
Polyamines constitute a major group of metabolites that are particularly rich in nitrogen and can accumulate in millimolar quantities in plant cells. The diverse roles of polyamines in plants range from being storage products of excess nitrogen, to reducing ammonia toxicity, to specific roles in interacting with cellular macromolecules to stabilize them and to regulate their activity, e.g. transcription and translation. Consequently, the up-regulation or down-regulation of polyamine biosynthesis must depend upon the availability of nitrogen in the medium. We studied the effect of different amounts of nitrogen in the medium on the metabolism of polyamines in a nontransformed (NT) and a transgenic cell line (2E) of Populus nigra x maximowiczii expressing a heterologous ornithine decarboxylase (odc) cDNA. The effects of three types of variations in the nitrogen content of the medium in the two cell lines were studied: (a) increased concentrations of ammonium nitrate and potassium nitrate, (b) reduction in the concentration of ammonium nitrate, and (c) complete absence of both ammonium nitrate and potassium nitrate. From the results obtained so far, it can be concluded that: (a) Total nitrogen content of the MS medium is not limiting for optimal production of polyamines in both the NT and the 2E cells, (b) a continuous supply of nitrogen in the form of both ammonium nitrate and potassium nitrate is required to maintain the homeostatic levels of putrescine and removal of either ammonium nitrate or total nitrogen from the medium causes a rapid reduction in cellular content of putrescine, (c) fluctuations in putrescine are not accompanied by parallel changes in spermidine and spermine, and (d) the responses of both the NT and the 2E cells to increased or decreased nitrogen in the medium are quite similar. It is proposed that upon increased utilization of ornithine in the transgenic cells, there is a coordinated increase in the metabolic pathway for ornithine biosynthesis, starting from an increased uptake and assimilation of nitrate and/or ammonium through the various steps leading to increased ornithine production, as long as ammonium and nitrate are available in sufficient quantities in the medium. Number of graduate students: 3

Impacts
The results provide an insight into the regulation of polyamine metabolism in plants and its relationship with the availability of nitrogen and its effects on metabolism of aminoacids.

Publications

  • Bhatnagar P, Glasheen BM, Bains SK, Long SL, Minocha R, Walter C, Minocha SC (2001) Transgenic manipulation of the metabolism of polyamines in poplar (Populus nigra x maximowiczii) cells. Plant Physiol 125: 2139-2153.
  • Quan, Yi. 2001. Transgenic approach to study the interaction of polyamines and ethylene metabolism in poplar and tomato, MS thesis, University of New Hampshire, Durham, NH, pp. 1-158.


Progress 01/01/00 to 12/31/00

Outputs
Polyamine metabolism has been shown to be involved in plant growth, development and stress responses. Transgenic manipulation of polyamine metabolism has thus become a target for evaluating the various roles of polyamines in plant cells. We studied the catabolism of putrescine in a nontransformed (NT) and a transgenic cell line (2E) of Populus nigra x maximowiczii expressing a mouse ornithine decarboxylase (odc) cDNA. The rate of putrescine turnover and the initial half- life of putrescine were determined in the two cell lines in two different ways: (1) by labeling the endogenous putrescine in cells using labeled ornithine and analyzing the disappearance of label from putrescine with time on removal of the label, and (2) by allowing the cells to accumulate exogenously supplied labeled putrescine and following its disappearance with time. The results show that: (1) transgenic cells contained several-fold higher levels of putrescine but only slightly higher levels of spermidine and spermine; (2) over the period of seven days of culture, the cellular putrescine content fluctuated somewhat on different days in both cell lines; (3) the transgenic cells showed higher rates of putrescine turnover than the nontransgenic cells; and (4) the conversion of putrescine into spermidine was higher in the 2E cells. Furthermore, the initial half- life of putrescine turnover was not significantly different in the two cell lines when fed with either labeled putrescine or ornithine. The activity of diamine oxidase, the key enzyme involved in putrescine catabolism, was not significantly different in the NT and 2E cell lines at any of the time periods tested. It is concluded that in these cells the rate of putrescine catabolism is keeping pace with the rate of its biosynthesis. 3 Graduate Students.

Impacts
The results provide an insight into the regulation of polyamine metabolism in plants and its relationship with the metabolism of aminoacids.

Publications

  • Minocha, S.C. 2000. Optimization of the expression of a transgene in plants. In: S.M. Jain and S.C. Minocha (eds.). Molecular Biology of Woody Plants, Vol. I. Kluwer Scientific Publishers, Dordrecht, The Netherlands.pp.1-30.
  • Minocha, S.C. and J.C. Wallace. 2000. Gene transfer techniques and their relevance to woody plants. In: Jain, S.M. and S.C. Minocha (eds.). Molecular Biology of Woody Plants Vol. II. Kluwer Scientific Publishers, Dordrecht, The Netherlands. pp. 1-24.
  • Jain, S.M. and S.C. Minocha (eds.). 2000. Molecular Biology of Woody Plants Vol. I. Kluwer Scientific Publishers, Dordrecht, The Netherlands. pp. 1-520.
  • Jain, S.M. and S.C. Minocha (eds.). 2000. Molecular Biology of Woody Plants Vol. II. Kluwer Scientific Publishers, Dordrecht, The Netherlands. pp 1-511.


Progress 01/01/99 to 12/31/99

Outputs
The presence of two alternate pathways, i.e. ODC (ornithine decarboxylase) and ADC(arginine decarboxylase) for putrescine production in many plant tissues complicates the situation regarding their metabolic regulation, particularly, when the substrates of the two pathways (ornithine and arginine) are also interconvertible. It is thus conceivable that metabolic effects of overexpression of odc or adc gene may be limited by the availability of the substrates for these enzymes. Furthermore, ADC and ODC activities may be subject to feed back regulation by polyamine concentrations in the cell. These hypotheses were tested by analyzing polyamine metabolism and its relationship to the metabolism of arginine, ornithine and glutamine in transgenic and nontransgenic cells of hybrid poplar (Populus nigra x maximowiczii). A plasmid containing a mouse odc cDNA regulated by a 2x35S CaMV promoter was used in transformation of poplar cells by biolistic bombardment. Several transgenic cell lines were characterized with respect to the presence of the mouse odc cDNA by PCR and Southern hybridization of the amplified PCR product. The activities of ODC and ADC were measured in cell homogenates as well as in intact cells using 1-14C-ornithine or 1-14C-arginine as substrates. Putrescine contents were generally 3-10 fold higher in the odc-transgenic cells on any given day as compared to the nontransformed (NT) cells. There was no consistent change in the contents of spermidine or spermine in the transgenic cells. The incorporation of label from U-14C-ornithine into the putrescine fraction was significantly higher in the transgenic cells as compared to the NT cells. The amounts of label present in spermidine and spermine fractions were also generally higher in the transgenic cells as compared to the NT cells. When the two cell lines were treated with a range of concentrations of arginine, ornithine, urea or glutamine, and the polyamine contents determined at 24 and 72 h, no further increase in putrescine, spermidine or spermine content was seen under any of the treatments. Methionine sulfoximine (MSX), a strong inhibitor of glutamine synthetase, caused a significant reduction in cellular putrescine within 24 h in both the NT and the transgenic cells. Ornithine caused a substantial (but never complete) reversal of the effect of this inhibitor. From the data obtained so far, it can be concluded that: (1) transgenic expression of a heterologous odc gene can be used to modulate putrescine metabolism, (2) overproduction of putrescine and its accumulation in high amounts does not inhibit native ADC activity and its contribution to putrescine production, (3) ornithine biosynthesis occurs primarily from glutamate and not from a catabolic breakdown of arginine, (4) ornithine biosynthesis is not a limiting factor for putrescine production in the transgenic cells, and (5) assimilation of ammonia into glutamine keeps pace with an increased demand for its utilization for putrescine production and possibly also for proline production. 1 Graduate Student.

Impacts
The study provides better clues to understanding the effect of genetic manipulation of one reaction on the metabolism of related biochemical pathways.

Publications

  • Minocha, R., Long, S., Maki, H. and Minocha, S.C. 1999. Assays for the activities of polyamine biosynthetic enzymes using intact tissues. Plant Physiol. Biochem. 37:597-603.
  • Minocha, S.C. and Minocha, R. 1999. Genetic transformation in conifers. In: S.M. Jain, P.K. Gupta, and R.J. Newton (eds.) Somatic Embryogenesis in Woody Plants. pp. 291-312. Kluwer Acad. Publ. Dordrecht, The Netherlands.


Progress 01/01/98 to 12/31/98

Outputs
Several transgenic cell lines of Populus nigra x maximowiczii expressing a mouse ornithine decarboxylase (ODC) cDNA under the control of a 2x35S cauliflower mosaic virus promoter were produced. A fast and reliable method for the quantification of ODC activity in live cells was devised and used to measure mouse ODC enzyme activity in several transgenic and nontransgenic cell lines. The activity of mouse ODC enzyme was detected in high amounts in the transgenic cell lines while it was undetectable in the control cells. Cellular putrescine in the transgenic cells was several-fold higher than that in the control or GUS-transformed cells, however, there was no significant change in the cellular content of spermidine or spermine. Further metabolic studies using radio-labeled arginine and ornithine showed that: (1) Higher amounts of labeled ornithine were incorporated into putrescine in the transgenic cells as compared to controls. (2) There was no significant differences in the rates of incorporation of labeled arginine, showing that the overproduction of putrescine by ODC did not affect the native ADC activity. (3) Supplementation of the medium with unlabeled ornithine or arginine at several different concentrations did not affect cellular putrescine levels in either control or the transgenic cells. 1 Graduate Student.

Impacts
(N/A)

Publications

  • No publications reported this period


Progress 01/01/97 to 12/31/97

Outputs
Cell cultures of Populus nigra x maximowiczii were analyzed for their polyamine content in response to water stress and salt treatment and those of Picea rubens (red spruce) were analyzed for their response to Al stress. Water stress caused a significant increase in putrescine in Populus cells, while Al caused an increase in putrescine and a decrease in Ca, K and Mg in red spruce cells. Based upon these results, we wanted to test if increased production of putrescine in these cells through transgenic expression of an ornithine decarboxylase (ODC) gene would make the cells tolerant to these forms of stress. An expression vector containing a mouse ODC cDNA under the control of 2x35S promoter was constructed and used to produce several cell lines of Populus. The activity of mouse ODC was detected in high amounts in the transgenic cell lines. Cellular putrescine in the transgenic cells was several-fold higher than that in the control or GUS-transformed cells. The metabolism of radiolabeled ornithine, arginine, putrescine and methionine is being studied in the transgenic cells. Using Polymerase Chain Reaction, we have isolated partial coding sequences of ADC(arginine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase) from red spruce and Populus tissues. Needles of red spruce trees growing at sites showing varying levels of Ca/Al ratios in the soil showed a strong negative correlation between soil Ca/Al ratios and cellular putrescine and spermidine levels. 2 Graduate students.

Impacts
(N/A)

Publications

  • MINOCHA, R., SHORTLE, W.C., LAWRENCE, G.B., DAVID, M.B. and MINOCHA, S.C. 1997. Relationships among foliar chemistry, foliar polyamines, and soil chemistry in red spruce trees growing across the northeastern United States. Plant and Soil 191: 109-122.


Progress 01/01/96 to 12/30/96

Outputs
Several transgenic plants of Populus tremuloides were analyzed for the presence and the expression of neomycin phosphotransferase (NPT) and s-adenosylmethionine decarboxylase (SAMDC) genes. While little NPT protein was seen in nontransgenic plants, all 9 of the transgenic plants were found to have high levels of NPT protein. The enzyme activity of SAMDC was higher in two of the transgenic calli as compared to the nontransgenic calli. Polyamine content of the calli and leaves of all nine transgenic plants was compared with the nontransgenic plants on different days of culture on the same medium. A small increase in spermidine but a significant decrease in putrescine was observed in several transgenic calli. In the leaves of transgenic plants, however, an increase in all three polyamines was observed in some cases. Exogenous supply of neither putrescine nor ornithine had any effect on spermidine content of the calli, indicating that the lack of increase in spermidine in the transgenic cells was not due to limiting quantities of these substrates. Further attempts with Agrobacterium strains containing the mouse ornithine decarboxylase gene have still not produced any calli or plants. This may be due some cytotoxic effects of overproduction of putrescine or a depletion of ornithine pools in the transgenic cells. 2 graduate students.

Impacts
(N/A)

Publications

  • MINOCHA, R., W.C. SHORTLE, D.J. COUGHLIN, JR., AND S.C. MINOCHA. 1996. Effects of aluminum on growth, polyamine metabolism, and inorganic ions on suspension cultures of red spruce (Picea rubens). Can. J. For. Res. 26: 550-559.
  • SUN, DONGYU 1996. Modulation of Polyamine Metabolism and Tolerance to Osmotic and Salinity Stress in Plants . M.S. Thesis, University of New Hampshire, Durham, NH pp. 1-115.


Progress 01/01/95 to 12/30/95

Outputs
Leaf discs of aspen (populus tremuloides) plants grown in culture were transformed with Agrobacterium tumefaciens containing a human S-adenosylmethionine decarboxylase (SAMDC) cDNA. The callus was selected on kanamycin and induced to produce shoots that were rooted to produce whole plants. The leaf tissues were tested for the presence of the foreign DNA, its expression at the transcriptional and translational levels, the activity of the human enzyme and the cellular levels of polyamines. While several transgenic plants expressed the foreign gene, there was no significant increase in the activity of SAMDC. Cellular levels of polyamines were also quite similar in the transgenic and nontransgenic plants. These results are quite different from those with the tobacco and carrot cells transformed with the same gene. The reasons for the lack of increased enzyme activity and increase in polyamines are being studied further. 2 graduate students.

Impacts
(N/A)

Publications

  • ZHOU, X., R. MINOCHA AND S. C. MINOCHA. 1995. Physiological responses of suspension cultures of Catharanthus roseus to aluminum: Changes in polyamines and inorganic ions. J. Plant PHysiol. 145:277-284.


Progress 01/01/94 to 12/30/94

Outputs
Age dependence of red spruce (PICEA RUBENS) suspension cultures to Al sensitivity was studied by adding three different concentrations of Al (0.2, 0.5, and 1.0 mM) at each day of a 7 day culture period and analyzing for changes in total cell mass, polyamines, polyamine biosynthetic enzymes, and inorganic ions after 24 h of incubation. All levels of A1 inhibited growth. Cellular putrescine levels and arginine decarboxylase (ADC) activity increased. Spermidine and spermine levels were either slightly elevated or not affected by Al. Cellular content of Ca, Mg, Mn, and K decreased, while Al and P increased in Al-treated cells. With few exceptions, the response to 24 h incubation with Al was independent of culture age. To study longer term effects, three-day-old cultures were treated with Al for up to 11 days. Growth rates generally decreased as the incubation time increased. Cellular putrescine levels remained elevated for the entire duration of the experiment. There was a sharp dose-dependent increase in spermine levels by the 7th day of incubation. An increase in the molar ratio of putrescine/Ca in these cells may be a potential marker for diagnosis of Al stress condition. Transgenic plants of POPULUS TREMULOIDES selected on kanamycin were tested for the presence of human S-adenosylmethionine decarboxylase (SAMDC) cDNA and mRNA and found to be positive. However, no significant increase in SAMDC activity or spermidine was observed.

Impacts
(N/A)

Publications

  • MINOCHA, R., SHORTLE, W.C., LONG, S.L. AND MINOCHA, S.C. 1994. A rapid and reliable procedure for extraction of cellular polyamines and inorganic ions from plant tissues. J. Plant Gr. Regul. 13:187- 193.
  • MINOCHA, S.C. AND MINOCHA, R. 1994. HPLC methods for polyamine analysis. LC.GC 12:506.
  • MINOCHA, R., MINOCHA, S.C. AND SIMOLA, L.K. 1994. Somatic embryogenesis and polyamines in woody plants. In: S.M. Jain, P.K. Gupta and R.J. Newton (eds.) Somatic Embryogenesis in Woody Plant Vol. 1, pp. 337-359. Kluwer Acad. Press; Netherlan
  • MINOCHA, S.C. AND MINOCHA, R. 1994. Historical aspects of somatic embryogenesis in woody plants. In: S.M. Jain, P.K. Gupta and R.J. Newton (eds.) Somatic Embryogenesis in Woody Plant Vol. 1, pp. 9- 22. Kluwer Acad. Press; Netherlands.


Progress 01/01/93 to 12/30/93

Outputs
Transgenic callus of carrot (Daucus carota L.) was obtained by transformation ofsomatic embryos with Agrobacterium tumefaciens strains containing mammalian cDNAs for ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). On transfer to an auxin-free medium, transgenic somatic embryos were regenerated in both cases. Suspension cultures of ODC-transformed cell lines contained significantly higher levels of putrescine compared to control cells. Spermidine and spermine were not affected. Cell lines transformed with SAMDC gene contained higher levels of spermidine with minimal effects on putrescine and spermine. The presence of respective foreign DNA in transgenic cells was confirmed by Southern hybridization and amplification by Polymerase Chain Reaction (PCR). The activities of ODC and SAMDC were higher in transformed cells than control cells in each case. Whereas the control cells are sensitive to difluoromethylarginine, an inhibitor of arginine decarboxylase, the ODC-transformed cells were resistant to this inhibitor. Transgenic somatic embryos with ODC gene were faster growing than the controls. Somatic embryos produced from SAMDC-transformed cells were stunted in growth but had thicker hypocotyl and root. One graduate student.

Impacts
(N/A)

Publications

  • No publications reported this period.


Progress 01/01/92 to 12/30/92

Outputs
Callus cultures of aspen (Populus tremuloides) were used to study the effects ofaluminum (Al) stress on cellular polyamine levels. It was observed that the major polyamine in aspen tissue is spermidine. In two of three experiments, a promotion in cellular putrescine was observed in the presence of Al within 24-48 h. Spermidine levels were always lower in Al treated cells. Spermine was present in very small quantities and showed a decrease in the presence of Al. Transformation experiments have been conducted using Agrobacterium tumefaciens strains containing either a mouse ODC or a human SAMDC cDNA and the NPTII (neomycin phosphotransferase) gene. These genes had been earlier tested for their expression in tobacco and were found to be active. Aspen leaf and petiole tissues have produced callus capable of growth on kanamycin, indicating the expression of the NPT gene. No shoots have yet been produced from either ornithine decarboxylase or S-adenosylmethionine decarboxylase transformed callus.

Impacts
(N/A)

Publications


    Progress 01/01/91 to 12/30/91

    Outputs
    A number of Agrobacterium tumefaciens strains containing either a mouse ornithine decarboxylase (ODC) or a human S-adenosylmethionine decarboxylase (SAMDC) cDNA under the control of 35S CAMV promoter were tested for expression in transgenic plants of tobacco (Nicotiana tabacum) before being used for transformation of aspen (Populus tremuloides). The plants transformed with the ODC cDNA selected on kanamycin showed high activity of neomycinphosphotransferase (NPT) as well as ODC. The presence of the mouse ODC protein was confirmed by its optimum pH of 6.8 (cf. plant ODC, pH 8.2), inhibition by difluoromethylornithine and immuno-precipitation on SDS-PAGE. The transformed plants and calli produced significantly higher levels of putrescine as compared to the controls. Likewise, tissues transformed with the human SAMDC cDNA showed the presence as well as the expression of this gene. The transformed calli and the plants showed significantly higher levels of SAMDC activity. These tissues possessed highly reduced levels of putrescine and elevated levels of spermidine. The same strains of A. tumefaciens have been used to produce kanamycin-resistant callus from aspen leaves. The callus is being cultured on shooting medium to regenerate transgenic plants. In the mean time, A. tumefaciens strains have been produced which contain the ODC and the SAMDC cDNAs under the control of two inducible promoters, namely, chalcone synthase and the small subunit of RubisCo.

    Impacts
    (N/A)

    Publications

    • NOH, E. W. 1991 Modulation of Polyamine Metabolism in Plants by Transfer of a Human Gene for S-Adenosylmethionine decarboxylase. Ph.D. Thesis, University of New Hampshire. pp. 1-134.
    • DESCENZO, R.A. 1991 Expression of a Murine Ornithine Decarboxylase cDNA in transgenic tobacco (nicotiana tabacum var. Xanthi). Ph.D. Thesis, University of New Hampshire, Durham, NH. pp. 1-210.


    Progress 01/01/90 to 12/30/90

    Outputs
    Agrobacterium tumefaciens strains containing a gene for kanamycin resistance (Neomycinphospho-transferase - NPTII) were used to transform leaf explants of Populus tremuloides and Eucalyptus tereticornis. Transformed plants of Populus selected on kanamycin were tested for (i) the presence of the NPTII gene by Southern hybridization, (ii) the presence of mRNA for NPTII by Northern hybridization, and (iii) the enzyme activity of neomycinphosphotransferase by a non-radioactive method. All tests were positive indicating stable transformation, incorporation of the foreign gene in the plant genome and the expression of the foreign gene. In the case of Eucalyptus, only callus growing on kanamycin was obtained. No regeneration of transformed shoots has been observed thus far. For future applications of the technique of gene transfer to modulate plant metabolism, a number of A. tumefaciens strains have been produced which contain a mouse gene for ornithine decarboxylase (ODC) and a human gene for S-adenosylmethionine decarboxylase (SAMDC). The cDNA coding for the respective enzyme has been recombined with either a constitutive promoter (Cauliflower Mosaic Virus - 35S) or an inducible promoter (Chalcone synthase - CHS) for controlled expression of the gene. Each strain also contains the NPTII gene under the control of a constitutive promoter to allow the selection of transformed cells on kanamycin. Results with transformed tobacco plants show that the mouse cDNA for ODC is expressed and produces active enzyme.

    Impacts
    (N/A)

    Publications

    • NOH, E.W. and MINOCHA, S.C. 1990. Pigment and isozyme variation in aspen shoots regenerated from callus culture. Plant Cell, Tissue, and Organ Culture 23:39-44.
    • SUBBAIAH, M.M. and MINOCHA, S.C. 1990. Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis. Plant Cell Reports 9: 370-373.


    Progress 01/01/89 to 12/30/89

    Outputs
    Transgenic plants of Populus tremuloides were produced using Agrobacterium tumefaciens strains containing the gene for neomycinphosphotransferase (NPTII) under the control of nopaline synthase promoter. Leaf segments of in vitro cultivated plants were treated with A. tumefaciens and grown on a callus induction medium containing kanamycin. Shoots were regenerated from the callus and rooted in the presence of kanamycin. The shoot tips of these plants were used to micropropagate the transgenic plants. The expression of NPTII gene was studied by enzyme assay and the presence of the gene confirmed by Southern hybridization using NPT gene segment as a probe. In the case of Eucalyptus teriticornis, regeneration of shoots was obtained from leaf and hypocotyl segment callus. These shoots were rooted and transferred to the greenhouse. Transformed callus was obtained by cocultivation of leaves with Agrobacterium and selection on kanamycin. This callus will be used to regenerate transgenic plants. A variety of Agrobacterium tumefaciens strains containing the mouse ornithine decarboxylase gene under the control of several plant promoters have been produced. Some of these strains have been used to produce transgenic tobacco plants in which the presence of the gene has been confirmed by Southern hybridization. Graduate students = 3.

    Impacts
    (N/A)

    Publications

    • NO PUBLICATIONS REPORTED THIS PERIOD.