Source: NEW MEXICO STATE UNIVERSITY submitted to
INCREASING METHIONINE LEVELS IN ALFALFA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0196980
Grant No.
2003-35318-13701
Project No.
NM-1-5-28248
Proposal No.
2003-02116
Multistate No.
(N/A)
Program Code
54.3
Project Start Date
Sep 1, 2003
Project End Date
Aug 31, 2005
Grant Year
2003
Project Director
Sengupta-Gopalan, C.
Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239
Performing Department
AGRONOMY & HORTICULTURE
Non Technical Summary
Legumes are among the most important nutritional source of protein for animal feeding all over the world. However, they are deficient in the sulfur amino acids, methionine and cysteine and the ruminant animals that eat them suffer from limitations in sulfur amino acids. Improving the S-amino acid contents of legumes is, therefore, of potentially great value and it has been a frequent target of breeding, and in recent years of plant metabolic engineering. We plan to hit this target in alfalfa by engineering a greater supply of methionine. We believe that this strategy will work because a number of studies in different species have shown that methionine levels can be raised by engineering metabolism. Our plan involves increasing free methionine levels by over-expressing cystathionine g- synthase, or by down regulating either threonine synthetase or S-adenosyl methionine synthase; analyzing changes in gene expression and metabolites in the methionine metabolic pathway resulting from increasing the free methionine pools by using microarray technology and proteomics. The results will have a potentially dramatic impact on the levels of dietary methionine available to ruminant animals fed alfalfa. This would alleviate a serious problem in animal nutrition. By analyzing the effects of the proposed transgenic strategies on gene expression and metabolic flux we expect to contribute to the understanding of regulation in the hard-to-study metabolic network surrounding methionine.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011640104050%
2061640104050%
Goals / Objectives
Legumes are among the most important nutritional source of protein for animal feeding all over the world. However, they are deficient in the sulfur amino acids, methionine and cysteine and the ruminant animals that eat them suffer from limitations in sulfur amino acids. The goal of this project is to genetically engineer alfalfa for increased methionine content. Met is synthesized from three components: the C skeleton is derived from Asp just as are Lys, Thr and Ile; the S moiety is derived from Cys and the methyl group is derived from methyltetrahydrofolate. The C skeleton pathway includes enzymatic steps in which the side chain of Asp is converted from a carboxyl to the phosphate ester form, O-phosphohomoserine (OPH). Biochemical and functional studies have shown that the carbon flow through O-phosphohomoserine (OPH) plays a role in regulating Met synthesis. Met biosynthesis branches from that of Thr and Ile at OPH, with Cystathionine-synthase (CGS) converting OPHS to Met while Thr synthase (TS) converts it into Thr. Over-expresssion of CGS in transgenic Arabidopsis results in an increase in the pool of free Met. Down-regulation of CGS results in Met auxotrophy Down-regulation of TS by either mutation or antisense gene constructs leads to increased Met content. Met is further metabolized into S-adenosylmethionine (SAM), the primary biological methyl group donor. The reaction is catalyzed by the enzyme SAM synthetase (SAMS). Mutation in the SAMS gene in Arabidopsis show increased accumulation of free Met. By blocking SAM synthetase, the major route for Met metabolism is reduced, and the reduced level of SAM, increases CGS expression. The objectives towards increasing the Met levels in alfalfa are to use genetic engineering methods to increase the transcript level for CGS or to decrease the transcript level for TS or SAMS. An additional goal of this project is to analyze changes in gene expression, both at the transcript and protein level resulting from increased Met levels in transformed alfalfa and to determine the effect of increased free Met pool on the metabolite profiles.
Project Methods
Three different approaches will be taken to increase the Met level in alfalfa: i.transforming alfalfa with gene constructs driving an Arabidopsis CGS gene behind CaMV 35S promoter. Since CGS is subject to feed back inhibition at the transcript level an additional construct containing the feedback insensitive form of the gene will also be engineered. ii. transforming alfalfa with antisense gene constructs for TS. The TS gene will be isolated from alfalfa by performing RT PCR.. iii. transforming alfalfa with antisense gene constructs for SAMS. The SAMS sequences for alfalfa will be isolated by RT PCR. Since SAMS is known to be encoded for by a gene family, efforts will be made to downergulate SAMS in a gene specific manner. Alfalfa transformation will be performed using the standard protocol using Agrobacterium tumefaciens. To check if increasing free Met pools has any effect on gene expression in alfalfa, we will perform transcript and protein analysis in the control and transformed plants. For transcriptome analysis, we will screen both microarrays and macroarrays. For analysing changes in protein profile due to increased Met levels, we will use the proteomic approach consisting of 2D gel electrophoresis followed by spot excision and identification of protein by mass spectrometry.

Progress 09/01/03 to 08/31/05

Outputs
1) We have isolated cDNA clones for MMT and HMT from alfalfa. 2) We have initiated making gene constructs to downregulate MMT. 3) We have initiated analysis of nodules. 4) We have introduced the double zein gene constructs in transformants overexpresing the CgS gene.

Impacts
Legumes supplied as forage, are among the most important nutritional source of protein for animal feeding all over the world. However, legumes are deficient in the sulfur amino acids, Met and Cys, and it has been shown that wool growth in sheep, milk production in dairy animals and meat production is limited by the availability of S-amino acids. Improving the S-amino acid contents of legumes is, therefore, of potentially great value and it has been a target of breeding and transgenic efforts. Efforts using conventional breeding to increase the S-amino acid content of alfalfa has met with limited success and thus the only viable approach to increase methionine content in forage legumes is by genetic engineering. This project specifically targets the Met primary metabolic pathway in alfalfa using a powerful combination of both 'source' and 'sink' derived transgenes to understand the flow of metabolites through this pathway, and the ultimate destinations of Met and its derivatives within the plant. Co-transformation with both 'source' and 'sink' transgenes represents a unique approach for study of Met pathway regulation, and preliminary results also show that this approach has great value in the goal of increasing alfalfa's nutritional properties.

Publications

  • Bagga, S., Ross, J., Potenza, C., Leustek, T., and C. Sengupta-Gopalan. 2005. Increasing methionine levels in alfalfa by co-expressing genes for Cystathionine gamma Synthase, a key enzyme in methionine biosynthsis and the methionine rich zein proteins. In vitro Cell. Dev. Boil - Plant 41 (6): 731-741.
  • Sengupta-Gopalan, C., Bagga, S., Potenza, C., and Ortega, J.L. 2005. New techniques for genetic improvement of legumes. Genetic Engineering for forage quality in alfalfa. In Press.
  • Klypina, N., Bagga, S., Potenza, C. Hanson, S., Sutton, D., Kemp, J. and Sengupta-Gopalan, C. 2006. Differential expression and accumulation of the b-zein does not result in a significant change in d-zein accumulation. In Vitro - plant. (In preparation).


Progress 01/01/04 to 12/31/04

Outputs
1) The AtCgS gene engineered behind the CaMV 35S promoter was introduced into alfalfa and the transformants were analyzed for both transcript and CgS protein. The transformants showed accumulation of both the transcript and the polypeptide corresponding to AtCgS. The transformants showed a 5 to 14 fold increase in free Met and infinite increase in the amount of SMM. 2) A alfalfa transformant with the b-zein driven by the CaMV 35S promoter was re-transformed with the AtCgS gene construct. Total RNA from alfalfa transformants containing the b-zein gene alone, CgS gene alone, or co-expressors of both genes, were subjected to northern analysis and probed with the b-zein, the AtCgS, and rRNA probes. AtCgS transcript levels, while showing variation from plant to plant showed no major differences between the AtCgS /b-zein and the AtCgS alfalfa transformants. However, there is a 2 to 3-fold increase in b-zein transcript accumulation in the co-expressors compared to the b-zein transformants. Protein isolated from the same plants was subjected to immunoblotting using the b-zein and AtCgS antibodies. While the AtCgS level was more or less similar in both the AtCgS overexpressors and the co-expressors, the b-zein levels were 4 to 6-fold higher in the co-expressors compared to the b-zein overexpressors. These results suggest that increasing the free Met pool by expressing the AtCgS gene in alfalfa increases the stability and the translatability of the b-zein RNA. 3) While the CgS overexpressors, in general, showed high levels of Met and SMM, the co-expressors showed reduced levels of both Met and SMM. The Met profiles of the b-zein plants were identical to control plants, but they showed elevated SMM levels. 4) The d-zein protein (18kD zein) has 27% Met content while the b-zein (15kD zein) has only 15% Met content. For maximizing the increase in Met level by transgenic methods, the 18kD d-zein is more appropriate as a transgene. However, the d-zein requires complexing with the b-zein for stabilization. We have demonstrated this by producing co-expressors of the two zein genes produced either by sexual crosses or by re-transformation. To keep the two zein genes in the same linkage group, the b- and d- zein genes have been engineered behind two different promoters and in the same T DNA construct and introduced into alfalfa. The level of accumulation of the d-zein is highly enhanced in plants containing this dual gene construct when compared to transformants with just the d-zein or the co-expressors produced by sexual cross. 5) We have used RT-PCR to isolate cDNA clones for Cystathionine g- synthase, threonine synthase, S-adenosyl methionine synthase, cystathionine b-lyase and methionine synthase from alfalfa and have used them as probes in northern analysis.

Impacts
Legumes supplied as forage, are among the most important nutritional source of protein for animal feeding all over the world. However, legumes are deficient in the sulfur amino acids, Met and Cys, and it has been shown that wool growth in sheep, milk production in dairy animals and meat production is limited by the availability of S-amino acids. Improving the S-amino acid contents of legumes is, therefore, of potentially great value and it has been a target of breeding and transgenic efforts. Efforts using conventional breeding to increase the S-amino acid content of alfalfa has met with limited success and thus the only viable approach to increase methionine content in forage legumes is by genetic engineering. This project specifically targets the Met primary metabolic pathway in alfalfa using a powerful combination of both 'source' and 'sink' derived transgenes to understand the flow of metabolites through this pathway, and the ultimate destinations of Met and its derivatives within the plant. Co-transformation with both 'source' and 'sink' transgenes represents a unique approach for study of Met pathway regulation, and preliminary results also show that this approach has great value in the goal of increasing alfalfa's nutritional properties.

Publications

  • Bagga, S., Armendaris, A., Endres, M., Klypina, N., Ray, I.M., Sutton, D., Kemp, J.D., and Sengupta-Gopalan, C. 2004. Genetic engineering for ruminal stable high methionine protein in alfalfa. Plant Science 166:273-283.


Progress 01/01/03 to 12/31/03

Outputs
To check if the beta-zein (15kD zein) had a stabilizing effect on the delta zein (10kD zein) in alfalfa, pMEZ (CaMV 35S delta- zein gene) transformants were re-transformed with Agrobacterium strains containing the pM10Z (CaMV 35S-10kD delta-zein gene) and the hygromycin phosphotransferase (HPT) gene cassette. A second maize zein gene encoding a Met-rich deltazein (18kD deltazein) that is distinct from but closely related to the 10kD delta-zein was also engineered behind the CaMV 35S promoter (pM18Z) and by retransformation introduced into pMEZ alfalfa transformants. Coexpressors of the delta- and beta-zeins were also obtained by sexual crosses. A comparison of the zein levels in the single transformants with the co-expressors, showed a 7 to 10 fold increase in the delta-zein levels in the co-expressors compared to transformants with just the delta-zein gene. However, comparison of the zein content in the alfalfa coexpressors compared to the levels in the tobacco coexpressors showed a 5 to 10-fold lower levels in alfalfa. To check if the internal Met levels in the cells is a limiting factor in the synthesis of the zein proteins, embryogenic cultures of alfalfa co-expressing the beta-zein and the delta-zein genes were supplemented with Met or other Met precursors. Preliminary results showed a significant increase in the level of zein proteins when the culture was supplemented with Met, suggesting that availability of free Met in the alfalfa cells limits the synthesis of zein proteins. Similar results were obtained when leaves from a beta- x delta-zein transformant were incubated in Met. A CGS cDNA clone from Arabidopsis was engineered behind the CaMV 35S promoter and introduced into alfalfa with and without the beta-zein gene construct. Re-transformed alfalfa plants were identified. RNA and protein analysis was performed on the beta-zein plants, the retransformants and alfalfa plants transformed with the CGS gene construct. The co-expressors showed higher levels (>3-fold) of both the transcript and protein for the beta-zein gene compared to the control beta-zein plants. These very preliminary data suggest that overexpression of CGS does enhance the accumulation of the high Met -zein protein. However, we do need to check for Met levels and CGS activity before we can attribute increased zein synthesis in the alfalfa retransformants to increased free Met pools.

Impacts
Legumes supplied as forage, are among the most important nutritional source of protein for animal feeding all over the world. However, legumes are deficient in the sulfur amino acids, Met and Cys, and it has been shown that wool growth in sheep, milk production in dairy animals and meat production is limited by the availability of S-amino acids. Improving the S-amino acid contents of legumes is, therefore, of potentially great value and it has been a target of breeding and transgenic efforts. Efforts using conventional breeding to increase the S-amino acid content of alfalfa has met with limited success and thus the only viable approach to increase methionine content in forage legumes is by genetic engineering.

Publications

  • Bagga, S., Ross, J., and Sengupta-Gopalan, C. 2003. Co-expression of methionine rich zein genes and the genes for cystathionine-synthase, a key enzyme in the methionine synthesis for increasing methionne levels in alfalfa. 7th International congress of plant Molecular Biology. Barcelona, Spain, June 23-28, 2003.
  • Cheng, R., Carbajal, L., Bagga, S., Ross, J., Potenza, C. Does overexpression of methionine rich zein proteins in transgenic plants modify the expression of enzymes in the methionine biochemical pathway. The Annual Biomedical Research Conference for Minority Students. San Diego, California, Oct 15-18, 2003.