"Ultra Vires" C#4

Light is an important environmental factor which controls growth and development in plants. Besides photosynthesis in which light is harvested by green plants and is converted- into chemical energy, there are numerous other plant responses to light such as phototropism, ger­mination of some light sensitive seeds e.g. lettuce, de-etiolation of monocot and dicot seed­lings etc., which are quite independent of photosynthesis and in which light just acts as envi­ronmental signal to bring about the particular photo-response. Most of these photo-responses control genetically defined structural development or morphogenesis (i.e., origin of form) of plants. The role of light in regulating morphogenesis is known as photo-morphogenesis. In plants, red and blue light are especially effective in inducing a photo-morphogenetic response. The effect of light in controlling morphogenesis can best be demonstrated by comparing a monocot (maize) or dicot (bean) seedling grown in light with one grown in darkness both of which have been reared from genetically identical seeds. Abundant reserve food in seeds elimi­nate the need for photosynthesis for many days. It can easily be noticed that dark grown seedling has become etiolated (i.e., pale and weak) while the one grown in light has stockier and green appearance with short stem and large leaf area (fig1. Since both etiolated and light grown seedlings were reared from genetically identical seeds, light must have altered the gene expression during germination so that the appearance or form of etiolated and light grown seedlings looks different. De-etiolation of light grown seedling can be done in very short period (hours) by placing it even in dim light. During de-etiolation, marked reduction in the rate of stem elongation, straightening of apical hook and development of green pigments can easily be noticed. The etiolated form of the seedling is thus gradually transformed to stockier green appearance and is the result of photo-morphogenesis. The development of seedling in darkness is called as skoto-morphogenesis (from Greek word Skotos = darkness). According to Hans Mohr (1983), there are two important stages of photo-morphogenesis: (i) Pattern specification, in which cells and tissues develop specific ability or competence to respond to light during certain developmental stage and (ii) Pattern realization, during which time the photo-response occurs. There are two main categories of plant responses to light signals: (i) Phytochrome medi­ated photoresponses and (ii) Blue-light responses or cryptochrome mediated photo-responses. (A) Phytochrome Mediated Photoresponses in Plants: Large number of photo-morphogenic responses in plants are known to be mediated by the proteinaceons pigment (chromoprotein) called phytochrome. This pigment acts as photorecep­tor and absorbs most strongly red and far-red light. It also absorbs blue light. The pigment phytochrome exists in two forms, (i) a red light absorbing form designated as PR form and (ii) another far-red light absorbing form designated as PFR form. These two forms are photochemically interconvertible. When PR form absorbs red light (650 – 680 nm), it is converted to PFR form. The PFR form absorbs far-red light (710 – 740 nm) and is converted to PR form. The PFR form of this pigment is believed to be physiologically active form. Absorption spectra of PR and PFR forms of phytochrome purified from etiolated Avena seedlings are given in Fig. 25.2. PR form shows a peak at 666 nm while PFR form at 730 nm. It is noteworthy that both forms of phytochrome also absorb in the blue region of spectrum of light. (The absorption maxima obtained in vitro mostly correspond with those in vivo provided that the native phytochrome is carefully purified and non-degraded). However, it is quite obvious from this figure that the absorption maxima of PR and PFR forms overlap considerably in the red region of the spec­trum of visible light and therefore, the phyto­chrome system cannot be quantitatively converted from PR to PFR. After irradiation with red light (or white light), there is an equilibrium between PR and PFR forms that depends upon the spectral composition of the light source. This equilibrium is called as photo-stationary equilibrium (ф) and is defined as ratio of the PFR conc. and the total phytochrome conc. (ρtotal) at a given wavelength. These values can be measured by difference spec­troscopy. ф = PFR/ PR+ PFR = PFR/Ptotal Most of the phytochrome mediated photoresponses in plants are reversible. These are induced by red light and reversed by far red light. A list of some of the photoreversible responses mediated by phytochrome in plants is given in table 25.1. Photoreversible responses mediated by phytochrome in plants One of the classical examples of photo-morphogenesis in plants induced by short red-far- red pulses is the germination of light sensitive seeds of lettuce (Lactuca sativa). In early 1930s, Flint and McAlister (1937) demonstrated that germination of lettuce seeds is not only stimu­lated by white light but also by red light (shorter than 700 nm) and inhibited by far-red light (greater than 700 nm). In 1950s, Borthwick and Hendricks and their associates obtained spectacular results by exposing lettuce seeds to alternating red and far-red treatments. They observed much higher per­centage of germination when the seeds received red light as the final treatment. Seed germination was markedly inhibited when seeds received final treatment with far-red light. (Table 25.2) Effect of alternating red/far Borthwick and his associates also predicted existence of the photoreceptor phytochrome in two different forms which was proved to be absolutely correct later on when this pigment was isolated in plant extracts for the first time by Butler et al in 1959 and its photo-reversibility was confirmed in vitro. Based on the amount of light required or the fluence (no. of photons absorbed per unit sur­face area), the phytochrome mediated photo-responses can be grouped into three main categories: (a) Very Low Fluence Responses (VLFRs): These responses are initiated by very low fluences (0.1 to 1 n mol m-2) saturating at 50 n mol m-2 and are non-photo reversible. For example, brief flash of red light with fluence as low as 0.1 n mol m-2 can stimulate the growth of coleoptile and inhibit growth of mesocotyl in oat seedlings that have been grown in dark. Similarly, red light with fluence of only 1-100 n mol m-2 is enough to stimulate seed germination in Arabidopsis. (In monocots, the elongated area of axis between coleoptile and root is called as mesocotyl) (b) Low Fluence Responses (LFRs): These responses require fluence of at least 1.0 nmol m-2 saturating at 1000 n mol m-2 and are photo-reversible. Most of the red/far-red photo-responses including the lettuce seed germi­nation belong to this category. (c) High Irradiance Responses (HIRs): These responses require continuous or prolonged exposure to light of relatively high irradiance saturating at much higher fluences (at least 100 times more) than LFRs and are non- photo-reversible. Examples are: (i) Anthocyanin synthesis in dicot seedlings and in apple skin, (ii) Ethylene production in sorghum, (iii) Induction of flowering in Hyoscyamus (a long day plant), (iv) Opening of plumular hook in lettuce, (v) Enlargement of cotyledons in mustard, (vi) Inhibition of hypocotyl elongation in many dicot seedlings etc. (B) Blue Light Responses or Cryptochrome Mediated Photoresponses: Apart from phytochrome mediated photo-responses, large number of photo-responses in plants are known which are controlled by blue light and are believed to be mediated through a group of yet unidentified pigments called crypto chrome (crypto from cryptogams), the latter acting as photoreceptor in such responses. Blue light responses have been reported in algae, fungi, ferns and higher plants. Some of the typical and most commonly known blue-light responses in plants are: (i) Phototropism (ii) Stomatal opening (iii) Inhibition of hypocotyl elongation (iv) Sun tracking by leaves (v) Phototaxis (vi) Movements of chloroplasts within the cells and (vii) Stimulation of synthesis of carotenoids and chlorophylls etc. Crypto chrome absorbs light rays mostly in violet-blue region of the spectrum (400 – 500 nm). It also absorbs long wave ultraviolet rays in UV-A region (320 to 400 nm). However, most photo-responses of plants caused by crypto chrome result from absorption in violet-blue region of the spectrum but they are simply called as blue-light responses Although phytochrome and some other photoreceptors also absorb blue light, but the typi­cal blue-light morphogenetic responses differ from photo-responses mediated by them in being insensitive to red light and there is no red/far- red reversibility. i. The action spectra of many blue-light re­sponses in higher plants such as phototropism, stomatal movement, inhibition of hypocotyl elon­gation etc. are similar and characteristic. They show three peaks in blue region (400 – 500 nm) of the spectrum of visible light. This three peaked, action spectrum is also known as three fingers action spectrum (because of its resemblance in shape with three fingers) and is typical of most blue light responses (Fig. 25.3). Three fingers action spectrum is not observed in phytochrome mediated photo-responses or photo-responses mediated by other photoreceptors other than crypto-chrome. Typical three peaked ii. Scientists have implicated roles of yellow pigment carotenoids or flavins as photoreceptors in blue-light responses of plants for a long time. However, the spectroscopy of blue-light responses is complex and it is not easy to distinguish between these two types of pigments by comparing available action and absorption spectra. Action spectrum for phototropism Fig. 25.4. Shows relationships between action spectrum for phototropism and absorption spectra of riboflavin and β-carotene. The strong peak in UV-region of the spectrum (360-380 nm) suggests riboflavin as the photoreceptor pigment, while three peaks in blue regions (400 – 500 nm) of the spectrum favours carotene. Nevertheless, accumulating evidences strongly favour flavin pigment to be the primary photoreceptor in phototropism. Schmidt (1984) has summarised arguments in favour of flavins or carotenoids as photoreceptor pigments in blue light responses of plants as follows: (a) Arguments in Favour of Flavins: (i) Action spectra show UV maximum between 350-400 nm. (ii) Primary steps of the blue light response are dependent on presence of O2. (iii) Flavin reactions are often redox reactions. (iv) Light can be substituted by oxidants while reductants suppress the blue light reaction. (v) Blue light reaction is inhibited by flavin inhibitors such as KI. (vi) Blue light action spectra resemble low temp, spectra of flavins. (vii) Neurospora mutant which is free of carotenoids shows blue light response. (viii) Half life of carotenoids in first excited singlet state is very short (10-13 seconds) (b) Arguments in Favour of Carotenoids: (i) Three peaked (three fingers) action spectra resemble absorption spectra of carotenoids. (ii) Small or no UV maximum in some action spectra. (iii) Energy transfer from UV absorbing pigment to carotenoids is feasible. (iv) Carotenoids from diatom mutant do not show blue light response. Earlier evidences suggested crypto chrome to be one or both of the yellow pigments, carotenoids (such as β-carotene, zeaxanthin) and/or flavins (such as riboflavin, FAD) which medi­ate blue-light responses in plants. However, with extensive researches done with mutants and transgenic plants and over expression studies beginning in early 1990s, the vexed problem of identification of blue-light receptors in plants has gradually been resolved now. The term crypto chrome is now applied specifically to flavoprotein photoreceptor that mediates inhibition of hypocotyl (stem) elongation caused by blue-light. Blue-light photoreceptor in phototropism and chloroplasts movements in plants is phototropin which is also a flavoprotein. The carotenoid zeaxanthin is blue-light photoreceptor involved in stomatal opening. Photoreceptors: A brief account of all these photoreceptors follows: 1. Crypto-chrome: The first protein with characteristics of blue-light receptor was isolated in 1993 from Arabidopsis. It was found that hy4 mutant of Arabidopsis had lost the capacity to respond specifically to blue-light in that it showed an elongated hypocotyl even on irradiation with blue-light (In the wild type, blue-light causes inhibition of hypocotyl elongation). Isolation of the hy4 gene (later named as cryl) showed that it encoded a 75 kDa protein called crypto-chrome 1 (CRY1) with remarkable sequence similarity (homology) to DNA photolyase in having two chromophores: a flavin adenine dinucleotide (FAD) and a pterin attached to the apoprotein (Fig. 25.5). This led to the establishment of cryptochrome to be a flavoprotein that was involved in inhibition of hypocotyl elongation in response to blue-light. (The structure of pterin is given in figure 25.6. For structure of FAD). Diagrammatic representation of the chromophere binding domains (DNA photolyase is a blue-light activated flavoenzyme which repairs UV-induced damage to mi­crobial DNA. Cryptochrome differs from photolyase mainly in two respects. Firstly, the cryptochrome does not show photolyase activity and secondly, unlike photolyase it has an extended carboxy-terminal domain (Fig. 25.5) with kinase activity). A second cryptochrome 2 (CRY2) also with two chro­mophores like CRY1, has also been isolated from Arabidopsis (Lin 2000). CRY2, mediates blue-light stimulated inhibition of hypocotyl elongation, increase in cotyledon expansion and anthocyanin production. It also has a role in determining flow­ering time. Both CRY1 and CRY2 appear to be ubiquitous in plant kingdom, but while CRY1 is stable in light grown seedling, CRY2 is rapidly degraded in light. Structure of pterin Mechanism of action of cryptochrome: The mechanism of action of crypto chrome remains elusive so far. The flavins are known to participate in oxidation-reduction reactions and photolyases repair damaged DNA (as a result of UV-radiations) by transferring electrons to pyrimidine dimers. Crypto chromes may act probably in a similar way through some electron transfer mechanism. 2. Phototropins: Phototropins are blue-light receptors that mediate phototropism and chloroplasts movements in plants. In late 1980s, it was found that blue-light stimulated phosphorylation of a 120 kDa pro­tein located on plasmamembrane of actively growing regions of etiolated seedlings. These regions were also most responsive to phototropic stimulus. Extensive biochemical and physiological studies showed this protein to be a kinase autophosphorylating in blue-light and which could be the photoreceptor for phototropism. Later on, a mutant nph1 (won phototropic hypocotyl 1) was isolated from Arabidopsis which lacked phototropic response in the hypocotyl and also the 120 kDa membrane protein. It was genetically independent of the hy4 mutant as it showed blue-light induced inhibition of hypocotyl elongation. The nph1 gene was cloned and it was found (as postulated) to encode a 120 kDa pro­tein nph1. The nph1 gene was renamed as phot1 and the protein encoded by it was named phototropin (Briggs and Christie, 2002). Phototropin is also a flavoprotein with two flavin mononucleotide (FMN) chromophores. The protein has a carboxy-terminal domain with a serine/threonine kinase activity. In the amino-terminal half, there are two domains called LOV domains (of about 100 amino acids each) to which are attached the chromophores (Fig. 25.5). (LOV domains are so called because they are characteristics of microbial proteins which regulate response to light, oxygen and voltage). Recent spectroscopic studies done by Swartz et al, 2000) have shown that in dark, FMN mol­ecules remain non covalently bound to LOV domains, but on irradiations with blue-light they become covalently bound to cysteine residues of the apoprotein through a sulphur atom forming a cysteine- flavin covalent adduct. The reaction is reversed in dark. A second gene called phot 2 has also been isolated from Arabidopsis which is related to phot 1. It is believed that phototropic response involves both phot 1 and phot 2. Mechanism of action of phototropins: The mechanism of action of phototropins is not clear. It has been observed that blue-light causes a transient increase in cytosolic calcium concentra­tion and there are indications that phototropin signalling chain may partly involve regulation of cytoplasmic calcium concentration. 3. Zeaxanthin: The carotenoid zeaxanthin has been shown to be blue-light receptor in guard cells that plays central role in blue-light stimulated stomatal opening. (See chapter 17 for structure of zeaxanthin). Following evidences strongly support role of zeaxanthin in stomatal opening: (i) The absorption spectrum of zeaxanthin closely resembles the action spectrum of blue- light stimulated stomatal opening. (ii) During stomatal opening in intact leaves, the incident radiation, zeaxanthin concentration in guard cell, and stomatal apertures have been found to be directly correlated. (iii) Blue-light sensitivity of guard cells increases with an increased concentration of zeaxanthin in guard cells. (iv) There is complete inhibition of blue-light stimulates stomatal opening by 3mM conc. of dithiothreitol (DTT) which is a potent inhibitor of the enzyme that converts violaxanthin to zeaxanthin. (v) In facultative CAM plant species such as MeSembryanthemum crystallinum, there is a shift from C3 to CAM mode of carbon metabolism in response to accumulation of salts. In C3 mode, the guard cells accumulate zeaxanthin and exhibit blue-light response. But, in CAM mode, neither there is accumulation of zeaxanthin in guard cells nor they respond to blue- light. (In CAM plants, stomata remain closed during the day). Mechanism of action of zeaxanthin: It is believed that the excitation of zeaxanthin by blue- light in guard cells starts a signal transduction pathway that includes: (i) Isomerization of zeaxanthin, (ii) Conformational changes in the apoprotein (iii) Transmission of blue-light signal across the chloroplast membrane by a secondary messenger (most probably Ca++, phosphatases, calcium binding protein calmodulin and inosi­tol triphosphate (IP3), (iv) Activation of H+-ATPases at the guard cell plasma membrane resulting in pumping of protons across the membrane and intake of K+ ions followed by Cl– ions. (v) Turgor build up in guard cell and stomatal opening. The blue-light stimulated stomatal opening can be reversed by green light. This may happen if green light is applied with blue-light in continuous light treatment or if a blue-light pulse (of about 30 seconds duration) is followed by a green light pulse. A second blue-light pulse after green-light can restore the stomatal opening. It has been suggested by various workers that green light reverses the isomerization of zeaxanthin resulting in regeneration of inactive zeaxanthin isomer. The latter is unable to mediate the blue-light response. (Besides phytochrome and cryptochrome, there are two other categories of photoreceptors which are known to affect photomorphogenesis in plants. They are, (i) protochlorophyllide-a, a pigment which absorbs red and blue light and is converted to chlorophyll-a and (ii) UV-B photoreceptor – one or more unidentified compounds which absorb short wave ultra­violet rays in UV-B-region (280-320 nm).