Gene Expression and Physiological Analyses to Study Metabolic Shifts in Medicago Truncatula Subjected to Biotic Stresses

Plants are frequently under attack from pests and deploy various defense mechanisms to fight off predators. Many plant defenses are induced following herbivory or pathogen infection. An investment in defense could potentially lead to a diversion of metabolic resources away from primary metabolism, including reduction in plant commitment to photosynthesis. In this thesis, changes in expression levels of representative genes for primary and secondary metabolism were studied using real-time polymerase chain reaction (PCR) in Medicago truncatula Gaertner subjected to beet armyworm (Spodoptera exigua H¨¹bner) damage or Phoma medicaginis Malbr. & Roum. infection. Photosynthetic yield was also measured using chlorophyll fluorescence to determine if changes in expression level of genes encoding photosynthetic machinery corresponded with changes in yield. In insect-damaged plants, transcripts encoding ¦Â-amyrin synthase (¦ÂAS), farnesyl pyrophosphate I (FPS1) and a cytochrome P450 monooxygenase (CYP716A12) were induced to levels significantly higher than in non-damaged control plants. Each of these genes is predicted to encode a product directly involved in the synthesis of secondary metabolites, so the strong levels of induction are consistent with activation of plant defenses. However, transcripts encoding protein products directly involved in photosynthesis were significantly suppressed. These included genes for chlorophyll a/b binding protein (CAB), ribulose-1,5-bisphosphate carboxylase small subunit (RubP) and oxygen-evolving enhancing protein (OEE). This observation, along with previous reports in the literature, is consistent with a shift of plant resource commitment away from primary metabolism. Transcript accumulation of genes encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and chalcone reductase (CHR) were also suppressed in insect-injured plants. In pathogen-inoculated plants, CHR transcripts were induced while those for ¦ÂAS, FPS1, CYP716A12, DXR, CAB and RubP were suppressed, and the expression level of OEE was unchanged. The CHR enzyme regulates a branch point in the biosynthesis of flavonoids and isoflavoids, which include antimicrobial phytoalexins in the Fabaceae. Its induction following pathogen infection suggests an activation of pathogen-specific responses by the plant. Changes in expression level for all members of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene family in M. truncatula were also assessed. Differential transcript accumulation for gene family members encoding HMGR, which catalyzes the first committed step in the mevalonate pathway leading to sesqui- and tri-terpenes in plants, is well established. Transcripts for all five M. truncatula HMGR isoforms were induced following insect herbivory. In fungal-inoculated plants, all transcripts were suppressed except HMGR2, which was induced by pathogen infection. The differential expression of the HMGR gene family members in M. truncatula demonstrated in this study is consistent with the varied expression profiles of this gene family observed in other plants. Photosynthetic yield was measured via leaf fluorescence as photosystem II operating efficiency. No significant difference in yield was observed among the treatments and all samples had yield values typical for unstressed plants. This was unexpected based on transcript data. Expression levels for photosynthetic genes were greatly suppressed in plants subjected to either treatment while defense genes were induced indicating a metabolic shift. Although photosynthesis measurements showed no change in yield, transcript data showed suppression of photosynthetic genes. This suggests that although photosynthetic-gene transcript levels are reduced, there must still be adequate levels to maintain photosynthesis in the time frame of these experiments. In this study, it was shown that there is not a direct link between molecular activity in the form of transcript accumulation and physiological activity following insect attack and pathogen infection in plants