PHOTOMORPHOGENESIS AND PLANT DEVELOPMENT

PHOTOMORPHOGENESIS AND PLANT DEVELOPMENT

9.   PHOTOMORPHOGENESIS AND PLANT DEVELOPMENT

Photomorphogenesis is the regulation of plant development by light. Light acts as a signal for the photoreceptors that selectively absorb a different wavelength of light. The absorption of light by photoreceptor leads to a cascade of events that result in a developmental response.
9.1.    Photoreceptors are classified as follows:
•    Phytochromes that absorbs lights of wavelength 660 nm and 735 nm. i.e red and far-red light. These have a role in every stage of development from seed germination to flowering.
•    Cryptochromes and phototropin absorb both blue (400-450 nm) and UV-A light (320-400 m).
Major roles of cryptochromes are observed during seedling development, flowering and resetting the biological clock. Similarly, phototropin is responsible for phototropic responses, or differential growth in a light gradient.
One more class of photoreceptors must also be present that mediate response to low levels of UV-B(280-320 nm).
9.1.1.    Chromoprotein 
Chromoproteins consist of a light absorbing group or chromophore i.e. attached to a protein with catalytic properties called apoprotein. Chromophores and apoprotein forms holoprotein.
9.1.2.    Phytochromes
Phytochromes are the pigments ubiquitous in plants. It exists in two states- one with absorption maximum in red (665 nm) region and other with an absorption maximum in the far-red region (730 nm).
There are three distinct phytochromes: phy A, phy B, phy C encoded by PHY A, PHY B, and PHY C. In Arabidopsis five types of phytochromes are found (phy A-E). The phytochromes are different because of the differences in protein. The chromophore is common to all members. The major phytochromes are phy A and phy B responsible for red and far-red responses.
9.1.2.1.    Photoreversibility of Phytochromes
When Pr light absorbs red light, it is converted to Pfr form, which is the physiologically active form for most of the responses. This is known as photoreversibility. One of the experiments to demonstrate the photoreversible control of seed germination of Lettuce seeds was conducted. Lettuce seeds were allowed to imbibe water in darkness for 3 hours and then subjected to various brief light treatments. After irradiation, the seeds were returned to darkness for 48 hours, and germinated seeds were counted. The red light was found to promote the high rate of germination but red light followed by far-red light maintains the germination at the dark level. Thus, it was concluded that germination is dependent on a switch that is switched on by red light and switched off by far-red light.
Various experiments were conducted with dark-grown etiolated seedlings and were found that they accumulate a relatively large amount of phytochrome and Pfr is relatively unstable. The concentration of Pfr declines when the tissue is returned to darkness after Pr is converted to Pfr. This reduces the total amount of phytochrome.
The loss is demonstrated by conjugation of Pfr with ubiquitin by immunochemical studies. This indicates degradation by ubiquitin/26 S proteasome system. It was found that Pfr suppresses the transcription of the phytochrome gene by feedback inhibition.
9.1.2.2.    Photoequilibrium between PR and PFR
It is found that Pfr absorbs some light at 660 nm and Pr absorbs a small amount at 735 nm (far red). Thus, at 660 nm i.e. when ‘pure’ red light is given, it is not possible to convert 100% of pigment to Pfr.
Thus, a dynamic equilibrium is established between Pr and Pfr. This equilibrium can be expressed by Pfr/PTOT where PTOT is the sum of Pr and Pfr.
In etiolated tissues, photoequilibrium established by red light is 0.8 and by far-red light 0.03. that means red light maintains about 80% Pfr and 20% Pr. While far-red light maintains 3% Pfr.
In natural conditions, sunlight contains red and far-red wavelength and produce an equilibrium mixture of the two wavelengths depending on the time of day and environmental conditions. In most cases, the proportion of Pfr determines the biological response. The proportion of Pfr depends on:
1.    The relative proportion of red and far-red wavelength in the light source. 
2.    The forward and reverse rates of photoconversion between Pr and Pfr.
3.    The rate of thermal reversion of Pfr to Pr.
9.2    Phytochrome mediated responses:
9.2.1.    Responses mediated by phytochromes are grouped into 3 categories :
1.    LFRs (Low Fluence Responses) – These are classical red, far-red responses discovered by Hendricks and Borthwick. LFRs are stimulated by light doses in the range of 1 micro m-2 to 1000 micro m-2. LFRs are FR reversible.
2.    VLFR (Very Low Fluence Responses)- These are the responses stimulated by light levels in the range 10-6 to 10-3 micromol-3. These low levels convert only 0.01% of the phytochrome. VLFR are not photoreversible.
3.    Plants in natural environments receive sunlight at relatively high fluence rates with high energy over long periods of time. The maximum expression of photomorphogenic responses is achieved and responses such as leaf expansion and stem elongation are observed. These responses are known as high irradiance reactions (HIRs).
9.2.2.    High irradiance responses depend on 
1.    A high proportion of far-red light along with the prolonged exposure of high irradiance fluence rate and duration.
2.    Fluence rate and duration.
The action spectra of VLF response is found to be similar to the absorption spectra of phytochrome. However, HIR may possess different action spectra depending on species or growth conditions. For example, etiolated seedlings, respond to blue, red and far-red light. As de-etiolation progresses, there is a shift from far red sensitive HIR to a red sensitive HIR.
9.2.3.    Cryptochrome
Various plant responses such as the inhibition of hypocotyls elongation and stimulation of cotyledon expansion, the opening and closure of stomata, are found to be regulated by blue and UV-A region of the spectrum. Action spectra for blue light responses closely resembled absorption spectra of flavin molecules, such as riboflavin.
Later, the blue light photoreceptor was isolated from Arabidopsis in 1993. Because of its ‘cryptic’ nature and blue light responses in cryptograms, or non-flowering plants, the pigment was referred to as cryptochrome.
The first cryptochrome to be isolated was crying 1. It was observed that cry 1 binds two flavin adenine dinucleotide (FAD). It was found to be responsible for elongation of hypocotyls in Arabidopsis. Another cryptochrome cry 2 is found to mediate blue light suppression of hypocotyls elongation, cotyledon expansion, Anthocyanin production in Arabidopsis.
9.2.4.    Developmental responses mediated by phytochrome and cryptochrome
9.2.4.1.    De-etiolation
It is a process, exhibited by dark-grown seedlings i.e. etiolated seedlings when exposed to light. This process is regulated by phytochromes and cryptochromes. Hypocotyls growth is arrested, a hook of plumule is straightened and epicotyl elongation is accelerated.
9.2.4.2.    Seed germination
Phytochrome regulates seed germination in positively photoblastic seeds (stimulate to germinate by light) as well as negatively photoblastic seeds (inhibited by light). Also, in seeds like lettuce that requires only brief exposure to light and in Lythrum salicara that require constant or intermittent light is regulated by light.
Other examples that regulate suppression of germination in negatively photoblastic seeds are Avena fatua that requires long term exposure at high fluence rates.
9.2.4.3.    Shade Avoidance
Plants that grow under the shade of neighbours or under a canopy adjust to the availability of light by either increasing the specific leaf area or adjusting the morphology to position their leaves avoiding the shade. Plants thus respond to shade light with increased elongation of a stem, a more upward orientation of leaves (hyponasty), reduced branching and reduced tillering. Thus, shading is responsible for early flowering and seed set so that it can escape shading by shortening generation time. Red and blue light can largely be absorbed by chlorophyll while far-red light is passed through chlorophyll.
Detection of these differences between the composition of shade-light and unfiltered daylight is regulated by phytochrome and cryptochrome. The effect of canopy shading is thus described as the ratio of red to far-red fluence rates (R/FR) i.e. expressed by ζ (zeta).
9.2.4.4.    Detection of dusk and dawn signals
A decrease in ζ is observed, when seen sits low on the horizon. An example of the response of pumpkin (Cucurbita pepo) reveals that the reduced proportion of phytochrome maintained as Pfr at the end of photoperiod changes its developmental pattern drastically. Attenuation of stem and petiole extension, leaf expansion and branching is reduced and chlorophyll content is lowered.
9.2.4.5.    Electrical properties of tissues
Some phytochrome-mediated responses are rapid such as bioelectric potential or ion flux. An experiment to demonstrate these responses was conducted by T. Tanada. He observed that dark grown barley root tips would float freely in a glass beaker with a specially prepared negatively charged surface. When brief irradiation was provided root tips adhered to the surface and subsequent far-red treatment would release root tips from the glass. These effects were found to be correlated to phytochrome induced changes in transmembrane potential. It is still not known whether these effects are due to the action of phytochrome directly or because of secondary messenger system.
9.2.4.6.    Anthocyanin biosynthesis
Biosynthesis of anthocyanin is a high irradiance response. Similarly, initiation of accumulation of anthocyanin is a classic phytochrome-dependent LFR. The action peak for continued anthocyanin accumulation shifts to far-red light. Thus indicating the requirement for maintaining a low level of Pfr over time.
9.2.4.7.    Detection of the presence of adequate light
Phytochrome accumulates in a large amount in some situations like in the seeds that require red light to germinate but do not germinate when buried deep in the soil and also germinated seedlings where phytochrome detect light as the seedling approaches the soil surface. 
Once the seed or seedling is exposed to adequate light, the excessive quantity of phytochrome disappears and allows more stable phy B to monitor R-FR ratio over time and thus direct the development.
9.3.    Mode of action of phytochrome and cryptochrome
9.3.1.    Chemical structure
Chromophore of phytochrome is called phytochromobilin as it is a linear tetrapyrrole structure similar to mammalian bile pigments. Pr chromophore and Pfr chromophore can be differentiated by a rotation of a double bond between two rings (Cis-trans isomerization). Phytochrome absorb red light that provides energy for rotation around double bond by overcoming the activation energy. When it absorbs FR light it returns back to the stable Pr configuration. 
Apoprotein is a small protein with molecular mass 125 kDa. Its N-terminal domain is globular and photosensory while the C-terminal domain or regulatory domain is extended and contains a subdomain that resembles a two-component system i.e histidine kinase-related domain.
9.3.2.    Signal transduction of phytochromes
Phytochromes regulate development of plants by phosphorylating different substrates and initiates different kinase cascades.
It was found that phytochrome is a cytosolic protein but when it was tagged by fusing with GFP (green fluorescent protein), it was found that Pr accumulates in the cytoplasm but is converted to Pfr and its nuclear localization sequence is unmasked and is recognized by nuclear import machinery.
Phytochromes arrive inside the nucleus and form aggregates called nuclear bodies or speckles.
Phytochromes interact with Phytochrome Interacting Factors (PIF) that are negative regulators and repress transcription by binding with the promoter of photoresponsive genes. (In dark phytochrome accumulates in the cytoplasm as inactive Pr. After irradiation it is converted to Pfr and is imported to the nucleus) .
In nucleus Pfr targets transcriptional regulators for degradation thus activates transcription of phytochrome-responsive genes.
Pfr recruits promoter-bound PIF for degradation by 26 S proteasome system. This relieves the repressor genes.
9.3.3.    Cryptochrome structure
Cryptochromes are 70-80 kDa. It has two domains. N-terminal domain is known as photolyase-related (PHR) domain because it resembles microbial photolyase (flavoenzyme that utilizes blue light and catalyzes UV-induced damage to microbial DNA. This PHR domain binds two chromophores, one is flavin adenine dinucleotide (FAD), second is 5,10-methyltetrahydrofolate (MTHF). Functions of the C-terminal domain are not clear.
9.3.3.1.    Signal transduction of Cryptochrome
Cryptochromes are located in the nucleus.
Cry 1 is located in the cytoplasm in the light but moves into the nucleus in the dark. While Cry 2 permanently resides in the nucleus.
In the nucleus, cryptochrome interacts either COP 1 (CONSTITUTIVELY PHOTOMORPHOGENETIC 1) i.e. an E3 ubiquitin ligase (COP 1 represses transcription factors).
When irradiated with blue light, cryptochrome undergoes a conformational change deactivating COP1.
Transcription factors essential for proper development accumulates.


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