Light quality as a developmental cue in plants

Plants respond to light

(i) Quantity (expressed in terms of fluence rate photon dose mmol sec^-1 m²)

(ii)

Quality (wavelengths)

(iii) Direction – phototropism.

Light quality changes during the day and if plants are shaded.

Light quality variation in due the increased atmospheric “thickness” at dawn and sunset tending to allow only longer light wavelengths to penetrate to the earth surface.

Hence, at ~noon Blue light dominates whilst either earlier or later Red/Far-Red wavelengths predominate i.e. light quality can act as a clock.

Day length varies over the year and this can also be detected using light quality detection systems, i.e. plants have a calendar.

Hence plants have evolved the ability to detect .......

(i) UV and Blue light – cryptochromes and phototropins

(ii) Red light – phytochromes

Blue light detection

Cryptochromes

Blue (390–500 nm) and ultraviolet-A (UV-A; 320–390 nm) light elicit a variety of physiological responses in plants. Of these,

Most maximize photosynthetic potential in weak light and prevent damage to the photosynthetic apparatus in excess light.

· Phototropism — bending towards the light — is one of the best known plant tropic responses,

· Light-induced opening of stomata (cooling of leaf)

· chloroplast migration in response to changes in light intensity (protection)

· solar tracking by leaves of certain plant species

Action spectrum typically observed for phototropin-mediated responses. Notice the presence of a major peak at 450 nm, a shoulder at 425 nm and a minor peak at 470 nm in the blue region of the spectrum. This fine structure is not observed in the broad absorption band at 365 nm in the ultraviolet region of the spectrum.

Despite being reported by Darwin and others, over a century ago to be specifically under the control of blue light, the photoreceptors have only just become known.

Blue-light photoreceptor from Arabidopsis, are named CRY1 and CRY2 (cyptochrome 1 and 2).

This photoreceptor is a flavoprotein that mediates numerous blue-light-dependent responses.

Arabidopsis, mutant plants lacking both the CRY1 and the CRY2 blue-light photoreceptors are deficient in the phototropic response.

Transgenic Arabidopsis plants overexpressing CRY1 or CRY2 show enhanced phototropic curvature.

Phototropins

Phototropins 1 (phot1) and 2 (phot2) - the most recently characterized blue-light receptors in plants, have spectral properties.

Both phot1 and phot2 mediate not only phototropism, after which they were named, but also blue-light-induced chloroplast migration and blue-light-induced stomatal opening In addition, the rapid inhibition of stem growth by blue light is probably mediated by phot1.

phot1 also plays a role in blue-light-mediated calcium uptake and might have a minor role in blue-light-induced membrane depolarization.

Arabidopsis mutants with an impaired phototropic response (designated nph for non-phototropic hypocotyl) led to the cloning and characterization of the first phototropin gene.

Four loci identified (NPH1–NPH4)

NPH1 protein is a classic serine/threonine kinase.

The N-terminal half of the protein contains two repeated domains of ~100 amino acids with ~40% amino acid sequence identity. These domains are regulated by light, oxygen or voltage and are given the acronym LOV. LOV domains constitute a subset of the PAS-domain superfamily, which is known to mediate both ligand binding and protein–protein interactions.

Crucially, the LOV domains are flavine mononucleotide (FMN) binding domains

Protein structures of the Arabidopsis blue-light receptors, phot1 and phot2 (996 and 915 amino acids, respectively). Light, oxygen or voltage (LOV) domains are shown in green. The kinase domains, which catalyse the phosphorylation of proteins on specific amino acid residues (threonine and serine in this case), are shown in red.

When irradiating isolated LOV domains with blue light, they undergo a complex spectral change. This They fail to absorb in the blue region of the spectrum though a new peak appears near 390 nm. These light-induced absorbance changes result in the formation of isosbestic points at 330 nm, 375 nm and 410 nm ( see below, arrows). The observed light-induced spectral changes are fully reversible in darkness.

These changes result from the formation of an adduct between a cysteine residue and the C(4a) carbon of the flavin.

Mutation of a highly conserved cysteine in LOV1 and LOV2 to alanine or serine completely abolishes their photochemical reactivity.

It is likely that blue-light mediated excitation of FMN to form FMNH₂ is a key step in the formation of this cysteine adduct.

Phytochromes

First discovered in the 1950s when it was found that a brief pulse with red-light

(i) Initiated seed germination in dark

(ii) De-etiolation

(iii) Inhibition of leaf elongation

(iv) Regulation of flowering

In fact - the red/ far red ratio (R:FR 660/730nm) is the vital factor in phytochrome mediated events.

Found to exist in two forms “Pr and Pfr” with differing spectral properties.

Note that Pr has a peak absorbance at 660nm whilst Pfr absorbs maximally at 730nm.

It was found that these represented interchangeable forms of the same protein.

The Pr is the inactive form which is converted to the Pfr form bt RED (660nm) light. The Pfr form is active but is converted to the Pfr form by far-red light (730nm).

Evidence – Consecutive 5min treatments

Five phytochromes have been characterised in the model plant Arabidopsis thaliana.

Two classes /types of phytochrome

Type I (Gene PhyA)

· Accumulates to high levels in etiolated seedlings

· Unstable in light – falling to 1 to 2% original levels in the light.

· Degraded using the ubiquitin – ligase pathway

Type II (Genes PhyB, C, D and E).

· Accumulated to low concentrations in green leaves

· Stable in light

How much light? Variable phytochrome responses.

Types of phytochrome response –

1. Very low fluence (VLF).

· Used in soil surface detection

· 0.001mmol threshold

· Not photoreversible

· PhyA mediated

2. Low fluence (LF).

· Used in seed germination, shade avoidance and stem elongation.

· 1mmol threshold

· Photoreversible

· PhyB (Type II)- mediated.

3. High irradiance (HIR).

· Used in Photoperiod detection

· 100 mmol threshold

· Not photoreversible

· Both phyB and phyA are involved.

Different modes of photoperception by phyto-chrome A (PhyA) and phytochrome B (PhyB). Phytochrome partners are represented by X, Y and Z, which are pathway-specific or shared in the pathways. Abbreviations: B, blue light; FR, far-red light; FR-HIR, far-red-light-mediated high irradiance response; LFR, low fluence response; PhyA_fr, PhyA in far-red-light-absorbing form; PhyA_r, PhyA in red-light-absorbing form; PhyB_fr, PhyB in far-red-light-absorbing form; PhyB_r, PhyB in red-light-absorbing form; R, red light; VLFR, very low fluence response; UV, ultraviolet light.

Phytochrome induced gene expression.

Microarray analysis has found

Photosynthetic genes encoding chloroplast proteins e.g.

Rubisco (Ribulose 1,5-bisphosphate

carboxylase – small subunit)

Developmental genes e.g. chalcone synthase – which is

involved in anthocyanin synthesis

(amongst others).

Glycolysis and TCA cycle

Suppresses

Cell wall –loosening enzymes

Water – channel forming proteins (aquaporins).

Molecular properties of phytochrome.

Phytochrome is an apoprotein + bilitriene chromophore → hytochromobilin.

The apoprotein is a homodimer (120→127kDa)

Phy A- E share 50%/80% identity

Key features

The chromophore – phytochromobin (PfB) is attached in an invariant cysteine. This

is identical in each phytochrome – ie only the apoprotein changes.

The spectral shift comes about due to the geometric isomerisation –

C15 double bond between pyrrole rings C and D.

- of a linear tetrapyrrole ring within the chromophore.

Note : PhyA remains active in far-red light – even though shows the same isomeration.

Thus the PhyA Pfr form is active!

Chromophore binding region (CBD)

PAS domains – A B exhibit homology to similar domains in mammalian systems. Allow intra-molecular interactions within the phytochrome molecule.

HERD1/2 – histidine kinase related domains BUT actually appears to be a serine threonine kinase.

Different phytochromes (PhyA and B) phosphorylate different substrates.

Downstream signaling from phytochrome.

Evidence form biochemical analyses.

Cytoplasmic signaling

(i) The switch to Pfr triggers a heterotimeric GTP binding protein

cGMP lead to the production of “anthocyanins” and

Calcium initiates chloroplast development.

However, what proteins calmodulin and cGMP interact with is still obscure.

The elucidatory role of Arabidopsis mutants

Some terminology –

Photomorphogenesis – basically light induced developmental changes but usually refers to de-etiolation.

Skotomorphogenesis – dark associated development – etiolation.

(i) Screen to for light grown plants which look like etiolated plants.

These isolated chromophore biosynthesis genes –

hy1, hy 2, cry1 and cry2.

As well as all five photoreceptors - phyA→E which are mutations in the apoprotein.

But NOTE -NO DOWNSTREAM MUTANTS

IN RED-LIGHT.

However, PhyA far-red mutants have been isolated (see below). Demonstrates that this form is active.

(ii) Screen for dark grown plants which look like light grown plants-

Aiming to isolate elements in a “suppressive complex”.

COP series → “Constitutive photomorphogenesis”

DET De-etiolation mutants

FUS Fusca

Comprise the COP9 signalosome (CSN) with eight sub-units.

The CSN proteins bind to Hy5 – a transcription factor involved in activating light dependent gene expression.

A recent model has suggested the CSN acts by targeting Hy5 for degradation to the 26S proteosome.

This is localised to the nucleus in the dark. In the light

Hy5 is “released”, the CSN complex is translocated to the cytoplasm - probably by interaction with the cytoplasm - and eventually degraded.

COP9 shows some similarity to the lid complex of the 26S proteosome.

Downsteam signalling 1 : Phytochrome as a kinase

Phytochrome – an atypical – light regulated- serine protein kinase.

Phosphorylates and autophosphorylates.

Substrates

(i) Phytochromes proteins .

PhyA has a higher autophosphorylation activity in the Pfr form.

(ii) CPR5 – translocation to nucleus

(iii) G-box BF – translocation

(iv) Nucleotide diphosphate kinase 2 (NDPK2). NDP →NTP.

(v) PKS1 –

Nucleotide diphosphate kinase 2 (ndpk2)

Nucleotide diphosphate kinase(NDPK) is a multifunctional protein.

The primary role of NDPK is synthesising (d) NTP fropm (d) NDP.

Many biological phenomena are associated with NDPK, however these cannot be explained by NDP kinase activity.

In Drosphila , the mutation in the ndpk gene (awd) results in abnormal wing development.

In humans, ndpk has been identified as a tumour suppressor, nm23.

Recently, a protein kinase function has been detected. NDPK can phosphorylate both serine/threonine and histidine/asparate residues.

Other functions, e.g. activating G-proteins, have also been suggested.

PKS1

PKS1 – phytochrome kinase substrate – 439aa

Identified via two hybrid screens using the PhyA C-terminus as a “bait”.

PKS1 seems to bind both Pr and Pfr forms.

But it is phosphorylated in a light dependent manner.

Anti-sense did not yield any phenotype – are other genes compensating? – functional redundancy?

However, if PKS1 was over-expressed – lines had elongated hypocotyls -

PKS1 is a PhyA(?)-mediated negative regulator of PhyB. PKS1-GFP fusions remain in the cytoplasm so PKS1 may act by inhibiting PhyB action in the cytoplasm or prevent translocation to the nucleus.

Downstream signaling 2 : Change in Cellular localisation

(i) Transcription factor mobilisation.

Some transcription factors appear to be sequested

in the cytoplasm and migrate to the nucleus in response to red-light.

· G-box binding (GACGTx) transcription factors which have been found to bind light- regulated promoters.

· CPRF2 – common promoter-binding transcription factor family.

This process appears to involve transcription factor phosphorylation – presumably by phytochrome serine threonine kinases.

Some phytochrome interactions with AUX/IAA proteins. These are induced by auxin – hormones which modulate plant development.

(ii) Mobilisation of phytochrome itself

In the Pr form, phytochrome proteins are found in ordered arrays along the plasma membrane.

PhyB-GFP showed nuclear localization in red-light which reversed in Far-red.

PhyA-GFP also exhibited red induced nuclear targeting but this could not be reversed by cFR but my pulsed FR.

Note – Differences in the number and size of

Phy A and B complexes.

Light-induced nuclear translocation of phyA. A phyA GFP fusion in transgenic seedlings is uniformly distributed throughout the cytoplasm (and therefore invisible!) in darkness. A five-minute pulse of red light induced a rapid translocation of phyA-GFP into the nucleus.

Mutation of the phy A signalling pathway.

A simplified genetic model for the phyA-mediated signalling pathway. The genes encoding all these components excpet FIN2 andFHY4 have been cloned.

A series of mutants have been isolated which show less sensitivity to continuous far-red light.

Of particular note

LAF1 (long after far-red light) myb-type transcription factors

LAF6 nuclear-localised ATP binding cassette involved in communications between plastids and the nucleus.

Suppressors.

Mutants show enhanced phyA-specific responses

SPA1 “suppressor of phyA”

EID1

Both are involved in targeting proteins to the 26S proteosome.

Nuclear Events: transcriptional activation

PIF3

PIF3 is a nuclear basic helix-loop-helix (H-L-H) transcription factors which binds as a dimer.

The binding site was determined and found to be a G-box motif.

Binds to DNA in the absence of phytohrome proteins

But will interacts with the PAS domain of PhyA and B only upon conversion to the Pfr form.

This interaction was also photo-reversible.

It has been proposed that PIF3 in conjunction with phytochrome will active light responsive gene expression.

PIF3 will not activate all light- responsive genes with G-boxes.

Anti-sense PIF3 only suppressed

CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)

LATE ELONGATED HYPOCOTYLS (LHY)

Both myb-like transcription factors are involved in regulating the circadian rhythms.

The hy5 mutant – (involved in detiolation) did not display any change in CCA1 and LHY

Thus, PIF3 may be detecting very low (night- phyA) and low (day –phyB) fluence reactions – PHOTOPERIOD.

A prediction of this model is that phyA and phyB can act antagonistically.

PhyA over-expression – (therefore present in daylight) suppresses day-length perception.

A model for phytochrome signal transduction built around selected cloned intermediates. The activation of light-regulated genes is mediated by a complex of Arabidopsis nuclear phytochrome B (phyB) in the phytochrome far-red form (phyfr) and phytochrome interacting factor3 (PIF3). Phytochrome A and phyB might operate by similar mechanisms. The light-regulated genes are under additional control by phyA-specific (FAR1, SPA1) or general regulators (HY5, COP1 and other COP/DET/FUS proteins). COP1 and HY5 are known to interact ^. Whether PIF3 operates via genes that are independent of COP/DET/FUS and HY5 proteins is not known. Unbroken arrows denote physical movement or chemical modification. Broken arrows indicate genetic interactions. Elements of the model without experimental support are depicted in gray.

Can be developed into a model for day-length detection.

SUMMARY

A simplified model of phyA-regulated transcriptional network.

It is proposed that a master set of rapidly responding transcription-factor genes (HY5–RPT2 here, with ^* representing those genes with a G-box motif in their promoter) are primary targets of phyA signalling through constitutively present transcriptional regulators (PIF3 and other potential factors that are indicated by question marks) that are direct recipients of incoming phyA signals. This master set of genes is proposed to encode transcriptional regulators that control one or more main branches of the transcriptional network that drives the various facets of photomorphogenesis, such as cell-wall expansion and chloroplast biogenesis.

Dashed arrows indicate potential regulation of G-box-containing genes by

· CAB, CHLOROPHYLL A/B-BINDING PROTEIN;

· CCA1, CIRCADIAN CLOCK-ASSOCIATED PROTEIN 1;

· CHS, CHALCONE SYNTHASE;

· CO, CONSTANS;

· DOF, H-PROTEIN PROMOTER-BINDING FACTOR-2A;

· GDCH, H-PROTEIN SUBUNIT OF GLYCINE DECARBOXYLASE;

· FT, FLOWERING LOCUS T;

· HY5, LONG HYPOCOTYL 5;

· LHY, LATE ELONGATED HYPOCOTYL;

· PIF3, PHYTOCHROME-INTERACTING FACTOR 3;

· RBCS, RIBULOSE BIPHOSPHATE CARBOXYLASE SMALL SUBUNIT;

· RPT2, ROOT PHOTOTROPISM 2;

· TOC1-L, TIMING OF CAB 1 EXPRESSION-LIKE