L4: Regulation of mitochondrial and chloroplast biogenesis

How do both mitochondria and plastids develop

• By division of pre-existing organelles

• i.e→ there is not de- novo synthesis

But theri functional state is affected by their

1. Developmental stage

2. Environmental conditions

Example of Environmental conditions affecting this

1. Oxygen

2. Light

Affect on mitochondrial with the absence of oxygen

• mitochondria electron tranfer cannot occur as O2 is the final electron acceptor

  • Catalysed by cytochrome oxidase (complex IV)

    • COX

Yeast show how mitochondria development can be alted by O2 levels

• Anaerobic conditions (or on glucose)→ use fermentaion and mitochondria are small with few internal membranes and no respiratory complexes→ PROMITOCHONDRIA

• Aerobic conditions→ Mitochondria are functional→ fully develop when O2 is supplied and/or all glucose consumed

Why does O2 have this affect on mitochondria?

• Because O2 is reuired by haem biosynthesis

• and haem is a cofactor for cytochromes

• therefore→ when there is oxygen→ mitochondria can develop fully

• note: Haem is produced in the mitochondria

Not only is there regulation of haem (found in the mitochondria) but Oxygen regulates the nucleur encodes genes for mitochondria→ e.g CYC1 what is it and how regulated

• Nuclear encodde CYC encoding cytochrome c

• Regulated by → HAP1 (haem-activator protein 1)

How does it set up a kinda of feedback cycle, affect by oxygen

1. Oxygen→ makes Haem

2. Haem binds to HAP1

3. HAP1 activates Upstream Activating Sequences (UASs) in the CYC promoter

4. This promotes CYC expression and the making of cytochrome c WHICH Haem binds to for mitochondrial function

i.e: Overall need oxygen for haem and then the haem can be used for its actual function as well as transcibed the protein that it needs for its function (cytochrome C)

How does oxygen regulate genes that are not directly require Heam for regulation?

e.g COXIV, V, VI

1. haem stimulates HAP1

2. then the HAP1 activates HAP2/3/4/5 complex

3. which then goes to promote the COXIV, V, VI

4. makes cytochrome c oxidase/subunit IV

i.e→ does not bind directly to haem but still responds to it via the levels of HAP1 activation of gene for HAP4

How does light change the function of chloroplasts

Flowering plants

• Dark→ do not synthesise chlorophyll in the dark→ no chloroplasts→ etioplasts

• Light→ make chlorophyll→ chloroplasts

Note: some land plants and algae in the dark…

• have an enzyme for dark chlorophyll synthesis

• dark-operative protochlorophyllide oxioreductase (DPOR)

• So are green in the dark

i.e→ still shows how chloroplast function can be altered by the environment

Chloroplast development from dark to light

• Etioplast→ Crystalline structure

  • Prolamellar body

  • para crystalline array

  • all the components needed for chloroplast but just not in correct formation

• Chlorolpast→ thylakoid membrane developed over 1-2 days

Even though there is no need for grene expression of phosotynethesis components in non plastids such as amyloplasts…

• genes are transcibed in all tissues

• there is no large differences in relative rates of transciption of individual genes in different tissues

• and

• no evidence for repressor or activator proteins for most individual genes

Then what are transcription levels dependent on?

• Levels of RNA polymerase

• The level of transcription of individual genes depends on promoter strength

But if transcription levels in amyloplasts and chloropasts are the same, why don’t amyloplasts contain photosynthetic machinery?

Chloroplast genes are regulated post-transciptionally:

• I.e even through there is transcription of all genes in all plastid types

• Level of transcription depends on level of RNA polymerase (leaf>>>root)

• The level of individual transcripts depends on ‘promoter strength’

Extensive translational regulation→ what does this require

• Positive regulation of translation:

  • i.e in the DARK→ there are transcripts but not translated

  • in the light→ the they get translated

• There are many ways for this to happen

Example: Redox-regulated translation-initiation of psbA→ What is it

• Forms the D subunit of the PSII core

• Light→ translated

• Dark→ not translated

• As an mRNA transcript→ the psbA has a stem-loop structure in the 5’ UTR

  • Chloroplast polyadenylate-binding protein (cPABP) specifically binds this and is essential for translation

  • and a protein disulfided isomerase (cPDI) modulates the binding of this by chaning redoc status

Example: Redox-regulated translation-initiation of psbA→ How is its translation regulated: in the light

1. Light

2. PSI redox reactions cause FD reduced

3. This reduced FDTR

4. this Reduced protein disulfide isomerase (cPDI)

   • through redox potential or adenosine 5’-diphosphate-dependent phosphorylation

5. this allows the chloroplast polyadenylate-binding protein cPABP to bind to the 5’UTR step loop

6. binding to stem loop turns translation ON

7. PsbA translation is ON

(in the dark)

1. The cPDI is oxidised

2. so no cPABP binding

3. not attached to stem loop

4. translation off

How we know that there is electron transfer/ redox reactions happening

• Expression is inhibited by:

• DCMU [N-(3,4- dichlorophenyl)-N-dimethylurea] (aka Diuron)

  • which inhibits electron transfer

Therefore what does this mechanism allows for

• a simple reversible switch regulating gene expression in the chloroplast

• translation of psbA is therefore linked to photosynthetic activty

• overall: Shows how the environmental cue can change the function of chloroplast

Light can also alter chloroplast function/plastid type by regulating what

• nuclear genes

• instead of the chloroplast genes

→ e.g the transciption of LHC controlled by light

What is the light-harvesting complex

(antenna complex or LHC)

• an array of protein and chlorophyll molecules

• embedded in the thylakoid membrane of plants and cyanobacteria

• transfer energy to one chlorophyll a molecules at the reaction centre of a photosystem

• The transcription of LHC is controlled by light

  • note: this is not post-transciptionally like above!

• its genes are encoded in the nucleus

• and so light must regulate its transcription

What aids this transciption regulation?

• phytochrome signalling

What are phytochromes

• Photoreceptors autocatalytically bound to a bilin prosthetic group

• they are made downstream of haem

  • note: in the dark when there is no chlorophyll made, haem feedback inhibits ALA synthesis

How do they act as photoreceptors

With light, changes the position of ring D

• red light→ activation→ Pfr cis isomerisation

• far red→ Pr→ inactivation→ transisolerisation?

How does overall Light regulate LHC expression

1. Activation by red light

2. Pfr conformation

3. can translocate fro the cytosol to the nucleus

4. interact with the repressive transciption factors (PIFs) that bind the promoter of the genes including LHC

5. Allows LHC transciption

overall: phytochoromes de-repress gene-expression

Coordintion of nuclear and organelle gene expression: complexes in oragnelles contain protein complexes that are encoed by

• Both the organelles and the nucleus

• e.g mitochondrial→ cytochrome c oxidase/subunit IV complex

• e.g Chloroplast→ cytochrome b6f complex

Therefore: it is important that the ratio of nuclear to chloroplast proteins is correct and coordinated correctly

Picture of distribution of nuclear vs chloroplast encoded genes

Problem with achieving this ratio

• There are different copy numbers of organelles and nuclear genes

• very large im blanace

• Example:

  • Mitochondria→ Yeast 50 mit DNA molecules : 1 haploid genome (50:1)

  • Human HeLa cells 8800 mit DNA molecules : 1 diploid genome (8800:1)

Why is the nuclear and mitochondrial expression genes not tightly coordinated in yeast?

• synthesis of nuclear-encoded subunits can occur in absence of mitochondrial gene

• e.g in petite p- mutatns (rho)

Example: Chloroplast protein Rubisco→ how is it encoded

• Ribulose 1,5-bisphosphate carboxylase

• L8S8 structure (large and small subunits)

  • Large subunit (LSU) (52 kDa; rbcL chloroplast gene: >5000 copies per leaf cell

  • Small subunit (SSU) (14 kDa); RbcS nuclear genes: 6-10 nuclear genes per cell

• but→ see that in the northern blot→ the levels of LSU to SSU are similar

  • therefore→ there must be some kind of regulation of the translation

Evidence that the expression of protein subunits is not tightly coordinated

1. Protein synthesis inhibitors in vivo

2. Antisense RNA inhibition of S unit synthesis in transgenic plants

i.e→ there is coordination but it is not tight!

1. Protein synthesis inhibitors in vivo

• cyclohexamide→ inhibits synthesis of SSUs (nucleus)

  • but rbcL transcription and LSU synthesis continues for some time

• Chloramphenicol→ inhibits synthesis of LSUs (plastid)

  • but RbcS transcription and SSU synthesis continues for a short time

  • HOWEVER→ SSU des not accumulate and is degraded in the chloroplast

so there is no affect on the translation BUT there is still some degredation which might be involved in this regulation?

2. Antisense RNA inhibition of S subunit synthesis in transgenic plants

• Tobacco plant produced containing antisense constrcut of RbcS cDNA

  • expressed from a consitutive promoter

• result→ degredation of the mRNA and the decreased SSU synthesis

• i.e it induced post-transciptional gene silencing

Summary of the Rubisco subunit reguation

• the amount of rubsico correlates with the amount of SSU:

• Reduction in SSU→ reduced accumulation os LSU, even if rbcL transcipt level is not affected

• What does this suggest

  • Assembly of Rubisco is driven by the availability of SSU and excess LSU subunits are degraded

How has this coordination been found to be achieved by

• post-transciptional process→ ‘Control of Epistasy by synthesis’ CES:

  • LSU that is not complexed into Rubisco yet self- regulates by binding rbcL mRNA

i.e: In the absence of the SUU→ there is a repressor which causes the degredation of the LUU

This repressor system accounts for fluctuations in gene expression

3. Nuclear factors for organelle biogenesis: Anterograde signalling→ nuclear genes are essential for organelle genetic machinery

• The include components of

  • RNA polymerase

  • Chloroplast sigma factors

  • Ribosomes

• They are synthesised with pre-sequences and imported via TIM/TOM or TIC/TOC

• THEREFORE: the nucleus does

• BUT they do not encode transcription factors for specific organelle genes

• then how is post-transcitional regulation of mitochondrial genes done by the nucleus?

Major point of organelle gene expression control by the nucleus

• Nucleus encoded proteins are also requred for the stability and translation of specific mRNAs in both mitochondria and chloroplasts

• including editing

• often, multilple nuclear genes are needed for a single organelle protein to be produced

Example 1 → nucleuar factors regulate the splicing of mitochondrial cox genes

• removal of intron 5b from COX1 requires 3 helicases

• Northern blot shows the relative sizes of RNA moieties of COX

• splice variances

• different splices due to different nuclear mutations

• therefore: nucleus has a role in ost-transiptional expression in the organelle

• Found that:

  • in the nucleus? there are three helicase proteins for the removal of intron 5b in COX1

Example 2→ nuclear factors regulate the translation of mitochondrial cox genes

Procedure:

• screen of yeast nuclear respiratory deficient mutants

result:

• >20 additional genes identified that are trans-acting factors needed for COX assembly

Two types of trans-acting factors

1. Enzymes for haem biosynthesis and Cu-homeostasis and insertion into complex

2. proteins need for expression of mitochondrial COX genes:

   • stability, splicing and translation

   • PPRs

What are PPR proteins

• bind and stabilise and help with splicing and translation

Example: petite mutant pet309

• Pet is a PPR protein

results

1. Impaired in translation of Cox I mRNA

2. Complementation→ restored function

3. But→ complentation wtih proteins lacking the Huam nPet309→ neurodegenerative Leigh syndrome

   • due to inability to assemble COX

Therefore: show the need for nuclear regulation and the proteins it makes to regulate the organelle proteins

Nuclear factors regulate the stability and translation of chloroplast transcipts

• mutant libraries of Chlamydomonas reinhardtii

• Two independent mutants impaired in phototrophic growth:

  1. Abnormal steady-state fluoresence levels

  2. acetate-requiring phenotype

• Result→ identifed loss of cytochrom f (PetA)

What was found to be the cause of these mutations

• Insertions found in PPR proteins:

  • MCA1→ stabilised petA mRNA

  • TCA1→ Facilitates petA translation

Signalling from organelles to the nucleus→ retrograde signalling compared to anterograde

• Anterograde→ nucleus affecting organelle gene expression

• retrograde→ signals from organelle to nucleus

  • transciption of nuclear genes for chloroplast and mitochondrial proteins is dependent on the functional state of the organelles

why do we know there is retrograde signalling

• In the absence of functional chloropast or mitochondria

• nuclear genes for organelle components are not transcibed

  • (or transcribed at basal levels)

Examples of this

1. Yeast p- mutants→ no mitochondria and so reduced expression of TCA cycle and respiratory complex genes

2. Barley mutants→ defects chloroplast ribosomes

   • → reduced RbcS and LHC expression

In what ways can organelles communicate to the nucleus

1. Positive regulators→ from functional organelles

2. Repress nuclear gene expression→ Dysfunctional organelles

Example 1: of investigating nuclear transcription regulation by chloroplasts: way to cause chloroplast damage

Inhibitors of chloroplast transcription and translation

• e.g Tagetitoxin→ inhibits chloroplast RNA polymerase

• e.g Linomycin and chloramphenicol→ chloroplast protein synthesis inhibitors

Results:

• prevent nuclear gene expression early in seedling development

Example 2: of investigating nuclear transcription regulation by chloroplasts→ way to cause chloroplast damage

Photooxidation of chloroplasts

• in carotenoid-deficient mutant plants or in plants treated with norflurazon

  • inhibitor of carotenoid biosynthesis

Results:

• Absence of carotenoids→ photobleaching of chlorophyll and destruction of normal chloroplast function

In these two examples→ the treatments lead to what

• reduced levels of transcipts derived from so-called photosyntheis-associated nuclear genes

  • PhANGs

  • Such as those for light-harvesting chlorophyll protein (Lhc) or Rubisco small subunit→ RbcS

Is the genes encoding products for mitochondrial and cytosolic componenets affected?

no

How was the actual retrograde signal found

• mutant libraries

• identified → ‘gun’ mutants

  • gun= genomes uncoupled mutants

• These contiued to express PhANGs even thorugh plastid development has been blocked by the treatments

• What did this identfify:

  • these genes are involved in tetrapyrrol biosynthesis→ which include haem

i.e: found mutatns that allowed genes to be expressed even thorugh the chloroplast was dyring

→ note: hard to work with→ have to work with seedlings coz can’t phoyosynthesise

so these mutants mean the nucleus encoded the chloroplast genes anyway, even though there was no retrograde signal from the chloroplast coz they were dying

What go gun mutants do

• encode proteins that affect tetrapyrole biosynthesis

examples:

1. GUN2-GUN5 + GUN6(OE)→ enzymes and regulators of tetrapyrrole biosynthesis→ essential for production of heme, chlorophyll and others

2. GUN1→ interacts with proteins involved in tetrapyrrole biosynthesis→ directly binds tetrapyrroles including heme

Summary

1. O2 supply impacts mitochondrial biogenesis

2. Light impacts chlorophyll biogenesis

3. • Functional organelles are required for eeicient nuclear gene expression

4. Anterograde signals: nuclear-encoded genes are required for organelle function, including PPR proteins for RNA stability and translation

5. Retrograde signals: - mitochondria: heme produced in mitochondria required for expression of nuclear-encoded cytochrome c - chloroplasts: tetrapyrrole biosynthesis (possibly heme) required for expression of photosynthesis-associated nuclear genes (PhANGs)

6. Signals identified by perturbing the expression of: - nuclear genes (e.g., antisense, overexpression, mutant libraries) - organelle genes (e.g., mitochondrial mutations; treating plants with norflurozon/lincomycin)

7. Organelle inheritance - coadaptation of nuclear and organelle genomes

8. Defects in mitochondria - linked with many diseases, and ageing, in humans - cytoplasmic male sterility in plants