Plant Stress

1. Plant Stress Friday, Feb 22: presenting an abiotic plant stressor, what is known about it, and how it might affect plants in ~ 10 minute presentation.

2. Light-independent (dark) reactions occur in the stroma of the chloroplast (pH 8) Consumes ATP & NADPH from light reactions regenerates ADP, Pi and NADP+

3. Light-independent (dark) reactions Overall Reaction: 3 CO2 + 3 RuBP + 9 ATP + 6 NADPH = 3 RuBP + 9 ADP + 9 Pi + 6 NADP+ + 1 Glyceraldehyde 3-P

4. Light-independent (dark) reactions 1) fixing CO2 2) reversing glycolysis 3) regenerating RuBP

5. Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi mechanism to regulate PS

6. Regenerating RuBP G3P has 2 possible fates 5 in 6 regenerate RuBP necessary to keep cycle going

7. Regenerating RuBP Basic problem: converting a 3C to a 5C compound must assemble intermediates that can be broken into 5 C sugars after adding 3C subunit

8. Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C 7C + 3C = 5C + 5C Uses 1 ATP/RuBP

9. Light-independent (dark) reactions build up pools of intermediates , occasionally remove one very complicated book-keeping Use 12 NADPH and 18 ATP to make one 6C sugar

10. Regulating the Calvin Cycle Availability of CO2 Demand is set by mesophyll, stomata control supply Ci is usually much lower than Ca A vs Ci plots tattle on the Calvin cycle

11. Regulating the Calvin Cycle A vsCi plots tattle on the Calvin cycle • In linear phase rubisco is limiting • When curves RuBP or Pi regeneration is limiting

12. Regulating the Calvin Cycle Currently Rubisco usually limits C3 plants Will increase plant growth until hit new limiting factor

13. Regulating the Calvin Cycle Currently Rubisco usually limits C3 plants Will increase plant growth until hit new limiting factor Free-Air CO2 Enrichment Experiments show initial gains, but taper off w/in a few years Now are limited by nutrients or water

14. Regulating the Calvin Cycle Rubisco is main rate-limiting step

15. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase : uses ATP to activate rubisco

16. Regulating the Calvin Cycle • Rubisco is main rate-limiting step • Rubisco must be carbamylated & bind Mg2+ to be active! • RuBP binds & inactivates uncarbamylatedrubisco • Rubiscoactivase removes this RuBP

17. Regulating the Calvin Cycle • Rubisco is main rate-limiting step • Rubisco must be carbamylated & bind Mg2+ to be active! • RuBP binds & inactivates uncarbamylatedrubisco • Rubiscoactivase removes this RuBP • In the dark many species phosphorylate carboxyarabinotol to form carboxyarabinitol 1-phosphate which binds the rubisco active site

18. Regulating the Calvin Cycle • Rubiscoactivase removes this RuBP • In the dark many species phosphorylate carboxyarabinotol to form carboxyarabinitol 1-phosphate which binds the rubisco active site • Rubiscoactivase also removes CA1P in the light • CA1P phosphatase then removes the PO4

19. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma

20. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH: rubisco is most active at pH > 8 (in dark pH is ~7.2)

21. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark

22. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark Mg2+ moves from thylakoid lumen to stroma to maintain charge neutrality

23. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+] c) CO2 is an allosteric activator of rubisco that only binds at high pH and high [Mg2+] also: stomates open in the light

24. Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma Several other Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also activated by high pH & [Mg2+]

25. Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark

26. SH SH light oxidized enzyme (inactive) S - S reduced thioredoxin 2Fdox 2e- PSI + PSII S - S SH SH oxidized thioredoxin reduced enzyme (active) 2Fdred Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme

27. Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme How light reactions talk to the Calvin cycle SH SH light oxidized enzyme (inactive) S - S reduced thioredoxin 2Fdox 2e- PSI + PSII S - S SH SH oxidized thioredoxin reduced enzyme (active) 2Fdred

28. PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

29. PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate Releases CO2 without making ATP or NADH

30. PHOTORESPIRATION Releases CO2 without making ATP or NADH Called photorespiration : undoes photosynthesis

31. PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon

32. PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon Both rxns occur at same active site

33. PHOTORESPIRATION C3 plants can lose 25%-50% of their fixed carbon phosphoglycolate is converted to glycolate : poison!

34. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes

35. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine produce H2O2

36. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria

37. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 Why photorespiration loses CO2

38. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome

39. Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome 6) peroxisome converts it to glycerate & returns it to chloroplast

40. Detoxifying Glycolate Why peroxisomes are next to cp and mito in C3 plants Mitochondrion

41. PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon Both rxns occur at same active site

42. Detoxifying Glycolate Why peroxisomes are next to cp and mito in C3 plants Mitochondrion

43. Detoxifying Glycolate • A recent study engineered 3 alternative pathways for detoxifying glycolate within the chloroplast • Idea is detox in chloroplast will improve CO2 recovery

44. Detoxifying Glycolate Glycolate detox from E. coli (a 5 step pathway) to form glycerate

45. Detoxifying Glycolate Glycolate detox from E. coli (a 5 step pathway) to form glycerate Arabidopsis glycolate oxidase, Pumpkin malate synthase, and E. coli catalase

46. Detoxifying Glycolate Glycolate detox from E. coli (a 5 step pathway) to form glycerate At glycolate oxidase, Pumpkin malate synthase, E. coli catalase Chlamydomonas glycolate dehydrogenase, Pumpkin malate synthase

47. Detoxifying Glycolate Glycolate detox from E. coli (a 5 step pathway) to form glycerate At glycolate oxidase, Pumpkin malate synthase, E. coli catalase Chlamydomonas glycolate dehydrogenase, Pumpkin malate synthase Also inhibited glycolate export

48. Detoxifying Glycolate 3 (Chlamydomonas glycolate dehydrogenase, Pumpkin malate synthase plus inhibiting glycolate export worked best

49. C4 and CAM photosynthesis Rubisco can use O2 as substrate instead of CO2 [CO2] is 1/600 [O2] _-> usually discriminate well

50. C4 and CAM photosynthesis Rubisco can use O2 as substrate instead of CO2 [CO2] is 1/600 [O2] Photorespiration increases with temperature