Plant lecture 2 - Manipulation of plant metabolism

Question 1

Analysis of plant metabolism

Answer 1

Difficulties

1. Measuring E activity
   - Tissue disruption = breaking tough cell wall
   - Enzyme inactivation - proteases + polyphenols in vacuole
   - Assay of enzyme - contains isozymes so have to compromise conditions
2. Measuring metabolites (metabolite levels)
   - Low amounts - cytosol could only occupy 2-5% of cell. Need ↑ tissue and have ↑ background noise
   - Breakdown extraction - phosphatases/hydrolases in vacuole = released. Mixes + distorts levels
3. Metabolic flux measurements
   - Low metabolic rates, difficult to measure substrate utilisation through depletion
   - Diversity of substrates/ complexity of pathways = ↑ end-products + pathways

Question 2

Plant transfer

Answer 2

1. Transfer w/o vector
   - Direct uptake into naked protoplast e.g. w/ electroporation (24,000V)
   - Biolistic gun, most popular, small gold particle coated w/ solution of DNA + fired into plant tissue
2. Virus mediated transfer
   - Either DNA or RNA viruses
   - Advantage = can simply infect plant cells + virus can replicate within infected tissue
   - Disadvantage = most viruses are RNA-based, handling difficult and ↑ levels of unpredictable recombinatino
3. Agrobacterium-mediated transfer
   - Technically simple, ↑ capacity for gene insertion

Question 3

Agrobacterium natural

Answer 3

• Moves to plant w/ cellular damage in response to phenolic compounds e.g. acetosyringone
• Then transfers tDNA into host
• Genes encoded in tDNA:
  1. E for growth regulators e.g. auxins + cytokines then → synthesis of auxins → de-deifferentiation + multiplication of infected cells → crown gall tumor
  2. Genes responsible for synthesis of opines. Used as source of N + respiration substrate in bacteria
  3. Vir genes (8 transcriptional units VirA-H e.g. VirA/6 encodes 2-component system responsible for detecting phenolic compounds)

Question 4

Agrobacterium technical

Answer 4

• tDNA region x have to be in same plasmid. Often vectors modified → binary vector system
• Vir genes maintained in separate large plasmid + tDNA region is excised to small plasmid (easier to manipulate)
• tDNA plasmid could tDNA region w/ genes for opines removed + replaced w/ kanamycin resistance + GOI
• Can select, transfer to different medium + grow to a good size

Question 5

Agrobacterium floral dip procedure

Answer 5

• Take immature Arabidopsis seedlings + submerge in medium w/ Agrobacterium
• Flowers fertilised + seeds spread on soil + germinated. Treat w/ kanamycin + select what grows

Question 6

How can Agrobacterium be used to manipulate enzymes of metabolism

Answer 6

1. PFP
   - Antisense inhibition
   - Coding region (PFP) inserted in reverse btw promoter + terminator that allows expression in cells
   - Catalytic capacity of PFP ↓ by 80-90% WT + x effect respiration
2. PFK
   - Coding region in sense orientation
   - 20x normal PFK in selected transformed lines
   - PFK = thought to be rate-limiting stage. But even though ↑ ↑ expression, x impact on rate

Question 7

Insertional mutagenesis

Answer 7

• Ablate expression of individual genes in plants
• Exploits agrobacterium + ability to insert v large pieces of foreign DNA into host
• E.G. used tDNA to ablate expression of glycolytic E like glycerate mutase (plastidic)
• → ↑ normal lipids (conversion of 3PGA-2PGA x essential)
• If ablate genes encoding plastid enzymes (2-PG→PEP), → normal lipid production (cytosolic pathway can supply PEP needed)
• x necessarily pathway used by WT but just plastid x essential