1. Differential Accumulation Pattern of Met-rich  -zein in Medicago sativa and Medicago truncatula Serina Padilla Dr. Champa Sengupta-Gopalan, Mentor Department of Plant and Environmental Science, New Mexico State University

2. Medicago sativa (alfalfa) and the Importance of Methionine Important forage legume for livestock feed Low levels of methionine  Methionine deficiency in animals limits wool growth, milk production, and meat production (Bagga et al, 2004) Livestock owners must supplement feed with methionine  Most of the methionine is consumed by rumen microorganisms

3. Increasing Methionine In M.sativa Cross-breeding Cell selection Genetic engineering approach: Introduction of genes encoding for high methionine protein.

4. Zeins Seed storage proteins of corn Four classes:   (19kD and 22kD) has low levels of methionine   15kD) 10% methionine   (16kD and 27kD) has low levels of methionine   10kD and 18kD) 22 and 27% methionine respectively (Bagga et al, 2004)  -zein ideal for introduction into M.sativa through a gene cassette

5. Medicago sativa Transformation 1.Leaf segments are inoculated with Agrobacterium engineered with -β zein gene construct. Callusing is observed in leaf segments within 2-3 weeks 2.Callus is placed on selection media (25mg/L kanamycin) 3.Green embryoids are observed in callus after 6-weeks and they are regenerated into plantlets 4.The transgenic M. sativa plants appear to be morphologically normal, produce flowers and can be nodulated

6.  -zein protein accumulation in M.sativa  - zein is a bypass protein (not broken down by rumen organisms) but methionine levels are not high enough to be of nutritional value (Bagga et al, 2004)

7. Working Hypothesis Make a comparison using a model Medicago species to find possible differences with regards to the amino acid composition and the rate of synthesis of Met rich proteins in the two legumes of the same species. An understanding of the basis for any differences between M.sativa and the model legume, M.truncatula, will allow us to increase the Met- containing proteins in M.sativa using a genetic engineering approach.

8. Medicago truncatula Transformation 1.M. truncatula leaf and root segments from seedings are inoculated with Agrobacterium engineered with β-zein gene constructs. Callusing is observed in leaf and root segments within 2-3 weeks 2.Callus is placed on selection media (25mg/L kanamycin) 3.Green embryoids are observed in callus after 6-weeks and they are regenerated into plantlets 4.The transgenic plants appear to be morphologically normal.

9.  zein protein in M.truncatula independent transformants The transformants with the  -zein gene construct show higher levels of  -zein accumulation. Pattern of protein accumulation of β-zein in transgenic M. truncatula leaves was perfomed with Western analysis: EtOH-soluble fractions (equivalent to 25 ug of PBS-soluble fraction) from M. truncatula leaves were subjected to SDS- PAGE followed by immunoblot analysis using  -zein antibodies. Lanes: M. truncatula transformants lanes 7 to 14; Tobacco transformant expressing  -zein

10. Comparison of  -zein Protein Accumulation Medicago truncatula shows ~10-20 fold higher levels of  -zein protein accumulation compared to Medicago sativa Pattern of protein accumulation of β-zein in transgenic M. sativa and M. truncatula leaves was perfomed with Western analysis: EtOH-soluble fractions (equivalent to 25 ug of PBS-soluble fraction) from M.sativa and M. truncatula leaves were subjected to SDS-PAGE followed by immunoblot analysis using  -zein antibodies. Lanes: M. sativa non-transformed control, M. sativa 7 and 10  -zein transformants, M. truncatula non-transformed control, M. truncatula 8 and 14  -zein transformants

11. Comparison of  - Zein Transcript Levels The M. truncatula transformants show approximately 20 fold higher levels of  -zein expression compared to M. sativa expressing the same gene. Comparison of transcript levels in M. sativa and M. truncatula was performed by Northern analysis: 20 ug of total RNA from M. sativa and 10 ug of total RNA from M. truncatula was seperated on the same 1.0% agarose formaldehyde gel and subjected to gel blot analysis using a 690 bp  -zein fragment as a probe. Blots were also hybridized to M. sativa Glutamine Synthase (GS2) probe to check the levels of GS2 transcript in M. sativa and M. truncatula. The third panal shows ethidium bromide stained ribosomal bands. Lanes: M. sativa not-transformed control, M. sativa 7 and 10  -zein transformants, M. truncatula non- transformed control, M. truncatula 8 and 14  -zein transformants.

12. Methionine Pathway

14. Conclusion Metabolite analysis shows M.sativa has high levels of S-adenosylmethionine (SAM) Down regulation of SAM synthase could increase  -zein production

15. Acknowledgements Dr. Suman Bagga Matt Barrow Omar Holguin References Bagga, S, Armendaris, A, Klypina, N, Ray, I, Ghoshroy, S, Endress, M, Sutton,D., Kemp, J.D., and Sengupta-Gopalan, C.(2004). Genetic engineering ruminal stable high methionine protein in the foliage of alfalfa. Plant Sci. 166, 273-283.

16. Questions