Advanced plant Physiology Dprof. Dr. Samih Tamimi

1. Advanced plant Physiology Dprof. Dr. Samih Tamimi

2. Biol 751 Phytochrome and Photomorphogenesis

6. Bean (Phaseolus vulgaris) seedlings grown under different light conditions for 6 days. Five minutes of dim red light per day is sufficient to prevent some of the symptoms of etiolation that appear under conditions of total darkness, such as reduced leaf size and maintenance of the apical hook. (Photo courtesy of H. Smith.)

8. Comparison of dark-grown (etiolated) and light-grown (de-etiolated) seedlings

9. Concept 1: Photomorphogenesis • Power of light: a developmental switch from dark to light growth • Light response: decrease stem elongation, beginning of apical-hook straightening, the initiation of the synthesis of pigments • …Photomorphogenesis • Light acts as a developmental trigger rather than a direct energy source • …chlorophyll is not present this time

10. Concept 2: Photoreversibility • Phytochrome:a protein pigment that absorbs red and far-red light most strongly. • Photoreversibility:the effects of red light (650-650nm) on morphogenesis could be reversed by a subsequent irradiation with light of longer wavelength (710-740nm).

11. Lettuce seed germination

16. Phytochrome response modes- photoreversibility

17. Concept 3: The Absorption Spectra of the Pfr and Pr Forms Overlap Blue light: 400-500nm • Photostationary state: the equilibrium between Pfr and Pr. • Both forms of phytochrome absorb light in the blue region of the spectrum.

18. Classical LFR reaction LFR=Low fluence red/far red response Phytochrome is synthesized as Pr i.e. seeds and seedlings grown in total darkness contain only this isomer. Responses promoted by a few minutes of dim light are prevented by subsequent brief exposure to dim far red light.

20. Phytochrome consists of chromophore and apoprotein • Pigment (chromophore).  • blue-green • open chain tetrapyrolle; called phytochromobilin • made in the plastids. • Protein (apoprotein). • glycoprotein • soluble • dimer (MW 240,000 D = 240 kD); each of the two peptides are identical with a MW ca. 124,000 D and comprised of ca. 1128 amino acids.   • gene(s) have been cloned and the amino acid sequence is known; large proportion of hydrophobic amino acids; suggests phytochrome is associated with membranes. • tetrapyrolle is covalently-bonded to the protein via a thioether linkage involving a cysteine. 

22. Phytochromes • 120 kDa protein family • TKDs transmitter kinase domains • HKLD prokaryotic histidine kinase like domain (Quail 2002)

24. Concept 5: Phytochrome is a Dimer Composed of Two Polypeptides • A dimer of two equivalent subunits. • Each subunit consists of two component: (a) Chromophore, a light-absorbing pigment molecule, and (b) Apoprotein, a polypeptide chain. • Assembly of the phytochrome apoprotein with its chromophore is autocatalytic.

25. 3. The Photochemical and BiochemicalProperties of Phytochrome • Phytochrome Can Interconvert between Pr and Pfr Forms

26. Red light Pr Pfr Far-red light Photoreversibility Red light: 650-680nm Far-red light: 710-740nm • In dark-grown plants, phytochrome is present in a red light-absorbing form, Pr. • Pr is converted by red light to a far-red-light-absorbing form, Pfr. • Pfr can be converted back to Pr by far-red light. • Pfr is the physiological active form of phytochrome.

28. Synthesized in plastids

29. Synthesized in plastids

30. Structure of the Pr and Pfr Forms of Phytochrome

31. Phytochrome = Blue Protein-Pigment(125 kDa) Pr = Red Light-Absorbing Form Pfr = Far-Red Light-Absorbing Form Phytochrome = PhotoReversible Blue Form (Pr) absorbs Red Light Blue Green Form (Pfr) absorbs Far Red Light  Blue Form (Pr) absorbs Red Light Blue Green Form (Pfr) absorbs Far Red Light  P660_____ P695_____P710_____P730 P730________P650______P660

32. Both the Chromophore and the Protein UndergoConformational Changes • Since the chromophore is what absorbs the light, conformational changes in the protein are initiated by changes in the chromophore. • Upon absorption of light, the Pr chromophore undergoes a cis–transisomerization by rotation around the double bond between carbons 15 and 16. • This change results in a more extended conformation of the tetrapyrrole.

33. Both the Chromophore and the Protein UndergoConformational Changes • During the conversion of Pr to Pfr, the protein moiety of phytochrome (the apoprotein) also undergoes a subtle conformational change. • Pr and Pfr differ in their susceptibilities to proteases and in their phosphorylation by exogenous protein kinases.

34. 2) Pfr is the Physiologically Active Form of Phytochrome • In general, the magnitude of the physiological response to red light is proportional to the amount of Pfr produced. • In some cases the magnitude of the response is proportional to the ration of Pfr to pr, or of Pfr to Ptot. • Phytochrome deficient (hy) Arabidopsis mutants have long hypocotyls in both darkness and white light. If the red light response were due to a lack of Pr, we would expect the opposite to be true, i.e. the hypocotyls would be short in both darkness and white light.

35. Note: • The absorbance spectra of Pr and Pfr overlap significantly in the red region of the spectrum, and the Pr form of phytochrome absorbs a small amount of light in the far-red region • As a consequence, a dynamic equilibrium exists between the two forms. The proportion of phytochrome in the Pfr form after saturating irradiation by red light is only about 85%. Similarly, an equilibrium of 97% Pr and 3% Pfr is achieved after saturating irradiation by far-red light . This equilibrium is termed the photostationary state.

36. Concept 7: Two types of phytochrome • Type I: • more abundant than type II in dark • PHYA • mRNA is unstable • PfrA protein undergoes degradation • Type II: • less abundant than type I in dark • PHYB, PHYC, PHYD, PHYE • mRNA stable • Pfr protein stable • PHY: the apoprotein by itself (without the chromophore) • phy: the holoprotein (with the chromophore)

37. 4. Two Types of Phytochrome Have Been identified Type I a) About 9X more abundant in dark-grown tissues. b) The Pfr form is rapidly degraded. c) The Pfr form feed-back inhibits its own synthesis. Type II a) Present in equal amounts with Type I phytochrome in light-grown tissues. b) The Pfr form is not degraded. c) Synthesis of Type II phytochrome is not feed-back inhibited by Pfr.

38. Phytochrome is Encoded By a Multigene Family • Arabidopsis has five structurally related phytochrome genes: PHYA, PHYB, PHYC, PHYD, and PHYE. • PHYA is the only Type I phytochrome • PHYB - PHYE are all Type II phytochromes

40. Distribution of phytochrome

41. Concept 6: Phytochrome is an Autophosphorylation Protein Kinase

42. Phytochrome functional domains Confers photosensory specificity to the molecule, i.e. whether it responds to continuous red or far-red light Transmits signal to proteins that act downstream of phytochrome

43. Phytochrome is an autophosphorylating serine/threonine kinase Phytochrome may phosphorylate other proteins (X)

44. Gibberellins and seed germination • Phytochromes mediate the effects of gibberellins on germination by influencing gibberellin biosynthesis and sensitivity. • Red light induces and far red light represses transcription of the GA4 and GA4H genes of Arabidopsis.

45. Interaction of phytochromes and gibberellin

47. The reduced R:FR ratio of light acts as asignal that other plants are nearby

49. What happens when PHYA/B move into nucleus? PIF = Phytochrome Interacting Factor PIC = pre-initiation complex MYB = transcription factor LHCB = Light-harvesting chlorophyll a/b-binding protein