Effect of light quality on chlorophyll accumulation and protein expression in wheat (Triticum aestivum L.) seedlings.
Plants sense many aspects of light in their environment including its wavelength, duration, intensity, and direction. Plants tend to adapt the structure of photosynthetic apparatus and pigment composition to light quality and quantity (Buschmann et al., 1978). Different fluences of light regulate growth and development of plants. Seedling growth is a photomorphogenic process that is most responsive to light. The temporal and spatial regulation of plant photomorphogenesis are regulated by light. Through photosynthesis light provides the energy source for plants and ultimately, for all living organisms. In response to fluctuating environment, the non motile plants must be able to sense varying light signals and to optimize growth and development. Higher plants possess sophisticated photosensory and signal transduction systems to monitor the direction, quantity and quality of the light signal and to adjust their growth and development through regulated gene expression at every stage of their life cycle as germination, seedling development, and flowering. These light regulated developmental processes are known as photomorphogenesis.
Chlorophyll (Chl) biosynthesis requires light and its biosynthetic intermediates are involved in the regulation of chloroplast biogenesis (Jilani et al., 1996; Hoober and Eggink, 1999; Eggink and Hoober, 2000). The coordination between chloroplast and nuclear genomes may be achieved by Chl biosynthetic intermediates and/or redox signaling events (Pfannschmidt et al., 2001; Strand et al., 2003). Light-dependent development of plants involve the combined action of several important photoreceptors including the red/far-red light-absorbing phytochromes (Quail, 1995), the blue/UV-A light absorbing cryptochromes (Ahmad and Cashmore, 1996), and distinct UV-A (Young et al., 1992) and UV-B (Beggs and Wellman, 1985; Christie and Jenkins, 1996) light photoreceptors.
These photoreceptors alone or in combination affect the overall growth and development of plants (Casal and Mazella, 1998; Hennig et al., 1999; Duke and Fankhauser, 2003; Montogomery and Lagarias, 2002). Phytochromes (PHY) are responsible for red-light-mediated responses and may interact with blue-light responsive cryptochromes (CRY) for certain photomorphogenic response such as, hypocotyls elongation and cotyledon expansion (Casal and Mazella, 1998; Henni et al., 1999). PhyA is necessary for CRY1/CRY2 to activate anion channels within the first few seconds of blue light and suppresses hypocotyl elongation for at least 120 min in Arabidopsis after a flash of red light (Folta and Spalding, 2001). A putative basic-helix-loop-helix transcription factor, HFR1, mediates both phytochrome and cryptochrome signaling in Arabidopsis (Duke and Fankhauser, 2003). A red light pretreatment enhances CRY1 mediated induction of chalcone synthase in Arabidopsis indicating coaction between CRY1 and PHYB (Wade et al., 2001). Also, different photoreceptors work under different fluences of light. PHYB responds to low fluences (Casal et al., 2003) whereas PHYA responds to very-low and high irradiance responses (Shinomura et al., 1996; Hamazato et al., 1997). Stimulation of hook opening of dicotyledonous seedlings by red light and low fluence blue light is far-red reversible and exhibits reciprocity, as is characteristic of many low fluence-dependent phytochrome-mediated responses. Far-red and high-fluence blue light appears to stimulate hook opening and cotyledon unfolding through high-irradiance-response systems during long-term light treatments (Liscum and Hangarter, 1993). Far-red light was shown to block greening in Arabidopsis. This loss in greening was accompanied by loss in expression of HEMA1 gene and was irreversible. This response of far-red light was shown to be mediated by phyA (McCormac and Terry, 2002). Plants are adapted to live in extremely different light conditions i.e., on forest floor, in deep ocean or in open fields of tropics. There are substantial differences in spectral distribution of light in these habitats. Plants tend to adapt the structure of photosynthetic apparatus and pigment composition to light quality and quantity. Light regulates Chl biosynthesis. Chl itself or Chl biosynthetic intermediates are involved in the regulation of chloroplast biogenesis. Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate. Exposure of wheat seedlings to red light (400 [micro]mol [m.sup.-2] [s.sup.-1]) resulted in yellowish white seedings (Tripathy and Brown, 1995).
In a preliminary study it was previously shown that exposure of roots of wheat to red light down-regulated the greening process. This near-etiolation was significantly reversed by far red light and blue light. The present investigation aims to study the role of light quality on plant growth and development using 2D-PAGE and western blot analysis.
Materials and methods
Wheat (Triticum aestivum cv HD 2329) seeds were used as experimental materials. The seeds were obtained from Indian Agricultural Research Institute, New Delhi.
Gallium-aluminum-arsenide light emitting diodes (LEDs), with high output in the red region of photosynthetic absorption and action spectra offer a tremendous technical advantage over conventional light sources for plant growth. The spectral output of red LEDs used in the present investigation have a peak wavelength at 670 nm and narrow bandwidth of 26 nm. This peak wavelength corresponds to the peak of photosynthetic action spectrum of plants (McCree, 1972). Blue LEDs had a peak wavelength at 475 nm having a bandwidth of 30 nm. LED arrays were obtained from Quantum devices, USA.
Plant Growth under Continuous Illumination
Wheat (Triticum aestivum cv HD 2329) seeds were grown in different light regimes, for six days. Seeds were germinated and grown under red light (400 [micro]moles [m.sup.-2] [s.sup.-1]) on germination paper with whole plant exposed (V-) or in vermiculite having their roots as well as root-shoot transition zones shielded from red light (V+). There was no increase in temperature in the plant growth area. Seeds were germinated and grown in white light (W) (400 [micro]mol [m.sup.-2] [s.sup.-2]), in dark (D), in red (400 [micro]moles [m.sup.-2] [s.sup.-1]) + far-red light (50 [micro]moles [m.sup.-2] [s.sup.-1]) (R+FR), far-red light alone (50 [micro]moles [m.sup.-2] [s.sup.-1]) (FR), and in red (400 [micro]moles [m.sup.-2] [s.sup.-1]) + blue (50 [micro]moles [m.sup.-2] [s.sup.-1]) (R+B) for 6 days.
Chlorophyll, Carotenoid and Protein Estimation
Chl and carotenoid contents were estimated as described previously (Porra et al., 1989; Wellburn and Lichanthaler, 1984). Protein was estimated according to Lowry et al., (1951) or Bradford method 1976.
Total protein isolation for two dimensional gel electrophoresis
Leaves were homogenized in liquid nitrogen and were grinded in mortar and pestle in extraction buffer consisting of 56 mM [Na.sub.2]C[O.sub.3], 5mM DTT, 10mM Isoascorbate, 12% Sucrose, 2mM EDTA, and 2% SDS. The samples were vortexed thoroughly. They were incubated at 80oC for 20 min and were centrifuged at 13000 rpm for 5 min, and supernatant was collected and protein estimation was done with Bradford's method.
Two dimensional PAGE
Sample preparation for 2D PAGE: Total protein samples after protein estimation were precipitated by using cold acetone containing 0.1% v/v 2-mercapto ethanol and was vortexed vigorously and left at -20[degrees]C for 1h. Then the samples were centrifuged at 16000 rpm for 30min at 4[degrees]C. Pellet was dried under reduced pressure and dissolved in 2D sample buffer containing 9.5M Urea, 4% NP-40, 5% (v/v) 2-mercaptoethanol, 2% v/v ampholytes of pH 5-8 and pH 3-10.
Iso electric focusing (IEF)
IEF was performed according to Biorad manual. The stock solution of acrylamide was prepared by mixing 30% acrylamide powder with 5.2% bis acrylamide, 10% NP-40, 10% CHAPS, 10% APS, 20mM NaOH as catholyte, 0.01[micro]M ortho phosphoric acid as anolyte, 8M urea and 250[micro]l of ampholyte as sample overlay. The gel solution was carefully mixed at 30[degrees]C and degassed for 10 min. After the run they were used for second dimensional run in 10% SDS-PAGE. After polymerization the IEF rods were carefully placed on stacking gel and molecular weight markers were loaded in the well. Gel was run either at constant voltage of 100 V or constant current of 20 mA. Silver staining was done after run.
SDS-PAGE (Jilani et al., 1996; Lamelli, 1970) was run to check the status of protein profile of second dimension after IEF and in broken plastids isolated from seedlings grown under different light regimes.
Western blot analysis of plastidic membrane proteins
Broken plastids were isolated as described previously (Tripathy and Mohanty, 1980). For isolating plastidic membranes pellet was suspended in 1 ml of TE buffer containing 0.01M Tris pH 7.5 and 1 mM EDTA. This was then kept on ice for 10-15 min and centrifuged at 1200g for 5 min. Pellet containing plastid membrane was suspended in suspension buffer containing 0.4 M sorbitol, 0.05 M tris pH 7.5, 1 mM Mg[Cl.sub.2] and 1 mM EDTA. An antibody for wheat protochlorophyllide oxidoreductase (POR) was gifted by Dr. W. T. Griffith. Antibodies for cytf of cyt bf complex were gifted from Dr. R. Malkin, USA (from Pea), antibody for Rubisco LSU and OEC33 (from Wheat) was obtained from Dr. G.S Singhal. After SDS-PAGE run, proteins were transferred to nitrocellulose membrane cut to the size of gel. Blots were stained for alkaline phosphatase using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Jilani et al., 1996; Lamelli, 1970; Towbin et al., 1979).
Plant growth under different light conditions
Wheat (Triticum aestivum cv. HD 2329) seedlings grown under red light (400 [micro]moles [m.sup.-2] [s.sup.-1]) (V+) in vermiculite were green and accumulated normal amounts of Chl and carotenoids (Fig.1). However seedlings germinated and grown on brown or white germination paper having their roots and shoots exposed to red light (400 [micro]moles [m.sup.-2] [s.sup.-1]) (V-) accumulated negligible amounts of Chl and carotenoids (Fig.1) and were yellowish white. Seedlings germinated and grown under white light (W) (400 [micro]moles [m.sup.-2] [s.sup.-1]) on germination paper were green and accumulated normal amounts of Chl and carotenoids (Fig.1).
[FIGURE 1 OMITTED]
Blue and far red light are required for photomorphogenesis. Red-light-induced inhibition of greening was found to be reversed by blue light (50 [micro]moles [m.sup.-2] [s.sup.-1]) when both were given simultaneously (400 [micro]moles [m.sup.-2] [s.sup.-1]) red + 50 ([micro]moles [m.sup.-2] [s.sup.-1]) blue (R+B) and seedlings accumulated normal amounts of Chl and carotenoid (Fig.1). When seedlings were grown under far red light alone (50 [micro]moles [m.sup.-2] [s.sup.-1]) (FR) and under red (400 [micro]moles [m.sup.-2] [s.sup.-1]) + far-red light (50 [micro]moles [m.sup.-2] [s.sup.-1]) (R+FR), were also green and accumulated Chl and carotenoids (Fig.1). However their Chl and carotenoid content was substantially reduced as compared to other green seedlings.
Total protein profile and two dimensional gel electrophoresis of seedlings grown under different light conditions
SDS-PAGE of total protein was done in seedlings grown under different light conditions as described before. SDS-PAGE revealed a 27kDa protein which was present in substantial amounts in V-, D, R+FR and FR seedlings. Its amount was substantially reduced in W, R+B and V+ -seedlings. A 25kDa protein band was abundantly present in V- -seedlings. It had reduced presence in etiolated, R+FR and FR seedlings. It was absent in W, R+B, V+ grown seedlings. Large subunit of Rubisco (53kDa) was absent in V- -seedlings (Fig. 2).
[FIGURE 2 OMITTED]
2-D gel electrophoresis of total proteins revealed that in dark grown seedlings, in acidic range (pH 3-5) several proteins having molecular weight of 81, 76, 71, 66, 41, 33, and 24kDa were present. These proteins were absent in white-light-grown green seedlings. In the basic range (pH 7-10) there were a few proteins having molecular weight of 51, 41, 39, and three proteins at 38kDa (having different PI's) were abundantly present (Fig.3A). These proteins were almost absent in white light grown seedlings (Fig.3B).
In white light grown seedlings, several new protein were expressed in the neutral range (pH 6.8-7.5) having molecular weights of 30and 32kDa. Of these most of the proteins were not clearly seen in dark grown seedlings. Large subunit of Rubisco (53kDa) and small subunit of Rubisco which appears as 2 bands (~14kDa) in the pH range of 7-7.6 were easily recognisable in white light grown seedlings (Fig. 3B).
Two dimensional gel analysis of total protein extracted from V- and V+ -seedlings showed the presence of many basic proteins in V--seedlings having molecular weights of 30, 28, 25, 22, 20, 18, 16, and 15kDa in the pH range of 6.5-8 (Fig.3C). Proteins of molecular weight 43 and 37kDa were present in V- -seedlings in the pH range of 3-5. These were not seen in V+ -seedlings. 24 and 27kDa proteins which were abundantly present in SDS-PAGE of V- -seedlings segregated in several protein bands in the 2D gels in the pH ranging from 6.8-8. These proteins were nearly absent in V+ -seedlings. Other high molecular weight proteins also which were present in green seedlings grown under different light regimes were also absent in V- -seedlings. LSU and SSU of Rubisco were present in V+ - seedlings and conspicuously absent in V- -seedlings (Fig.3D). Few protein of molecular weight 16 (pH-3-4), 24 (pH- 7-8), and 42 (pH-5-7) were present in V+ -seedlings and were absent in white light grown green seedlings (Fig.3B and Fig.3D). There were 26 and 28kDa proteins in the pH range of 3.5-5.0 in W -seedlings, these were absent in V+ -seedlings (Fig.3B).
These two dimensional gel analysis of seedlings grown under R+FR showed some similarities with white light grown seedlings i.e., the presence of large and small subunits of Rubisco. However R+FR showed the presence of many proteins in the acidic range (pH 3-5) having molecular weights of 60, 59, 45, 29, 28, 26, 24, 14, 11, and 8kDa, these were absent in W -seedlings. In the neutral range also R+FR showed the presence of proteins having molecular weights of 23, 22, 18, 17, and 15kDa (Fig.4A). All these were neither seen in W, nor in V+ -seedlings. These were farred light inducible as they were nearly absent from white light as well as red light grown green seedlings (V+). Seedlings grown under FR light alone showed the presence of many proteins of 60, 59, 29, 28, 17, 15, 14, 11, and 8kDa which were not present in any other green seedlings, but they were common with R+FR grown seedlings, showing that they were far-red inducible (Fig.4B). Protein bands of large and small subunit of Rubisco were easily identifiable in far-red alone grown green seedlings. There were few proteins of 26, 28, 32, and 38kDa which were present only in white light grown seedlings and were absent in seedlings grown under R+FR as well as FR seedlings (Fig.3B).
Two dimensional gel analysis of seedlings grown under R+B and white light grown seedlings showed the presence of many common protein bands, including large and small subunit of Rubisco, and many other proteins were present in both light conditions. But few bands of 45, 56, 57, 77, and 90 kDa in 3-4.5 pH range and of 21, 23, 25, 26, 39, 42kDa in 4-7 pH range were present in only R+B light grown seedlings showing that these are positively regulated by blue light (Fig. 4C).
The extra proteins present in V- -seedlings probably were not the same proteins which were present in dark grown seedlings though V- -seedlings also show near etiolation response. Those are red-light inducible proteins.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Polypeptide profile of plastidic proteins
Polypeptide profile of plastids isolated from seedlings grown under different light conditions was studied by SDS-PAGE. Protein profile of plastids isolated from excised leaves of seedlings grown in dark and different light regimes revealed that most of the high molecular weight proteins (<60kDa) and low molecular weight proteins (14-20 kDa) were present in V-, D, R+FR, FR, V+, R+B and W seedlings. In dark (D) samples LHCPII (25kDa), LHCPI (21-24kDa), and OEC-33 (33kDa) were absent. The POR (36kDa) was present in substantial amounts in plastids isolated from dark grown seedlings. It disappeared in V- -seedlings and was present in small amount in seedlings grown under different light regimes. In thylakoids membranes of green seedlings i.e., seedlings grown under R+FR, FR, V+, R+B, and W the prominent bands of LHCPII, LHCPI and OEC-33 were observed. V- -seedlings, lacked OEC33, LHCPII and LHCPI. Two additional bands of low molecular weight i.e., 16 and 18 KDa were prominently present in V- -seedlings (Fig. 5).
[FIGURE 5 OMITTED]
Western blot analysis of plastidic proteins
To study the protein expression western blot analysis of plastidic proteins was done in seedlings grown in dark or above mentioned light regimes.
Rubisco large subunit: The large subunit (53kDa) of Ribulose 1,5-bis phosphate carboxylase/oxygenase that functions in the dark reaction of photosynthesis is the most abundant protein. Its western blot analysis showed its presence in etiolated and light-grown green seedlings i.e., W, R+FR, FR, V+, R+B, it was downregulated in red light grown (V-) non-green seedlings (Fig. 6).
[FIGURE 6 OMITTED]
OEC33: OEC-33 is 33 kDa protein of oxygen evolving complex present in thylakoid lumen and is associated with photosystem-II. OEC33 was absent in dark grown seedlings and its expression increased in response to W, R+FR, FR, V+, R+B. It was absent in red light grown non -green seedlings (V-) (Fig. 6).
POR: This is protochlorophyllide oxido reductase, which is involved in the conversion of protochlorophyllide to chlorophyllide (Chlide). This is the only light requiring reaction in Chl biosynthesis. The POR is light labile. Western blot analysis showed that this was highly expressed in dark grown seedlings but with light exposure its expression declined. This is present in very low amounts in seedlings grown under W, R+FR, FR, V+, R+B. This protein was highly downregulated in red-light-grown non-green seedlings (Fig.6).
Cyt f: This protein is associated with cytochrome b6/f complex and is involved in the electron transfer through plastocyanin. Its expression was small in dark grown seedlings and increased in green seedlings grown under different light regimes as W, R+FR, FR, V+, R+B. In red-light-grown non-green V- -seedlings cyt-f was highly downregulated (Fig. 6).
These results show that light is differentially as well as specifically regulating plant protein expression.
As sessile organisms, higher plants are characterized by a high degree of developmental plasticity in response to environmental cues, thereby optimizing their development in a way that maximizes their chances of survival and reproduction. Light is an important environmental factor for plant growth and development (Kendrick and Kronenberg, 1994; Deng and Quail, 1999; Neff et al., 2000).
Wheat (Triticum aestivum cv. HD 2329) seedlings germinated and grown on brown/white germination paper having their roots and root-shoot-transition zones exposed to red light (400 [micro]moles [m.sup.-2] [s.sup.-1]) did not accumulate chl and carotenoid (Fig.1) and looked yellowish white. However, seedlings germinated and grown in vermiculite having their roots and root-shoot-transition zones shielded from red light were green and synthesized significant amounts of chl and carotenoids (Fig.1). Seedlings grown in white light either on germination paper or vermiculite accumulated chl (Fig.1) and were green.
Blue and far-red components of the white light spectrum are known to play a significant role in gene expression and photomorphogenesis (Senger et al., 1980; Reymond et al., 1992; Kaufman, 1993; Short et al., 1994). The-blue-light-supplemented seedlings were green and accumulated Chl (Fig.1) to the same extent as those grown under white light or with seedlings grown in vermiculite (V+). Supplementing the red-light with far-red light resulted in partial greening of V- seedlings. Seedlings grown in far-red alone also had lesser accumulation of chl (Fig.1). Since plants need light, why would light energy antagonize plant development? One need not search farther than the far-red portion of the electromagnetic spectrum to answer that question. Simple yet elegant experiments by Borthwick et al. (1952) illustrated that these far-red wavebands, inefficient for photosynthesis, impart potent environmental information. Generally, far-red light counters the developmental processes initiated by red light, and the ratio of red to farred dictates the activity of molecular, biochemical and morphological processes (Quail, 2002; Devlin et al., 2003; Chen et al., 2004; Casal and Yanovsky, 2005). This is a prime example of how a light quality almost useless to plant metabolism potently adjusts plant form, composition, and adaptive strategy to optimize light capture when quantities and/or qualities are unfavourable.
In molecular terms, red light induced the steady state expression of many proteins as revealed by 2D gel electrophoresis (Fig.3C). Etiolated seedlings expressed several proteins, which were not present in W as well as other light grown seedlings (Fig.3A). V- -seedlings showed the lack of most of the proteins and induction of 11 new proteins (Fig.3C). In V+ -seedlings three new proteins of 16, 24, and 42kDa appeared which were absent in green as well as V- -seedlings (Fig.3D). In R+FR- and FR seedlings, lots of new proteins appeared (Fig. 4A, 4B) of which a few of these were common in R+FR and FR, showing their induction by far-red light. R+FR seedlings also showed a few proteins which were not present in V+ or R+B seedlings. The 2D protein profile of R+B -seedlings showed a few proteins of 45, 56, 57, 77, and 90 kDa in 3-4.5 pH range and of 21, 23, 25, 26, 39, 42kDa in 4-7 pH range which were blue light-induced proteins, not present in seedlings grown in other light regimes (Fig.4C).
Previous studies have revealed probably more than 100 genes whose expression is controlled by light (Terzaghi and Cashmore, 1995; Fankhauser and Chory, 1997; Kuno and Furuya, 2000). However, the dramatic developmental transition during plant photomorphogenesis is likely to involve a much larger number of genes. Recent results suggest that plant photomorphogenesis involves a regulated change in the expression of up to 30% of the genes in the Arabidopsis genome (Ma et al., 2001), and this massive change in gene expression likely is the result of a transcriptional cascade (Tepperman et al., 2001). Therefore, the contrasting developmental patterns are mediated primarily by coordinated changes in light-regulated gene expression (Terzaghi and Cashmore, 1995; Puente et al., 1996; Ma et al., 2001; Tepperman et al., 2001). Furthermore, different light signals seem to be perceived by distinct photosensory systems and transduced by their signaling pathways to achieve the control of expression of a largely common fraction of the genome (Ma et al., 2001). These results clearly demonstrate that light quality either enhances or represses the expression of several proteins. In a previous study, it was found that at least 26 fundamental cellular processes or metabolic pathways were regulated by light. Some of these were activated by light, whereas others were repressed by light (Ma et al., 2001).
The light quality modulates plant development via expression of several proteins which may have adaptive role for plant growth and metabolism in a certain light environment. These altered gene and thus protein expression must be sustaining plants to grow in all kinds of environments. However, numerous genes with probable regulatory roles, such as protein kinases and phosphatases and cell skeleton proteins, also have been found to be regulated by light. These light-regulated proteins undoubtedly play important roles in various pathways.
Although morphologically both etiolated and V- -seedlings were similar, but their protein expression pattern were not identical. Induction of several new proteins in V-seedlings suggest that they may have been expressed in response to the near etiolation response induced by red light perceived by the plant. Further purification and characterization of these induced-proteins are required to assign the importance of these proteins.
SDS-PAGE and Western blot analysis of several plastidic proteins showed the down-regulation of LHCII, OEC33, D1, Rubisco large subunit, POR, and Cyt-f etc. in red light-grown non-green seedlings. Some of these proteins are the integral components of photosystem, cyt b/f complex or oxygen evolving complex and their absence suggested that their assembly to form a functional photosynthetic apparatus was affected in V- seedlings.
In conclusion light can modulate plant gene expression and thus protein expression very efficiently. The behavior of plants in light qualities is beautifully reviewed by Folta and Maruhnich, 2007 where they have shown that differential effect of different light qualities make sense in the context of normal plant growth in natural settings. In this way plants use the full spectrum and the relative ratios of energies within to adjust their form, composition, and physiology to best exploit prevailing conditions. For most of the light treatments, there is a small fraction of proteins whose expression seems to be distinct. It is possible that it is this small fraction of proteins whose expression may be important to define the characteristic physiological and developmental patterns associated with each light condition. These results substantiate a coordinated regulation of all pathways for the proper regulation of cell expansion in changing light environments.
This work was supported by a competitive grant from the Department of Science and Technology, Govt. of India (DST/SP/SO/A-49/95).
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Varsha Gupta (1) and Baishnab C. Tripathy (2)
(1) Department of Biotechnology Chhatrapati Shahu Ji Maharaj University, Kalyanpur, Kanpur, U.P., India
(2) School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India