Modulation of Chlorophyll Biosynthesis by Water Stress in Rice Seedlings During Chloroplast Biogenesis

1 Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast 2 biogenesis1

3 Vijay K. Dalal and Baishnab C. Tripathy*

4 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

6 *corresponding author

7 Supported by a grant from the Department of Biotechnology, Government of India grant 8 (BT/PR14827/BCE/08/841/2010), University Grants Commission capacity build up funds, and 9 Department of Science and Technology purse grant from Jawaharlal Nehru University, New 10 Delhi. Article 11

12 Running title: Modulation of chlorophyll biosynthesis by drought

14 Address correspondence to:

15 Baishnab C Tripathy 16 School of Life Sciences 17 Jawaharlal Nehru University 18 New Delhi 110067 19 India 20 Phone: +91-11-26704524 21 FAX: +91-11-26742558

This article has been accepted for publication and undergone full scientific peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/j.1365-3040.2012.02520.x

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5 Abstract

6 To understand the impact of water stress on the greening process, water stress was applied to six- 7 day old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1 8 by immersing their roots in 40 mM PEG 6000 (-0.69 MPa) or 50 mM PEG 6000 (-1.03 MPa) 9 dissolved in half-strength MS-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent + 10 incandescent light. Chlorophyll accumulation substantially declined in developing water-stressed 11 seedlings. Reduced Chl synthesis was due to decreased accumulation of chlorophyll Article 12 biosynthetic intermediates i.e. glutamate-1-semialdehyde, 5-aminolevulinic acid, Mg- 13 protoporphyrin IX monomethylester and protochlorophyllide. Although, 5-aminolevulinic acid 14 synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its 15 synthesis, glutamate-1-semialdehyde aminotransferase increased, suggesting its crucial role in 16 the greening process in stressful environment. The biochemical activities of Chl biosynthetic 17 enzymes i.e. 5-aminolevulinic acid dehydratase, Porphobilinogen deaminase, 18 Coproporphyrinogen III oxidase, Porphyrinogen IX oxidase, Mg-chelatase and 19 Protochlorophyllide oxidoreductase were down-regulated due to their reduced protein 20 abundance/gene expression in water-stressed seedlings. Down regulation of Protochlorophyllide 21 oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis 22 of early intermediates i.e. glutamate-1-semialdehyde and 5-aminolevulinic acid could modulate 23 the gene expression of later enzymes of Chl biosynthesis pathway. 24

25 Key words: Chlorophyll biosynthesis, Chloroplast biogenesis, Photosynthesis, Rice, Water- 26 stress.

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2 Plant growth, development, photosynthesis and plant productivity are severely affected 3 due to environmental stresses, particularly during early seedling growth. When seeds germinate 4 beneath the soil, their seedlings remain in near-darkness for a while. In angiosperms, the 5 differentiation of etioplast to chloroplast does not take place in dark as protochlorophyllide 6 oxidoreductase (POR) enzyme (EC 1.3.33.1) requires light as a substrate to photo-transform 7 Pchlide via trans addition of hydrogen across the C17–C18 double bond of the D-ring to form 8 Chlide (Apel et al. 1980; Griffiths 1978; Santel & Apel 1981). Therefore, etiolated rice seedlings 9 do not synthesize chlorophyll (Chl) and contain a special form of plastids called etioplasts or 10 etiochloroplasts. As seedlings come out of soil, they are exposed to light and light-mediated 11 chlorophyll biosynthesis and other associated greening processes are initiated resulting in 12 transformation of etioplasts to chloroplasts (Waters & Pyke 2005). Chloroplast development Article 13 involves the biosynthesis of components of photosynthetic apparatus involving synthesis of Chl, 14 carotenoids, lipids and proteins which involves a coordinated function of chloroplast and nuclear 15 genomes (Gray et al. 2003; Leon et al. 1998; Nott et al. 2006). Most steps of Chl biosynthesis 16 are well understood (Bollivar 2006; Goslings et al. 2004; Meskauskiene et al. 2001; Tanaka & 17 Tanaka 2007; Tripathy & Rebeiz 1986, 1987, 1988; Manohara & Tripathy 2000; Mohapatra & 18 Tripathy 2002, 2007; Pattanayak et al. 2002, 2005; Pattanayak & Tripathy 2011; Wang et al. 19 2010; Wu et al. 2007; Shalygo et al. 2009; Peter et al. 2010; Hedtke et al. 2007).

20 Chloroplast development is influenced by external and internal factors such as light 21 quality, salt, temperature, nutrition, leaf age and leaf water potential etc. (Dutta, Mohanty & 22 Tripathy 2009; Bhardwaj & Singhal 1981; Bengston et al. 1978; Mohanty & Tripathy 2011; 23 Virgin 1965). Chill-, heat- or salt-stressed seedlings have impaired Chl biosynthesis due to 24 down-regulation of gene expression and protein abundance or due to post-translational 25 modification of several enzymes involved in tetrapyrrole metabolism (Eskins et al. 1986; Sood, 26 Tyagi & Tripathy 2004; Sood, Gupta & Tripathy 2005; Tewari & Tripathy 1998, 1999; 27 Mohanty, Grimm & Tripathy 2006; Lay et al. 2000, 2001; Satpal & Tripathy unpublished).

28 Water stress is another very important stress frequently encountered by plants due to 29 scanty rainfall. It is also an important factor influencing the chloroplast development. Drought

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© 2012 Blackwell Publishing Ltd 1 substantially alters plant metabolism i.e. plant growth, photosynthesis and yield (Boyer 1982; 2 Gimenez et al. 1992; Munns 2002; Loggini et al. 1999; Lawlor & Cornic 2002; Lawlor & Tezara 3 2009; Chaves et al. 2009). Many studies have been already performed to know the impact of 4 water stress on photosynthesis in developed leaves (Loggini et al. 1999; Giardi et al. 1996; 5 Galmes et al. 2007; Massacci et al. 2008). Two principal mechanisms are invoked for decreased

6 photosynthesis, i) restricted diffusion of CO2 into the intercellular spaces of leaves, caused by 7 stomatal closure (Cornic 2000; Cornic & Briantais 1991), and ii) metabolic inhibition (Tezara et 8 al. 1999; Gimenez et al. 1992). Metabolic down-regulation in developing green leaves could 9 result from the reduced and improper assembly of photosynthetic complexes in thylakoid 10 membranes due to reduction in Chl biosynthesis under water stress (Bhardwaj & Singhal 1981; 11 Bengston et al. 1978). Photosynthetic proteins require Chl and carotenoids for their correct 12 folding, assembly and insertion into thylakoid membranes (Kim et al. 1994; Horn & Paulsen

13 2002). Because of reduced photosynthetic CO2 fixation in water-stressed plants, most of the Article 14 NADPH is not utilized resulting in a highly reduced electron transport chain (ETC) prone to 15 oxygen attack (Noctor et al. 2002; Chaves et al. 2009; Cornic & Briantais 1991; Lawlor & - 16 Tezara 2009). After extracting electrons from over-reduced ETC, O2 is converted to O2 that 17 could potentially damage the photosynthetic apparatus and other cellular components (Chaves & 18 Oliveira 2004). Singlet oxygen is also generated under such conditions. ROS could down- 19 regulate the synthesis of tetrapyrroles intermediate Pchlide probably due to impaired MPE 20 cyclase activity (Aarti et al. 2006; Stenbaek et al. 2008), that results in reduced Chl synthesis in 21 plants.

22 Most of the pigment biosynthesis and their assembly to pigment-protein complexes occur 23 in de-etiolating seedlings and stress severely affects early seedling growth. Therefore, the impact 24 of water-stress on early seedling development and Chl biosynthesis was monitored to understand 25 the mechanism of regulation of the greening process and Chl biosynthesis during light-induced 26 chloroplast development. In the present study, we investigated the effect of water stress on 27 pigment synthesis, gene and protein expression and activities of enzymes involved in Chl 28 biosynthesis in drought-sensitive rice cultivar, PB-1 during light-induced chloroplast 29 development. Our results demonstrate that the Shibata shift, observed during very early light- 30 induced chloroplast development is impaired and Chl biosynthesis is substantially down-

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© 2012 Blackwell Publishing Ltd 1 regulated due to water stress. It further reveals that Chl biosynthesis is down-regulated to 2 prevent the accumulation of harmful singlet oxygen generating tetrapyrroles at a very early step 3 i.e. ALA synthesis, due to reduced gene expression of early enzymes of Chl biosynthesis 4 pathway. 5

6 Materials and Methods

7 Plant Material

8 Drought-sensitive rice (Oryza sativa L.) cultivar Pusa Basmati-1 (PB-1) was used as 9 experimental material. Seeds of PB-1 were obtained from Indian Agricultural Research Institute 10 (I.A.R.I.), Pusa, New Delhi.

11 PlantArticle Growth Conditions and application of water-stress.

12 Seeds were washed with tap water and soaked in water for 24 h. Seeds were grown on 13 germination paper using half-strength Murashige and Skoog (MS) liquid media having no agar 14 or vitamins. Seeds were grown first in complete darkness for six days at 28 0C before giving 15 water stress. To study the regulation of chlorophyll biosynthesis by water stress, different 16 concentrations of polyethylene glycol 6000 (PEG 6000) dissolved in half strength MS growth 17 medium [40 mM PEG (osmotic potential, -0.69 MPa) or 50 mM PEG 6000 (osmotic potential, - 18 1.03 MPa)] (Michel & Kaufmann 1973) were added in dark to the roots of seedlings maintained 19 at 28 0C, 16 h prior to transfer to cool-white-fluorescent + incandescent light (100 µmoles 20 photons m-2 s-1). After every 12 h, water was added to compensate for the evaporation from 21 growth medium. 22 23 Chlorophyll, carotenoid and protein estimation

24 Chl and carotenoid contents were estimated as described by Porra, Thompson & 25 Kriedemann (1989) and Welburn & Lichtenthaler (1984). Protein was estimated according to 26 Bradford (1976).

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© 2012 Blackwell Publishing Ltd 1 2 Calculation of correction factors 3 Several parameters, i.e. Chl content, protein contents etc. were expressed on per g fresh 4 weight basis. However, due to water stress, there was severe loss of moisture that resulted in 5 reduction in relative water contents of plants. To correct for the loss of moisture, the pigment or 6 protein determined on fresh weight basis was multiplied by simultaneously measured correction 7 factor, determined as DW of 100 mg of control samples/DW of 100 mg of stressed samples.

8 GSA Content

9 GSA content was measured as described by Kannangara & Schouboe (1985) and Sood et 10 al. (2005). Leaf samples (200 mg) were taken, weighed and one set was processed immediately 11 for GSA estimation (0 h); another set was incubated in 500 µM gabaculine in 0.1 M MES (pH 12 7.0) for 4 h in light. The tissues were hand homogenized in pre-chilled mortar and pestle in 5.0 Article 13 mL of 0.1 N HCl and centrifuged at 10000 rpm for 10 min at 4 0C. Supernatant was taken for 14 GSA estimation. Reaction mixture contained: 400 µL of supernatant, 360 µL of 0.1 N HCl, 80 15 µL of 2% 3-methyl-2-benzothiazolinonehydrazone (MBTH). This was then incubated in boiling 16 water bath for two minutes and cooled rapidly. Boiling was done in capped tubes. Reference 17 cuvette contained 400 µL of 0.1 N HCl and no supernatant was added to it. After cooling 760 µL

18 of distilled water and 40 µL of 20% FeCl3 was added to it, mixed by vortexing and read at 620 19 nm. Extinction coefficient used was 16.9 mM-1. Net GSA synthesized during 4 h period was 20 measured by subtracting the 0 h GSA from 4 h GSA content.

21 ALA Content

22 ALA content was measured according to Harel & Klein (1972); Tewari & Tripathy 23 (1998). Leaf samples (200 mg) were taken at different time points. One set was kept in 60 mM 24 levulinic acid (LA) for 4 h in 100 µmoles photons m-2 s-1 light and another set was processed 25 immediately for ALA estimation (0 h). Tissues were hand homogenized in a pre-chilled mortar 26 and pestle in 5 mL of 1 M sodium acetate buffer (pH 4.6). The homogenate was centrifuged at 27 10000 rpm for 10 min and supernatant was taken for assay. The assay mixture consisted of 1 mL 28 of supernatant, 4 mL of distilled water and 250 µL of acetylacetone. The assay medium was 29 mixed properly and heated in a boiling water bath for 10 min. Then the extract was cooled at

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© 2012 Blackwell Publishing Ltd 1 room temperature and an equal volume of modified Ehrlich’s reagent was added to it and 2 vortexed for 2 min. After 10 min of incubation, absorbance of the extract was measured at 555 3 nm. ALA content was determined from the standard curve prepared from known concentration 4 of ALA (Sood et al. 2005). Net ALA synthesized during 4 h period was measured by subtracting 5 the 0 h ALA from 4 h ALA content.

6 Pchlide Content

7 Pchlide contents were estimated according to Tewari & Tripathy (1998) and Hukmani & 8 Tripathy (1992). Briefly, leaf tissues were homogenized in 90% chilled ammoniacal acetone (10 9 mL) in a pre-chilled mortar and pestle. The homogenate was centrifuged at 10000 rpm for 10 10 min at 4 0C. Supernatant was taken and hexane extracted acetone residue solvent mixture 11 (HEAR) was prepared according to Chakraborty & Tripathy (1992). Emission spectra E440, 12 E400 of HEAR were recorded from 580 nm to 700 nm and corrected for photomultiplier tube Article 13 response.

14 ALA Dehydratase (ALAD, EC 5.4.3.8)

15 Preparation of Ehlrich reagent: Ehlrich reagent was prepared as described by 16 Mauzerall & Granick (1956). For preparing Ehlrich reagent 2 g of dimethyl amino benzaldehyde 17 (DMAB) was dissolved in 30 mL of glacial acetic acid and 16 mL of 70% perchloric acid (4 N) 18 was added to it. Final volume was made upto 50 mL with glacial acetic acid. 19 ALAD assay was performed according to Hukmani & Tripathy 1994; Tewari & Tripathy 20 1998. Three replicates of 200 mg leaves were taken from seedlings grown in white light. Leaves 21 were homogenized at 4 0C in 5 mL of 0.1 M Tris (pH 7.6), 10 mM 2-mercaptoethanol in a pre- 22 chilled mortar and pestle. The homogenate was centrifuged 10000 rpm for 10 min at 4 0C. 23 Supernatant was taken for enzyme assay, performed as described by Shemin (1962) and Tewari 24 & Tripathy (1998). PBG formed was calculated using the absorption coefficient of 6.2 x 104 M-1 25 cm-1.

26 Porphobilinogen Deaminase (PBGD, EC 2.5.1.61)

27 Three replicates of 200 mg seedlings were hand homogenized in mortar and pestle in 5.0 28 mL of phosphate buffer (pH 8.0) and 0.6 mM EDTA at 4 °C and passed through 4 layers of

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© 2012 Blackwell Publishing Ltd 1 cheese cloth. Homogenate was centrifuged at 4 °C. Supernatant was taken for assay. Enzyme 2 assay was performed according to Hukmani & Tripathy (1994) and Tewari & Tripathy (1998). 3 Enzyme activity was assayed as amount of porphyrin synthesized in 3.0 mL of reaction mixture 4 having following composition as final concentrations: freshly prepared 0.6 mL PBG (550 µM), 5 90 µL EDTA solution (0.6 mM), 428 µL phosphate buffer (pH 8.0), 1.8 mL of enzyme extract 6 and 82 µL distilled water. In blank distilled water was added instead of enzyme. The reaction 7 mixture was incubated at 37 °C for 1 h. 750 µL of this reaction mixture was taken to which 1.7 8 mL of TCA (7.1%) was added to stop the reaction. Centrifuged at 10000 rpm for 10 min in 9 sorvall SS-34 tubes at 4 °C. Then 10 µL of iodine solution (1%) was added and incubated for 5 10 min at 37 oC. Finally 20 µL of freshly prepared sodium thiosulphate (2%) was added to reduce 11 iodine. Absorbance was measured at 406 nm. Extinction coefficient used at 406 nm for 12 porphyrin was 5.48 x105 M-1 cm-1. Absorbance of all samples was taken from 400-700 nm to 13 check for a peak at 405.5 or 406 nm. Article

14 Preparation of Coprogen III, and Protogen IX

15 Sodium amalgam was prepared according to Jacobs & Jacobs (1982). Reduction of 16 porphyrins was carried out according to the method of Poulson & Polglosse (1974) with some 17 modifications. Porphyrinogens were freshly prepared by reducing porphyrins, namely 18 coproporphyrin III, or Proto IX. Porphyrins were dissolved in 20% ethanol containing 0.1 N 19 KOH and centrifuged at room temperature. Supernatant was taken for performing reduction. 20 Oxygen present in the solution was removed by flushing the solution with nitrogen gas. To this 21 solution 5 g of 3% sodium amalgam was added with continuous flushing of solution with 22 nitrogen gas. The reduction was carried out for 2 to 3 min; the solution becomes colorless during 23 this time. Reduction was continued for 2 to 3 min more to ensure complete reduction of the 24 porphyrin. Immediately, the contents were filtered. The pH of the porphyrinogen solution was 25 carefully adjusted to 7.5-7.8 with 40% orthophosphoric acid.

26 Coproporphyrinogen oxidase (CPO, EC 1.3.3.3)

27 Leaves were taken from seedlings after 24 h of greening and hand homogenized in pre- 28 chilled mortar and pestle in isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1

29 mM MgCl2, 1 mM Na2EDTA, 0.2% BSA (w/v). Homogenate was passed through 8 layers of

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2 suspended in lysis buffer containing 0.01 M Tris-HCl (pH 7.7), 20 mM MgCl2, and 2.5 mM 3 EDTA and kept on ice for 10 min. This was then centrifuged at 4 0C. Supernatant was taken for 4 assay, done as described by Tewari & Tripathy (1998). Synthesis of protoporphyrin IX from 5 reduced coporphyrinogen was measured by spectrofluorimetry (Hukmani & Tripathy 1992; 6 Tewari & Tripathy 1998).

7 Protoporphyrinogen oxidase (protox, EC 1.3.3.4)

8 Protox assay was performed as described by Tewari & Tripathy (1998). Leaves were 9 taken from seedlings after 24 h of greening and hand homogenized in prechilled mortar and

10 pestle in isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1 mM MgCl2, 1 mM

11 Na2EDTA, 0.2% BSA (w/v). Homogenate was passed through 8 layers of cheese cloth, one layer 12 of mira cloth and centrifuged at 4 0C. Pellet containing plastids was suspended in lysis buffer Article

13 containing 0.01 M Tris-HCl (pH 7.7), 20 mM MgCl2, and 2.5 mM EDTA and kept on ice for 10 14 min. This was then centrifuged at 4 0C. Supernatant was taken for assay and synthesis of 15 protoporphyrin IX from reduced protoporphyrinogen was measured by spectrofluorimetry 16 (Hukmani & Tripathy 1992; Tewari & Tripathy 1998.

17 Mg-chelatase (EC 6.6.1.1)

18 Assay was done as described by Tewari & Tripathy (1998). Leaves were taken from 19 seedlings after 24 h of greening and hand homogenized in pre-chilled mortar and pestle in

20 isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1 mM MgCl2, 1 mM

21 Na2EDTA, 0.2% BSA. Homogenate was passed through 8 layers of cheese cloth, one layer of 22 Mira cloth and centrifuged at 4 0C. The pellet was gently suspended in suspension buffer

23 containing 0.5 M Sucrose, 0.2 M Tris-HCl, 20 mM MgCl2, 2.5 mM Na2EDTA, and 20 mM ATP, 24 at room-temperature (pH 7.7). The synthesis of MP(E) from protoporphyrin IX was monitored as 25 described before (Tewari & Tripathy 1998; Hukmani & Tripathy 1992).

26 Protochlorophyllide oxidoreductase (POR, EC 1.3.33.1)

27 POR activity was measured as described elsewhere (Tewari & Tripathy 1998). Water-stress was 28 applied to etiolated rice seedlings 16 h prior to transfer to light for 72 h. Five replicates of 50 mg

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© 2012 Blackwell Publishing Ltd 1 leaves were harvested from the seedlings under illumination and were transferred to 6 h dark and 2 subsequently transferred to light (100 µmoles photons m-2 s-1) for 1 h. Five batches of 50 mg 3 leaves were excised from the seedlings before the end of 6 h dark period and immediately after 1 4 h photo-period. Leaves were immediately homogenized in 90% ammoniacal acetone, acetone:

5 0.1 N NH4OH=9:1), in dark under safe green light and their Pchlide contents were estimated by 6 spectrofluorimetry (Hukmani & Tripathy 1992). The difference between the net amount of 7 Pchlide synthesized in dark and the same remaining after 1 h of light treatment is the amount of 8 Pchlide phototransformed from Pchlide to Chlide. POR activity was measured as relative 9 percent phototransformation of Pchlide, accumulated during 6 h dark period, to Chlide as 10 follows: [(Net amount of Pchlide synthesized in 6 h of dark period - Pchlide content remaining 11 after 1 h of light exposure)/Net amount of Pchlide synthesized in 6 h of dark period] x 100. 12

13 RNA Article Isolation, RT-PCR Analysis 14 RNA was extracted from fresh, control and water stressed rice seedlings using Tri® 15 reagent (Sigma, USA) according to the manufacturer’s instructions. RNA quantification was 16 done using Nanodrop spectrophotometer (ND 1000, USA) and confirmed by applying equal 17 amounts of total RNA to an agarose gel using Ethidium bromide staining. First-strand cDNA 18 was synthesized using 2 µg of total RNA using RevertAidTM H Minus M-MuLV Reverse 19 Transcriptase (Fermentas, USA) in a 20 µL reaction according to manufacturer’s protocol. 20 Semiquantitative RT-PCR was performed as described by Burch-Smith et al. (2006). After 21 synthesis, the cDNA was diluted 1:10, and 2 µL of cDNA was used as a template for PCR 22 amplification in a 20 µL reaction mixture. 23 PCR was performed for 26 to 29 cycles within a linear range of amplification for all 24 genes. The number of cycles and annealing temperature were optimized for each specific primer 25 pairs. Gene specific primers used in the study are provided in Supplimentary Information Table 26 S1. Ten µL of the PCR products were loaded and separated on 0.8-1% agarose Tris-acetate 27 EDTA gel. Ethidium bromide-stained PCR products were quantified using an Alpha Imager 28 3400. RT-PCR for each gene was performed in triplicates. The average values were determined 29 using Alpha Ease FC software. 30

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2 Plastids were isolated as described by Chakraborty & Tripathy 1992. Transfer of proteins 3 from polyacrylamide gels to nitrocellulose (NC) membranes was carried out in a semi-dry 4 Transblot apparatus (ATTO Corp., Tokyo, Japan), as recommended by the manufacturer. Blots 5 were stained with alkaline phosphatase using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 6 nitroblue tetrazolium (NBT) (Jilani et al. 1996). The Western-blot analysis was performed twice. 7 Polyclonal and heterologous antibodies were used (Supplimentary Information Table S2) for 8 some major enzymes of Chl biosynthetic pathway. 9

10 Fluorescence spectra of leaves for Shibata shift

11 Water stress was given in dark to six-day old etiolated rice seedlings. After 16 h of water-

12 stress-treatmentArticle etiolated leaves were excised and were given a red light flash. Subsequently,

13 they were frozen in liquid N2 after 0, 1 or 15 min of dark incubation and their fluorescence 14 emission spectra (77K) were recorded at excitation wavelength of 440 nm and emission 15 wavelength of 600-740 nm. Spectra were not corrected for photomultiplier tube response.

17 Results

18 Plants growth:

19 A drought-sensitive and high yielding cultivar of rice, [Oryza sativa L. cv Pusa Basmati- 20 1, (PB-1)] was taken for study. Rice seeds were germinated and grown in polyvinyl chloride 21 boxes on moist germination paper at 28 0C in dark. Six-day old etiolated rice seedlings were 22 treated with 40 mM or 50 mM PEG 6000 dissolved in half-strength MS salt solution for 16 h and 23 subsequently transferred to cool-white-fluorescent + incandescent light (100 µmoles photons m-2 24 s-1) at 28 0C for 24-72 h. After water-stress was applied, seedlings progressively rolled their 25 leaves. As the greening progressed, water-stressed seedlings showed reduced greening and 26 stunted growth (Fig. 1).

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2 As compared to their respective controls, the shoot and root length of water-stressed 3 seedlings (50 mM PEG-treated) declined by 35% and 10% respectively after 72 h of stress 4 treatment (Fig. 1E).

5 Shoot Dry Weight

6 The shoot dry weight (DW) of control seedlings increased by 19% and 32% after 48 h 7 and 72 h of light exposure (Fig. 2) compared to 24 h DW. However, in water-stressed seedlings, 8 shoot dry weight declined as the greening of leaves was substantially down-regulated. As 9 compared control, after 72 h of stress, their dry weight was reduced by 22% and 28% due to 40 10 mM or 50 mM PEG-treatment respectively.

11 Protein content Article 12 The protein content of stressed seedlings determined after 24-72 h of greening was 13 multiplied by simultaneously measured correction factor to take into account the loss of moisture 14 due to water stress. Protein content was 21.05 mg gFW-1 in control seedlings. It declined to14.94 15 mg gFW-1 (-29%) and 11.31 mg gFW-1 (-46%) in 40 mM or 50 mM PEG treated seedlings 16 respectively (Fig. 2).

17 Chlorophyll

18 Chl content of control and water-stressed seedlings is shown in Fig. 3. Shoots of control 19 seedlings had maximum Chl biosynthesis after 72 h of greening. The Chl content of stressed 20 seedlings determined after 24-72 h of greening was multiplied by simultaneously measured 21 correction factor to take into account the loss of moisture due to water stress. Chl content, 22 corrected for the loss of moisture in stressed-seedlings, decreased from 1.054 mg gFW-1 in 23 control to 0.931 mg gFW-1 (-12%) or 0.607 mg gFW-1 (-42%) in 40mM or 50 mM PEG-treated 24 rice seedlings respectively after 72 h of continuous de-etiolation (Fig. 3).

25 Carotenoids

26 Carotenoids contents, corrected for the loss of moisture as described for Chl, of rice 27 seedlings increased with increase of greening period and decreased in response to water stress

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3 Chl Biosynthesis intermediates 4 Chloroplast biogenesis and Chl accumulation was down-regulated in water-stressed 5 seedlings. To understand the mechanism of down-regulation of Chl accumulation, the steady 6 state levels of Chl biosynthetic intermediates were measured.

7 Glutamate 1-semialdehyde (GSA): GSA is the precursor of ALA in chlorophyll biosynthesis. 8 To prevent the conversion of accumulated GSA to ALA, gabaculine, an inhibitor of GSA-AT, 9 was added to the incubation medium. GSA accumulation, corrected for the loss of moisture as 10 described for Chl, decreased in response to water stress. After 72 h of greening in the presence of 11 50 mM PEG, GSA accumulation declined from 8.84 µmols gFW-1 in control seedlings to 3.98 12 µmols gFW-1 in stressed seedlings i.e. by 55% (Fig. 4A). Article

13 δδδ-Amino levulinic acid (ALA): After GSA, next early intermediate of chlorophyll biosynthesis 14 is ALA. Net accumulation of ALA was measured in the presence of levulinic acid (LA), an 15 inhibitor of ALA dehydratase. ALA accumulation, corrected for the loss of moisture as 16 described for Chl, decreased drastically in response water stress. In control seedlings, after 72 h 17 of greening, ALA content was 251.33 nmols gFW-1, however in stressed samples it was reduced 18 by 70% to only 75.46 nmols gFW-1 (Fig. 4B).

19 Mg-protoporphyrin IX monomethyl ester [MP(E)]: Later metabolic products i.e. Mg- 20 protoporphyrin IX and its monomethyl ester [MP(E)], corrected for the loss of moisture as 21 described for Chl, were measured after 72 h of light exposure in control and 50 mM PEG-treated 22 seedlings. MP(E) contents were reduced to 47% of control (3.28 nmols gFW-1) in stressed 23 seedlings (1.53 nmols gFW-1) (Fig. 4C).

24 Protochlorophyllide (Pchlide): MP(E) is further metabolized to Pchlide during greening 25 process. After 72 h of greening, the steady state Pchlide content corrected for the loss of moisture 26 as described for Chl, declined by 66% (Fig. 4D) from 14.48 nmols gFW-1 in control seedlings to 27 5.40 nmols gFW-1 in water-stressed seedlings. 28

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2 To understand the mechanism of impairment of Chl biosynthesis and that of Chl 3 biosynthetic intermediates due to water stress, different enzymes involved in Chl biosynthesis 4 were studied.

5 ALA dehydratase (ALAD): 6 Enzyme activity of ALAD that converts two molecules of ALA to PBG was determined 7 in 50 mM PEG-induced water-stressed seedlings after 72 h of greening. In stressed seedlings, 8 ALAD activity decreased by 33% (Fig. 5A). ALAD activity was 31.93 nmols mg protein-1 h-1 in 9 control seedlings and 21.26 nmols mg protein-1 h-1 in treated seedlings.

10 Porphobilinogen deaminase (PBGD): 11 The next step in the Chl biosynthesis is the conversion of four molecules of

12 porphobilinogenArticle to uroporphyrinogen, which is catalyzed by PBGD. The enzyme activity was 13 estimated by measuring the amount of porphyrin synthesis from porphobilinogen (PBG). After 14 72 h of greening the PBGD activity declined by 32% in stressed seedlings (Fig. 5B). PBGD 15 activity was 5.03 nmols mg protein-1 h-1 in control seedlings and 3.44 nmols mg protein-1 h-1 in 16 water-stressed seedlings.

17 Coproporphyrinogen III oxidase (CPO): 18 This enzyme catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen 19 IX. Coupled activity of CPO was estimated by measuring the amount of proto IX formed from 20 coproporphyrinogen III. Its activity is high during early plastid development and chlorophyll 21 biosynthesis. The CPO activity, measured at 24 h of greening, was reduced by 33% in response 22 to water stress (Fig. 5C). Its enzymatic activity reduced from 53.93 nmols 100 mg protein-1 h-1 in 23 control seedlings to 35.66 nmols 100 mg protein-1 h-1 in treated seedlings.

24 Protoporphyrinogen IX oxidase (protox): 25 Protox catalyzes the conversion of protoporphyrinogen IX to protoporphyrin IX. Its 26 activity was estimated by measuring the amount of protoporphyrin IX formed from 27 protoporphyrinogen IX after 24 h of greening. As compared to control, the protox activity was 28 reduced by 38% in water-stressed seedlings (Fig. 5D) to 1.38 µmols 100 mg protein-1 h -1.

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2 Mg-chelatase is the first enzyme in the Mg branch of the Chl biosynthetic pathway that 3 inserts Mg into Proto IX to form Mg-protoporphyrin IX (Mg-Proto IX). Proto IX was used as a 4 substrate and Mg-chelatase activity was measured as the amount of Mg-Proto IX and its 5 monomethylester [MP(E)] synthesized in control and stressed samples. MP(E) synthesizing 6 capacity of Mg-chelatase was reduced by half from 2.46 nmols 100 mg protein-1 h-1 in control 7 seedlings to 1.22 nmols 100 mg protein-1 h-1 in water-stressed seedlings (Fig. 5E) after 24 h of 8 stress.

9 Protochlorophyllide oxidoreductase (POR): 10 Protochlorophyllide (Pchlide) accumulates in dark and conversion of Pchlide to 11 chlorophyllide (Chlide) is catalyzed in angiosperms by a light-dependent enzyme 12 protochlorophyllide oxidoreductase (POR). The POR activity was estimated by measuring the Article 13 photo-transformation of Pchlide to Chlide. After 72 h of greening seedlings were transferred to 14 dark for 6 h, a few leaves were harvested and homogenized in dark. Subsequently, seedlings 15 were transferred to light (100 moles photons m-2 s-1) for 1 h and their leaves were harvested and 16 homogenized. Pchlide contents of dark incubated and 1 h light-exposed seedlings were estimated 17 as described in materials and methods. POR activity, calculated as relative percent photo- 18 transformation of Pchlide, accumulated during 6 h dark period to Chlide, was 38% of control in 19 stressed seedlings (Fig. 5F).

21 Shibata shift:

22 To understand the impact of water stress during very early seedling development the 23 Shibata shift was monitored in 50 mM PEG-treated etiolated seedlings. Six-day old etiolated 24 control or water-stressed seedlings were illuminated by a pulse of red light (1500 µmoles 25 photons m-2 s-1) of 0.2 seconds and were frozen in liquid nitrogen either immediately after red 26 flash or after 1 min or 15 min of dark incubation subsequent to flash illumination. Fluorescence 27 emission spectra (E440) of leaves were recorded before as well as after the red flash, at low 28 temperature (77K). Before flash illumination fluorescence spectra showed a characteristic peak 29 at 632 nm due to non-phototransformable Pchlide and a peak at 657 nm due to

Accepted 15

© 2012 Blackwell Publishing Ltd 1 phototransformable Pchlide. The non-phototransformable to phototransformable Pchlide ratio i.e. 2 E440F632:E440F657 increased from 0.1 in control etiolated leaves to 0.15 in water-stressed 3 etiolated leaves. In control samples, following flash illumination, the 657 nm peak declined due 4 to photo-transformation of Pchlide with simultaneous appearance of peak at 691 nm (Fig. 6A). 5 After 1 min of dark incubation, peak shifted to a longer wavelength at 694 nm. After 15 min of 6 dark incubation of flash-illuminated samples, the 694 nm-peak shifted to a lower wavelength i.e. 7 680 nm with a concomitant increase of peak at 657 nm due to regeneration of 8 phototransformable Pchlide (Fig. 6A).

9 Leaves from stressed seedlings exhibited a peak at 692 nm with a concomitant decrease 10 of phototransformable peak at 657 nm, immediately after flash illumination (Fig. 6B). After 1 11 min of flash, the peak shifted to 694 nm. After 15 min of dark incubation following red flash, the 12 peak at 694 nm shifted to 690 nm and only a hump appeared around 680 nm (Fig. 6B). The peak

13 at 657Article nm due to regeneration of phototransformable Pchlide was substantially reduced in 14 stressed samples.

16 Gene expression of chlorophyll biosynthetic enzymes:

17 To correlate the enzymatic activities with the gene expression, RT-PCR analysis was 18 performed after 24 h and 72 h of greening in response to water stress (Fig. 7).

19 HemA1 encodes Glutamyl-t-RNA reductase 1 that catalyses the conversion of Glutamyl- 20 t-RNA into Glutamate-1-semialdehyde (GSA) in photosynthetic tissues. In stressed seedlings, 21 HemA1 expression was reduced by 26-30% after 24-72 h of greening (Figs. 7 A & B). In the 22 same vein, the GSA synthesis declined in stressed seedlings.

23 Although, the ALA synthesis decreased, the gene expression of gsa was highly up- 24 regulated i.e. by 230% at 24 h and 90% at 72 h of stress treatment (Figs. 7 A & B).

25 The message abundance of Alad declined by 34% and 65% after 24 h and 72 h of stress- 26 treatment respectively (Figs. 7 A & B).

27 In water-stressed seedlings, transcript levels of pbgd decreased by 50% after 24-72 h of 28 greening (Figs. 7 A & B).

Accepted 16

3 Coprox/CPO transcript abundance decreased in stressed samples by 20% after 24-72 h of 4 greening (Figs. 7 A & B).

5 The gene expression of PPX-1 that codes for protporphyrinogen oxidase, declined in 6 water-stressed seedlings by 17% and 28% after 24 h and 72 h of water stress treatment 7 respectively (Figs. 7 A & B).

8 The Chlorina 1 (ChlD) that encodes for subunit D of Mg-chelatase was down-regulated 9 by 40% after 24 h and by 59% after 72 h of stress treatment (Fig. 7 A & B).

10 The transcript levels of Chlorina 9 (ChlI) decreased in stressed seedlings after 24-72 h of 11 stress application by 26-28% as compared to that of control (Figs. 7 A & B).

12 Article The expression of HemH2 that encodes for ferrochelatase 2, the first enzyme of heme 13 biosynyhetic pathway, shares substrate (Protoporphyrin IX) with Mg-chelatase and is mostly 14 expressed in green tissue, decreased after 24 h of stress by 35% (Figs. 7 A & B).

15 The transcripts abundance of rice homolog of Xantha-l (Chl27) that encodes for the 16 membrane-bound subunit of MPE cyclase decreased by 36% after 24-72 h of stress treatment 17 (Figs. 7 A & B).

18 In stressed seedling, Por B expression was lesser than control at both time points. Por B 19 transcript abundance decreased by 45-55% after 24-72 h of water stress (Figs. 7 A & B).

21 Immunoblot analysis of chlorophyll biosynthesis pathway enzymes:

22 To correlate the enzymatic activities and gene expression with that of protein abundance, 23 western blot analysis of certain enzymes involved in Chl biosynthesis was performed in control 24 and stressed seedlings with plastidic protein (30 µg). Equal amount of protein was loaded in each 25 lane as shown in Fig. 8A.

26 Gluamate-1-semialdehyde aminotransferase (GSA-AT, EC 5.4.3.8):

Accepted 17

© 2012 Blackwell Publishing Ltd 1 In agreement with that of gene expression, the protein abundance increased in control 2 seedlings during greening. Due to stress treatment, its protein abundance was up-regulated by 3 35% after 72 h of greening (Figs. 8 B & C).

4 Uroporphyrinogen III decarboxylase (UroD EC 4.1.1.37):

5 Protein abundance of UROD which catalyses the conversion of uroporphyrinogen III to 6 coproporphyrinogen III deceased greatly in water-stressed seedlings at 24 h (by 35%) and 72 h 7 (by 38%) of greening (Figs. 8 B & C).

8 Coproporphyrinogen III oxidase (CPO):

9 Abundance of CPO which catalyses the conversion of coproporphyrinogen III to 10 protoporphyrinogen IX, deceased by 45-50% after 24-72 h of greening in water-stressed 11 seedlings (Figs. 8 B & C).

12 ProtoporphyrinogenArticle oxidase:

13 The protein abundance of plastidic protoporphyrinogen oxidase I (PPX1), decreased 14 drastically in water-stressed seedlings by 65-70% after 24-72 h of stress treatment (Figs 8. B & 15 C).

16 Magnesium chelatase subunit I (Chl I):

17 ChlI protein abundance was only partially reduced in water-stressed seedlings after 24 h 18 and 72 h of greening (Figs. 8 B & C).

19 Protochlorophyllide oxidoreductase (POR):

20 POR protein abundance declined in stressed seedlings by 87-89% after 24-72 h of stress 21 treatment (Figs. 8 B & C).

22 Geranyl-geranyl reductase (Chl P, EC 1.3.1.83):

23 In water-stressed seedlings Chl P declined severely after 24 h and 72 h of stress (Figs. 8 24 B & C) by 62-68%.

26 Discussion:

Accepted 18

© 2012 Blackwell Publishing Ltd 1 Results demonstrate that during the de-etiolation of control rice seedlings, Chl contents 2 per gram fresh weight increased with period of light exposure and saturated at 72 h of 3 illumination (Fig. 3). In response to water-stress, Chl biosynthesis was down-regulated (42%). 4 Almost similar response during de-etiolation was seen in cucumber, wheat and other seedlings in 5 response to temperature and salt stress (Mohanty et al. 2006; Tewari & Tripathy 1998, Satpal & 6 Tripathy unpublished). 7 The reduced Chl synthesis in stressed seedlings was mostly due to down-regulation of 8 early intermediates of Chl biosynthesis i.e. GSA and ALA (Figs. 4A & 4B). In the same vein, 9 the down-regulation of GSA/ALA contents was observed in other stresses (Table 1) during early 10 seedling development in salt- (Satpal & Tripathy unpublished), chill- and heat-stressed 11 rice/maize/cucumber/Pinus seedlings (Hodgins and Van Huystee 1986 Tewari & Tripathy 1998; 12 Hodgins and Oquist 2006). 13 Reduced GSA synthesis in water-stressed rice seedlings was due to down-regulation of Article 14 HemA1 transcript abundance (Fig. 7). Similarly, its expression was down-regulated in chill- and 15 heat-stressed cucumber and wheat seedlings. In contrast, in response to salt-stress, the gene 16 expression of HemA1 was partially up-regulated in salt-tolerant and was not affected in salt- 17 sensitive rice genotype. HemA1 is mainly expressed in photosynthetic tissue. HemA2 is 18 predominantly expressed in roots and non-photosynthetic tissue of plants (Tanaka et al. 1996; 19 Nagai et al. 2007). Glutamyl t-RNA reductase is known to be post-translationally regulated by 20 phytochrome, heme, Pchlide and FLU (Vothknecht et al. 1998; Pontoppidan & Kannangara 21 1994; Goslings et al. 2004; Meskauskiene et al. 2001; McCormac et al. 2001) and also could be 22 similarly regulated by environmental stresses. 23 In water-stressed seedlings, although the protein/transcript abundance of the next enzyme 24 involved in ALA biosynthesis i.e. GSA-AT increased, the ALA contents declined. This suggests 25 that GSA-AT enzyme involved in ALA biosynthesis may be inactivated by post-translational 26 modification during stress condition. Similar increase in gsa transcript/protein abundance and 27 reduction in ALA contents were reported in response to salt stress and heat stress in rice or wheat 28 (Mohanty et al. 2006; Satpal & Tripathy unpublished; Table 1). The reduced ALA synthesis was 29 most likely partly due to limiting amounts of the substrate GSA. These results demonstrate that

Accepted 19

© 2012 Blackwell Publishing Ltd 1 Chl biosynthesis pathway is down-regulated at the early steps under stress conditions in order to 2 prevent the accumulation of harmful singlet oxygen generating tetrapyrroles. 3 Similar to decline in ALAD activity and its gene expression observed in salt-stressed rice, 4 chill- and heat-stressed cucumber or wheat seedlings (Mohanty et al. 2006; Satpal & Tripathy 5 unpublished; Table 1), the ALAD activity was reduced in water-stressed rice seedlings due to the 6 down-regulation of its transcript abundance. Limitation of ALA, a substrate for ALAD probably 7 reduced its gene expression in water stress and other stress conditions. These results suggest that 8 the increased or decreased availability of the substrate of the enzyme could positively or 9 negatively regulate the gene expression of the enzyme. The enzymatic activity of PBGD that 10 deaminates PBG to form Urogen was reduced due to down-regulation of its transcript abundance 11 in water-stressed rice seedlings (Figs. 5B & 7B). Similarly, PBGD enzyme activity and its 12 transcript abundance were down-regulated in rice/cucumber by salt or temperature stress (Satpal 13 & Tripathy unpublished, Tewari & Tripathy 1998, 1999; Table 1). Article 14 The subsequent enzyme in the porphyrin biosynthesis pathway is UROD, responsible for 15 copropoporphyrinogen synthesis. UROD protein abundance decreased in water-stressed 16 seedlings, which well correlates with the declined message abundance of UroD in response to 17 water stress. The UROD protein and transcript abundance also declined in salt-stressed and chill- 18 stressed rice/wheat seedlings (Satpal & Tripathy unpublished; Mohanty et al. 2006). This is in 19 contrast with our previous observations in cucumber and wheat where UROD activity and its 20 transcript/protein abundance increased in response to heat-stress (Mohanty et al. 2006; Tewari & 21 Tripathy 1998). Next two enzymes involved in protoporphyrin IX biosynthesis are 22 coproporphyrinogen oxidase and protoporphyrinogen oxidase. Similar to salt-, chill-stressed 23 rice/cucumber seedlings (Satpal & Tripathy unpublished, Tewari & Tripathy 1998; Table 1), the 24 enzyme activity of CPO and protox decreased in water-stressed seedlings (Figs. 5C & 5D) due to 25 down-regulation of their gene/protein abundance (Fig. 7 and Fig. 8).

26 All the steps in the biosynthesis of Mg-porphyrins and Fe-porphyrins are shared up to 27 protoporphyrin IX. Mg chelatase performs the Mg insertion step in porphyrin ring. It has three 28 subunits, Chl D, Chl H and Chl I (Gibson et al. 1995, 1999; Willows et al. 1998; Jensen et al. 29 1996). In the present study, Mg-chelatase activity decreased by 50% in water-stressed seedlings 30 (Fig. 5E). In the same vein, the gene/protein expression of ChlI/Chlorina9 and ChlD/Chlorina1

Accepted 20

© 2012 Blackwell Publishing Ltd 1 subunits of Mg-chelatase (Zhang et al. 2006) partially declined in water-stressed seedlings (Figs. 2 7 & 8). Chl I1 subunit is also post-translationally regulated by chloroplastic thioredoxin 3 (Ikegami et al. 2007) and therefore could have impaired function in altered redox environment in 4 water-stressed seedlings. Stoichiometric imbalance among the subunits of Mg-chelatase has 5 previously been shown to decrease the Mg-chelatase activity as seen in Chl I over-expressing or 6 under-expressing transgenic Arabidopsis plants (Papenbrock et al. 2000). Inadequate proportion 7 of all subunits is known to hamper the correct assembly of active Mg-chelatase (Guo et al 1998; 8 Hansson et al. 1999; Jensen et al. 1999). We have also previously shown that in salt- and chill- 9 stressed rice/cucumber seedlings, the Mg-chelatase activity and gene/protein expression were 10 down-regulated (Satpal & Tripathy unpublished; Tewari & Tripathy 1998; Table 1). ChlH was 11 proposed as a receptor of ABA, although there are reports to the contrary (Muller and Hansson 12 2009; Shen et al. 2006; Wu et al. 2009). ABA synthesis increases due to water stress. Increased 13 ABA could bind to ChlH and might down-regulate Chl biosynthesis. However, this needs Article 14 experimental verification. 15 16 MPE cyclase performs a very complex reaction of fifth ring formation in tertrapyrrole 17 biosynthesis. It is proposed to have two soluble subunits and a membrane subunit i.e. Xantha-l in 18 barley and Chl27 in Arabidopsis (Rzeznicka et al. 2005; Tottey et al. 2003). Transcripts level of 19 membrane subunit of MPE cyclase, encoded by AtChl27/Xantha-l homolog in rice, showed a 20 marginal decline in rice seedlings under water stress (Fig. 7). Its expression is also down- 21 regulated by salt-stress (Satpal & Tripathy unpublished; Table 1). MPE cyclase activity was 22 found to be protected by NADPH-dependent thioredoxin and 2-Cys peroxiredoxins system that

23 removes the H2O2 from the vicinity of MPE cyclase to protect its activity (Stenbaek et al. 2008). 24 Decreased gene expression of xantha-l homolog and its post-translational regulation due to the 25 oxidizing environment in stressed seedlings could have resulted in the down-regulation of MPE 26 cyclase activity and Pchlide accumulation. 27 Protochlorophyllide (Pchlide) is converted to chlorophyllide (Chlide) by 28 protochlorophyllide oxidoreductase (POR). In Arabidopsis, three POR iso-enzymes have been 29 reported namely POR A, POR B and POR C encoded by POR A, POR B and POR C genes 30 respectively (Armstrong et al. 1995; Pattanayak et al. 2002). However, several plants including

Accepted 21

© 2012 Blackwell Publishing Ltd 1 rice have only two iso-enzymes i.e. POR A and POR B. POR activity, measured as relative 2 percent phototransformation of Pchlide to Chlide, decreased by 60% in response to water stress 3 (Fig. 5F) after 72 h of greening. In the same vein, the POR protein abundance (Fig. 8) and PorB 4 expression (Fig. 7) were substantially down-regulated in stressed seedlings. A decrease in POR 5 activity/protein abundance and transcript expression of PorB was also reported in chill- and salt- 6 stressed cucumber/rice seedlings (Satpal & Tripathy unpublished; Table 1). Although plants 7 down-regulate the POR upon transfer from dark to light, the POR abundance is not affected by 8 light in seedlings exposed to low temperature likely due to the down-regulation of POR 9 degrading enzymes (Mohanty et al. 2006; Table 1).

10 The ChlP (geranyl-geranyl reductase) is responsible for phytol synthesis. Its protein 11 levels were highly down-regulated in seedlings due to water-stress treatment (Fig. 8). Similar 12 results were reported in salt-stressed rice seedlings (Satpal & Tripathy unpublished). Article 13 Table 1 examines the responses of Chl biosynthetic enzymes to various environmental 14 stresses. These stresses broadly down-regulate most of the enzymes of Chl biosynthesis 15 pathway. However, gene/protein expression of a certain enzyme i.e. GSA-AT is up-regulated in 16 most stresses, i.e. heat, water, salt etc. The expression of UroD is up-regulated in high- 17 temperature. As GSA-AT is a crucial enzyme involved in the last step of synthesis of ALA, 18 plants most likely up-regulate its expression to compensate for the reduced expression of earlier 19 enzymes of the ALA biosynthesis.

20 Shibata shift:

21 When etiolated leaves are subjected to a flash of light, the large aggregates of POR– 22 Phlide–NADPH ternary complexes are converted to POR–Chlide–NADPH complexes. Such 23 ternery complexes have higher emission and are slowly dissociated into smaller complexes 24 accompanied by the progressive release of Chlide from POR catalytic site. This leads to a large 25 blue shift in absorption and emission maxima of Chlide, and is called Shibata shift. The process 26 ends with the formation of Chlide absorbing at 672 nm and emitting at 682 nm (Chlide682). 27 Crosslinking experiments have shown that C672-682 is partly composed of Chlide still bound to 28 POR complexes and partly by Chlide bound to other proteins (Ryberg et al. 1992; Wiktorsson et

Accepted 22

© 2012 Blackwell Publishing Ltd 1 al. 1993). Shibata shift is followed by formation of photoactive photosystem II (PSII) units 2 (Franck 1993). 3 During plant development in dark, both the photo- and non-photo active pools of Pchlide 4 accumulate at different proportions, which is reflected in the modifications of fluorescence 5 spectra. The Photoreduction of Pchlide to Chlide is mediated by several short lifetime 6 intermediates, e.g. semireduced Pchlide radical species formed by hydrogen transfer from 7 NADPH (Belyaeva et al. 1988; Lebedev & Timko 1999) and characterized by their very low 8 fluorescence yield (Schoefs 2001). In contrary to the formation of first intermediate, which 9 requires light, the transfer of the second hydrogen ion would not need light and spontaneously 10 occurs at temperatures higher than 193K. In vitro experiments have shown that Chlide absorbing 11 at 676 nm and emitting at 690 nm has same organization as that of photoactive Pchlides i.e. 12 Chlide-LPOR-NADP complex (Oliver & Griffiths 1982; Wiktorsson et al. 1993) indicating that 13 this form of Chlide originated from photoactive Pchlides. Chlide 676-690 undergoes molecular Article 14 modifications at room temperature depending on the release of Chlide from LPOR active sites 15 along with formation of large aggregates of POR-NADP complexes (Chlide 670-675) (Sirnoval 16 et al. 1968). 17 Since the POR gene expression and protein abundance declined in water-stressed rice 18 seedlings, it was imperative to assess any changes in Shibata shift that is mediated by this photo- 19 enzyme. The etiolated control seedlings had a smaller emission fluorescence peak (77K) at 637 20 nm due to non-phototransformable Pchlide and a larger peak at 657 nm due to 21 phototransformable Pchlide (Fig. 6A). The non-phototransformable Pchlide peak is due to 22 monomeric Pchlide complex or esterified Pchlide i.e. protochlorophyll (Lindsten et al. 1988),

23 which spontaneously dimerizes to form (POR-Pchlide-NADPH)2. The short-wavelength, 24 monomeric Pchlide is not flash-photoactive; instead it regenerates the long wavelength Pchlide 25 forms (Schoefs & Franck 1993; He et al. 1994; Schoefs et al. 1994, 2000a, 2000b). The dimer 26 has the absorption maximum at 638 nm and emission maximum at 645 nm (Ouazzani Chahdi et 27 al. 1998; Lebedev & Timko 1999). The dimeric POR-Pchlide-NADPH complex further 28 polymerizes to form 16-mer or larger aggregates of POR-Pchlide-NADPH complex i.e. (POR-

29 Pchlide-NADPH)n having absorption maximum at 650 nm and emission maximum at 657-658 30 nm (Böddi et al. 1989; Wiktorsson et al. 1993) and is flash photoactive (Böddi et al. 1991).

Accepted 23

© 2012 Blackwell Publishing Ltd 1 Upon 16 h of stress treatment, the etiolated seedlings displayed an emission fluorescence peak 2 (77K) at 632 nm due to non-phototransformable Pchlide and a peak at 657 nm due to 3 phototransformable Pchlide (Fig. 6B). However, as compared to control, the ratio of non- 4 phototransformable/phototransformable Pchlide (F657/F632) increased from 0.10 to 0.15 in 5 stressed seedlings suggesting an impairment of aggregation of monomeric POR-Pchlide-NADPH

6 to 16-mer or larger aggregates of POR-Pchlide-NADPH complex i.e. (POR-Pchlide-NADPH)n. 7 This may be due to reduced availability of NADPH in water-stressed etiolated seedlings or due 8 to degradation of polymeric complexes in the stressed environment.

9 The flash-induced phototransformation and Shibata shift leading to chloroplast 10 biogenesis was substantially affected in 16 h water-stressed samples. Upon red light flash 11 illumination (0.2 sec) of control leaves the phototransformable Pchlide peak at 657 nm

12 emanating from large aggregates of polymeric (POR-Pchlide-NADPH)n complexes almost

13 disappearedArticle due to photo-reduction of Pchlide to Chlide and a new peak appeared at 691 nm due 14 to formation of Chlide-LPOR-NADP+ complexes (Oliver & Griffiths 1982; El Hamouri 1981; 15 Franck 1993; Franck et al. 1999; Wiktorsson et al. 1993). Transformation of Pchlide658 into 16 Chlide692 was previously observed by exposing the leaf primordia of common ash (Fraxinus 17 excelsior L.) and Hungarian ash, Fraxinus angustifolia Vahl. (Solymosi et al. 2006), wheat 18 (Franck et al., 1999) and that of Horse chestnut (Aesculus hippocastanum) (Solymosi et al. 2006) 19 to light flash. One min after flash, 691 nm-peak shifted to 694 nm (Fig. 6A) in control leaves due 20 to the formation of Chlide-LPOR-NADPH complexes (Oliver & Griffiths 1982; El Hamouri 21 1981; Franck et al. 1999). Subsequently, this peak blue-shifted to 680 nm after 15 min post- 22 flash incubation of control leaves (Fig. 6A) due to the release of Chlide from the active site of 23 LPOR and disaggregation of multimeric complexes a process called Shibata shift (Böddi et al. 24 1990; Shibata 1957; Franck 1993).

25 In stressed leaves, upon red light flash illumination of etiolated leaves the 26 phototransformable Pchlide peak at 657 nm disappeared and a new peak appeared at 692 nm 27 (Fig. 6B) due to formation of Chlide-LPOR-NADP+ complexes (Oliver & Griffiths 1982; El 28 Hamouri 1981) demonstrating that phototransformation of Pchlide to Chlide could still take 29 place in 16 h water-stressed samples. After 1 min post-flash incubation this peak shifted to 694 30 nm due to the formation of Chlide-LPOR-NADPH complexes (Fig. 6B). In water-stressed

Accepted 24

© 2012 Blackwell Publishing Ltd 1 leaves shift to lower wavelengths was substantially delayed. A shoulder appeared at 680 nm 2 after 15 min of dark incubation, in contrast to complete shift to 680 nm in control seedlings, 3 suggesting a slow release of Chlide from the active site of LPOR (Böddi et al. 1990; Shibata 4 1957). In a non-physiological environment i.e. after dessication of detached barley leaves a 5 slowdown of Shibata was earlier reported (Lay et al. 2000, 2001). In the same vein, arrest of 6 Shibata shift was demonstrated in heat-stressed (Mohanty & Tripathy 2011) and salt-stressed 7 (Abdelkader et al. 2007) wheat seedlings. On the contrary, in chill-stress the Shibata shift was 8 partially affected (Mohanty & Tripathy 2011). Arrest of Shibata shift in heat-stressed wheat 9 seedlings was due to disaggregation of polymeric Pchlide-POR-NADPH molecules which 10 delayed the conversion of non-phototransformable Pchlide to its phototransformable form 11 resulting in belated development of the core antenna protein complex CP47 of Photosystem II 12 (PS II) (Mohanty & Tripathy 2011). Upon 15 min of dark incubation after flash illumination, a 13 good amount of phototransformable Pchlide (F657) was regenerated in control seedlings (Fig. Article 14 6A) and substantially less in stressed seedlings (Fig. 6B) demonstrating the down-regulation of 15 synthesis of Pchlide and its conversion to phototransformable form.

16 The extent of down-regulation of ALA biosynthesis matches with that of Chl 17 biosynthesis in water-stressed seedlings implying that reduced gene expression and activity of 18 later enzymes of Chl biosynthesis pathway i.e. UROD, CPO, protox (PPX1), Mg-chelatase 19 (ChlI), POR etc. could be regulated by the abundance of early intermediates GSA or ALA. In the 20 same vein reduced expression and activity of later enzymes of Chl biosynthesis pathway i.e. Chl 21 synthase, Chl27 and Chl M in antisense plants caused a feedback-inactivation of the initial step 22 of the pathway leading to down-regulation of the metabolic flow to Chl (Shalygo et al. 2009; 23 Alawady & Grimm 2005; Peter et al. 2010; Bang et al. 2008; Rzeznicka et al. 2005; Pontier et 24 al. 2007). Observations from our laboratory in relation to PORC over-expression (Pattanayak & 25 Tripathy 2011), those of GUN4 (Peter & Grimm 2009) and ChlM (Alawady & Grimm 2005) led 26 to a general activation of the enzymes of Chl biosynthesis and consequent increase in Chl 27 contents. These studies along with our present observation of down-regulation of later enzymes 28 of Chl biosynthesis by reduced ALA synthesis in water-stressed seedlings demonstrates a 29 regulatory network of genes involved in tetrapyrrole biosynthesis.

Accepted 25

© 2012 Blackwell Publishing Ltd 1 In conclusion the Chl biosynthesis is substantially down-regulated due to water stress 2 during seedling development. Chl biosynthesis is down-regulated at a very early step i.e. ALA 3 synthesis, due to reduced gene expression of early enzymes of Chl biosynthesis pathway under 4 stress conditions that prevents the accumulation of harmful singlet oxygen generating 5 tetrapyrroles. Down-regulation of Chl content could act as a regulatory mechanism in plants to 6 resist drought. Minimization of light absorption by reduced amounts of Chl would down-regulate 7 the electron transport to reduce the ROS production. The increased gene expression and protein 8 abundance of GSA-AT coupled with our previous observation of up-regulation of gsa gene 9 expression in heat-stressed and salt-stressed seedlings suggest that it may play a crucial role in 10 tolerance to abiotic stresses. Sense and antisense expression of GSA-AT will shed light on the 11 role played by GSA-AT under stress conditions.

13 Acknowledgements:Article We thank C.G. Kannangara (Washington State University, USA) for the 14 gift of barley antibodies for glutamate-1-semialdehyde aminotransferase, W.T. Griffiths 15 (University of Bristol, UK) for wheat antibody for POR and B. Grimm, Humboldt University, 16 Berlin for UROD, CPO, PPOX1 and ChlP tobacco antibodies used for Western-blot analysis.

20 References:

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Accepted 26

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Accepted 27

© 2012 Blackwell Publishing Ltd 1 Böddi B., Ryberg M. & Sundqvist C. (1991) The formation of a short-wavelength chlorophyllide 2 form at partial phototransformation of protochlorophyllide in etioplast inner membranes. 3 Photochemistry Photobiology 53, 667-673. 4 Bollivar D.W. (2006) Recent advances in chlorophyll biosynthesis. Photosynthesis Research 90, 5 173-94. 6 Bradford M.M. (1976) A rapid and sensitive method for the quantification of microgram 7 quantities of protein utilizing the principle of protein-dye binding. Analytical 8 Biochemistry 72, 248-254. 9 Burch-Smith T.M., Schiff M., Liu Y. & Dinesh-Kumar S.P. (2006) Efficient virus-induced gene 10 silencing in Arabidopsis. Plant Physiology 142, 21-27. 11 Chakraborty N. & Tripathy B.C. (1992) Involvement of singlet oxygen in 5-aminolevulinic acid- 12 induced photodynamic damage of cucumber (Cucumis sativus L.) chloroplasts. Plant 13 Physiology 98, 7-11. Article 14 Chaves M.M., Flexas J. & Pinheiro C. (2009) Photosynthesis under drought and salt stress: 15 regulation mechanisms from whole plant to cell. Annals of Botany (London) 103(4), 551- 16 560. 17 Chaves M.M. & Oliveira M.M. (2004) Mechanisms underlying plant resilience to water deficits: 18 prospects for water-saving agriculture. Journl of Experimental Botany 55, 2365–2384. 19 Cornic G. (2000) Drought stress inhibits photosynthesis by decreasing stomatal aperture – not by 20 affecting ATP synthesis. Trends in Plant Science 5, 187–188. 21 Cornic G., Ghashghaie J., Genty B. & Briantais J.M. (1992) Leaf photosynthesis is resistant to a 22 mild drought stress. Photosynthetica 27, 295–309.

23 Cornic G. & Briantais J. M. (1991) Partitioning of photosynthetic electron flow between CO2

24 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.) at different CO2 concentrations and 25 during drought stress. Planta 183, 178–184. 26 Dutta S., Mohanty S. & Tripathy B.C. (2009) Role of temperature stress on chloroplast 27 biogenesis and protein import in pea. Plant Physiology 150, 1050-1061.

28 El Hamouri B., Brouers M. & Sironval C. (1981) Pathway from photoinactive P663-628

29 protocholorphyllide to the P696-682 chlorophyllide in cucumber etiplast suspensions. Plant 30 Science Letters, 21, 375—379.

Accepted 28

© 2012 Blackwell Publishing Ltd 1 Eskins K., McCarthy S.A., Dybas L. & Duysen M. (1986) Corn chloroplast development in 2 weak fluence rate red light and in weak fluence rate red plus far-red light. Physiologia 3 Plantarum 67, 242–246. 4 Franck F. (1993) On the formation of photosystem II chlorophyll-proteins after a short light flash 5 in etiolated barley leaves, as monitored by in vivo fluorescence spectroscopy. Journal of 6 Photochemistry and Photobiology B: Biology, 18, 35-40. 7 Franck F., Bereza B. & Böddi B. (1999) Protochlorophyllide-NADP+ and protochlorophyllide - 8 NADPH complexes and their regeneration after flash illumination in leaves and etioplast 9 membranes of dark-grown wheat. Photosynthesis Research 59, 53–61. 10 Galmés J., Medrano H. & Flexas J. (2007) Photosynthetic limitations in response to water stress 11 and recovery in Mediterranean plants with different growth forms. New Phytologist 175, 12 81–93. 13 Giardi M.T., Cona A., Geiken B., Kuˇcera T., Maojídek J. & Mattoo A.K. (1996) Long-term Article 14 drought stress induces structural and functional reorganization of photosystem II. Planta 15 199, 118–125. 16 Gibson L.C., Willows R.D., Kannangara C.G., von Wettstein D. & Hunter C.N. (1995) 17 Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of 18 activity by combining the products of the bchH, -I and -D genes expressed in Escherichia 19 coli. Proceedings of the National Academy of Sciences of the United States of America 20 92, 1941-1944. 21 Gibson L.C.D., Jensen P.E. & Hunter C.N. (1999) Magnesium chelatase from Rhodobacter 22 sphaeroides: initial characterization of the enzyme using purified subunits and evidence 23 for a BchI-BchD complex. Biochemical Journal 337, 243–251. 24 Gimenez C., Mitchell V.J. & Lawlor D.W. (1992) Regulation of photosynthesis rate of two 25 sunflower hybrids under water stress. Plant Physiology 98, 516-524.

Accepted 29

© 2012 Blackwell Publishing Ltd 1 Goslings D., Meskauskiene R., Kim C., Lee K.P., Nater M. & Apel K. (2004) Concurrent 2 interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of 3 metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown 4 Arabidopsis plants. Plant Journal 40, 957-67. 5 Gray J.C., Sullivan J.A., Wang J.H., Jerome C.A. & MacLean D. (2003) Coordination of plastid 6 and nuclear gene expression. Philosophical Transaction of Royal Society B: Biological 7 Sciences 358, 135-144. 8 Griffiths W.T. (1978) Reconstitution of chlorophyllide formation by isolated etioplast 9 membranes. Biochemical Journal 174, 681-692. 10 Guo R., Luo M. & Weinstein J.D. (1998) Magnesium-chelatse from developing pea leaves. Plant 11 Physiology 116, 605-615. 12 Hansson A., Kannangara C.G., von Wettstein D. & Hansson M. (1999) Molecular basis for 13 semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of Article 14 magnesium chelatase. Proceedings of the National Academy of Sciences of the United 15 States of America 96, 1744-1749. 16 Harel E. & Klein S. (1972) Light dependent formation of 5-aminolevulinic acid in etiolated 17 leaves of higher plants. Biochemistry and Biophysics Research Communications 49, 364- 18 370. 19 He Z.H., Li J., Sundqvist C. & Timko M.P. (1994) Leaf development age controls expression of 20 genes encoding enzymes of chlorophyll and heme biosynthesis in pea (Pisum sativum L.). 21 Plant Physiology 106, 537-546. 22 Hedtke B., Alawady A., Chen S., Bornke F. & Grimm B. (2007) HEMA RNAi silencing reveals 23 a control mechanism of ALA biosynthesis on Mg chelatase and Fe chelatase. Plant 24 Molecular Biology 64, 733–742. 25 Hodgins R.R. & Van Huystee R.B. (1986) Delta-aminolevulinic acid metabolism in chill stressed 26 maize (Zea mays L.). Journal of Plant Physiology 126, 257-268. 27 Hodgins R.R. & Oquist G. (2006) Porphyrin metabolism in chill-stressed seedlings of Scots pine 28 (Pinus sylvestris). Physiologia Plantarum 77, 620-624. 29 Horn R. & Paulsen H. (2002). Folding in vitro of lightharvesting chlorophyll a/b protein is 30 coupled with pigment binding. Journal of Molecular Biology 318, 547–556.

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© 2012 Blackwell Publishing Ltd 1 Hukmani P. & Tripathy B.C. (1992) Spectrofluorometric estimation of intermediates of 2 chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and 3 protochlorophyllide. Analytical Biochemistry 206, 125-30. 4 Hukmani P. & Tripathy B.C. (1994) Chlorophyll biosynthetic reactions during senescence of 5 excised barley (Hordeum vulgare L. cv IB 65) Leaves. Plant Physiology 105, 1295-1300. 6 Ikegami A., Yoshimura N., Motohashi K., Takahashi S., Romano P.G.N., Hisabori T., Takamiya 7 K.I. & Masuda T. (2007) The CHI1 subunit of Arabidopsis thaliana magnesium 8 chelatase is a target protein of the chloroplast thioredoxin. Journal of Biological 9 Chemistry 282, 19282–19291. 10 Jacobs N.J. & Jacobs J.M. (1982) Assay for enzymatic protoporphyrinogen oxidation, a late step 11 in heme synthesis. Enzyme 28, 862-866. 12 Jensen P.E., Gibson L.C., Henningsen K.W. & Hunter C.N. (1996) Expression of the chlI, chlD, 13 and chlH genes from the cyanobacterium Synechocystis PCC6803 in Escherichia coli and Article 14 demonstration that the three cognate proteins are required for magnesium protoporphyrin 15 chelatase activity. Journal of Biological Chemistry 271, 16662–16667. 16 Jensen P.E., Gibson L.C.D., Shephard F., Smith V. & Hunter C.N. (1999) Introduction of a new 17 branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes 18 encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium 19 protoporphyrin methyltransferase. FEBS Letters 455, 349–354. 20 Jilani A., Kar S., Bose S. & Tripathy B.C. (1996) Regulation of the carotenoid content and 21 chloroplast development by levulinic acid. Physiologia Plantarum 96, 139-145. 22 Kannangara C.G. & Schouboe A. (1985) Biosynthesis of δ-ALA in greening barley leaves. VII. 23 Glutamate-1-semialdehyde accumulation in gabaculine treated leaves. Carlsberg 24 Research Communications 50, 179-191. 25 Kim J., Lutz A., Eichacker L.A., Rudiger W. & Mullet J.E. (1994) Chlorophyll regulates 26 accumulation of the plastid-encoded chlorophyll proteins P700 and D1 by increasing 27 apoprotein stability. Plant Physiology 104, 907-916. 28 Lawlor D.W. & Cornic C. (2002) Photosynthetic carbon assimilation and associated metabolism 29 in relation to water deficits in higher plants. Plant Cell & Environment 25, 275–294.

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© 2012 Blackwell Publishing Ltd 1 Le Lay P., Eullaffroy P., Juneau P. & Popovic D. (2000) Evidence of chlorophyll synthesis 2 pathway alteration in desiccated barley leaves. Plant & Cell Physiology 41, 565-570. 3 Le Lay P., Boddi B., Kovacevic D., Juneau P., Dewez D. & Popovic P. (2001) Spectroscopic 4 analysis of dessication-induced alterations of the chlorophyllide transformation pathway 5 in etiolated barley leaves. Plant Physiology 127, 202-211. 6 Lebedev N. & Timko M.P. (1999) Protochlorophyllide oxidoreductase B-catalysed 7 protochlorophyllide photoreduction in vitro: insight into the mechanism of chlorophyll 8 formation in light-adapted plants. Proceedings of the National Academy of Sciences of 9 the United States of America 96, 9954-9959. 10 Leon P., Arroyo A. & Mackenzie S. (1998) Nuclear control of plastid and mitochondrial 11 developments in higher plants. Annual Review of Plant Physiology & Plant Molecular 12 Biology 49, 453-480. 13 Lindsten A., Ryberg M. & Sundqvist C. (1988) The polypeptide composition of highly purified Article 14 prolamellar bodies and prothylakoids from wheat (Tritium aestivum) as revealed by silver 15 staining. Physiologia Plantarum 72, 167-176. 16 Loggini B., Scartazza A., Brugnoli E., & Navari-Izzo F. (1999) Antioxidative defense system, 17 pigment composition, and photosynthetic efficiency in two wheat cultivars subjected to 18 drought. Plant Physiology 119, 1091–1099. 19 Manohara M.S. & Tripathy B.C. (2000) Regulation of protoporphyrin IX biosynthesis by 20 intraplastidic compartmentalization and adenosine triphosphate. Planta 212, 52-59. 21 Mauzerall D. & Granick S. (1956) The occurrence and determination of delta-amino-levulinic 22 acid and porphobilinogen in urine. Journal of Biological Chemistry 219, 435-46. 23 Massacci A., Nabiev S.M., Pietrosanti L., Nematov S.K., Chernikova T.N., Thor K. & Leipner J. 24 (2008) Response of the photosynthetic apparatus of cotton (Gossypium hirsutum) to the 25 onset of drought stress under field conditions studied by gas-exchange analysis and 26 chlorophyll fluorescence imaging. Plant Physiology & Biochemistry 46, 189-195. 27 McCormac A.C., Fischer A., Kumar A.M., Soll D. & Terry M.J. (2001) Regulation of HEMA1 28 expression by phytochrome and a plastid signal during de-etiolation in Arabidopsis 29 thaliana. Plant Journal 25, 549–61.

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© 2012 Blackwell Publishing Ltd 1 Meskauskiene R., Nater M., Goslings D., Kessler F., op den Camp R. & Apel K. (2001) FLU: a 2 negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proceedings of 3 the National Academy of Sciences of the United States of America 98, 12826-31. 4 Mohanty S., Grimm B. & Tripathy B.C. (2006) Light and dark modulation of chlorophyll 5 biosynthetic genes in response to temperature. Planta 224, 692-9. 6 Mohanty S. & Tripathy B.C. (2011) Early and late plastid development in response to chill stress 7 and heat stress in wheat seedlings. Protoplasma 248(4), 725-736. 8 Mohapatra A. & Tripathy B.C. (2002) Detection of protoporphyrin IX in envelope membranes of 9 pea chloroplasts. Biochemistry & Biophysics Research Communications 299(5), 751-754. 10 Mohapatra A. & Tripathy B.C. (2007) Differential distribution of chlorophyll biosynthetic 11 intermediates in stroma, envelope and thylakoid membranes in Beta vulgaris. 12 Photosynthesis Research 94(2-3), 401-410. 13 Michel B.E. & Kaufmann M.R. (1973) The osmotic potential of polyethylene glycol 6000. Plant Article 14 Physiology 51, 914-916. 15 Muller A.H. & Hansson M. (2009) The barley magnesium chelatase 150-kD subunit is not an 16 abscisic acid receptor. Plant Physiology 150, 157–166. 17 Munns R. (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment 18 25, 239–250. 19 Nagai S., Koide M., Takahashi S., Kikuta A., Aono M., Sasaki-Sekimoto Y., Ohta H., Takamiya 20 K-I. & Masuda T. (2007) Induction of isoforms of tetrapyrrole biosynthetic enzymes, 21 AtHEMA2 and AtFC1, under stress conditions and their physiological functions in 22 Arabidopsis. Plant Physiology 144, 1039-1051. 23 Noctor G., Veljovic-Javanovic S., Driscoll S., Novitskaya L. & Foyer C.H. (2002) Drought and 24 oxidative load in the leaves of C3 plants: a predominant role for photorespiration. Annals 25 of Botany 89, 841–850. 26 Nott A., Jung H.S., Koussevitzky S. & Chory J. (2006) Plastid-to-nucleus retrograde signaling. 27 Annual Review of Plant Biology 57, 739-759. 28 Oliver R.P. & Griffiths W.T. (1982) Pigment-protein complexes of illuminated etiolated leaves. 29 Plant Physiology 70, 1019-1025.

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© 2012 Blackwell Publishing Ltd 1 Ouazzani Chahdi A.M., Schoefs B. & Franck F. (1998) Isolation and characterization of photoactive 2 complexes of NADPH Protochlorophyllide oxidoreductase. Planta 206, 673-680. 3 Papenbrock J., Pfundel E., Mock H.P. & Grimm B. (2000) Decreased and increased expression 4 of the subunit CHL I diminishes Mg chelatase activity and reduced chlorophyll synthesis 5 in transgenic tobacco plants. Plant Journal 22, 155-164. 6 Pattanayak G.K., Biswal A.K., Reddy V.S. & Tripathy B.C. (2005) Light-dependent regulation 7 of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco 8 plants. Biochemistry & Biophysics Research Communications 326, 466-71. 9 Pattanayak G.K. & Tripathy B.C. (2002) Catalytic function of a novel protein 10 protochlorophyllide oxidoreductase C of Arabidopsis thaliana. Biochemistry & 11 Biophysics Research Communications 291, 921-924. 12 Pattanayak G.K. & Tripathy B.C. (2011) Overexpression of protochlorophyllide oxidoreductase 13 C regulates oxidative stress in Arabidopsis. PLoS One 6(10), e26532. Article 14 Peter E. & Grimm B. (2009) GUN4 is required for posttranslational control of plant tetrapyrrole 15 biosynthesis. Molecular Plant 2, 1198-210. 16 Peter E., Rothbart M., Oelze M.L., Shalygo N., Dietz K.J. & Grimm B. (2010) Mg 17 protoporphyrin monomethylester cyclase deficiency and effects on tetrapyrrole 18 metabolism in different light conditions. Plant & Cell Physiology 51, 1229–1241. 19 Pontier D., Albrieux C., Joyard J., Lagrange T. & Block M.A. (2007) Knock-out of the 20 magnesium protoporphyrin IX methyltransferase gene in Arabidopsis–effects on 21 chloroplast development and on chloroplast-to-nucleus signaling. Journal of Biological 22 Chemistry 282, 2297–2304. 23 Pontoppidan B. & Kannangara C.G. (1994) Purification and partial characterisation of barley 24 glutamyl-tRNA(Glu) reductase, the enzyme that directs glutamate to chlorophyll 25 biosynthesis. European Journal of Biochemistry 225, 529-537. 26 Porra R.J., Thompson W.A. & Kriedemann P.E. (1989) Determination of accurate extinction 27 coefficients and simultaneous equations for assaying chlorophylls a and b extracted with 28 four different solvents: verification of the concentration of chlorophyll standards by 29 atomic absorption spectroscopy. Biochimica et Biophysica Acta 975, 384-394.

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© 2012 Blackwell Publishing Ltd 1 Poulson R. & Polglasse W.J. (1974) Aerobic and anaerobic coproporphyrinogen oxidase 2 activities in extracts from Saccharomyces cerevisiae. Journal of Biological Chemistry 3 249, 6367-6371. 4 Ryberg M., Artus N., Böddi B., Lindsten A., Wiktorsson B. & Sundqvist C. (1992) Pigment– 5 protein complexes of chlorophyll precursors. In: Regulation of chloroplast biogenesis. 6 (eds, J.H. Argyrudi-Akoyunoglu), pp. 217–225. Plenum, New York,NY, USA. 7 Rzeznicka K., Walker C.J., Westergren T., Kannangara C.G., von Wettstein D., Merchant S., 8 Gough S.P. & Hansson M. (2005) Xantha-l encodes a membrane subunit of the aerobic 9 Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis. 10 Proceedings of the National Academy of Sciences of the United States of America 102, 11 5886-5891. 12 Santel H.J. & Apel K. (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare 13 L.): The effect of light on the NADPH:protochlorophyllide oxidoreductase. FEBS Article 14 Journal 120, 95-103. 15 Schoefs B. (2001) The protochlorophyllide–chlorophyllide cycle. Photosynthesis Research 70, 16 257–271. 17 Schoefs B., Bertrand M. & Franck F. (2000a) Spectroscopic properties of protochlorophyllide 18 aggregational state of protochlorophyllide. Photosynthtica 29, 205-218 19 Schoefs B., Bertrand M. & Franck F. (2000b) Photoactive protochlorophyllide regeneration in 20 cotyledons and leaves from higher plants. Photochemistry & Photobiology 72, 660–668. 21 Schoefs B. & Franck F. (1993) Photoreduction of protochlorophyllide to chlorophyllide in 2-d- 22 old dark-grown bean (Phaseolus vulgaris cv. Commodore) leaves. Comparison with 10- 23 d-old dark-grown (etiolated) leaves. Journal of Experimental Botany 44, 1053-1057. 24 Schoefs B., Garnir H.P. & Bertrand M. (1994) Comparison of the photoreduction 25 protochlorophyllide to chlorophyllide in leaves and cotyledons from dark-grown beans as 26 a function of age. Photosynthesis Research 41, 405-417. 27 Shalygo N., Czarnecki O., Peter E. & Grimm B. (2009) Expression of chlorophyll synthase is 28 also involved in feedback-control of chlorophyll biosynthesis. Plant Molecular Biology 29 71, 425-36.

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© 2012 Blackwell Publishing Ltd 1 Shemin D. (1962) 5-aminolevulinic acid dehydratase from Rhodo-pseudomonas spheroids. 2 Methods in Enzymology 5, 883-884. 3 Shen Y.Y., Wang X.F., Wu F.Q., et al. (2006) The Mg-chelatase H subunit is an abscisic acid 4 receptor. Nature 443, 823–826. 5 Shibata K. (1957) Spectroscopic studies on chlorophyll formation in intact leaves. Journal of 6 Biochemistry 44, 147-173. 7 Sirnoval C., Brouer M., Michel J.M. & Kuiper Y. (1968) The reduction of protochlorophyllide 8 into chlorophyllide: I. Kinetics of the P657–647 to P688–676 phototransformation. 9 Photosynthetica 2, 268–287. 10 Sood S., Gupta V. & Tripathy B.C. (2005) Photoregulation of the greening process of wheat 11 seedlings grown in red light. Plant Molecular Biology 59, 269-87. 12 Sood S., Tyagi A.K. & Tripathy B.C. (2004) Inhibition of Photosystem I and Photosystem II in 13 wheat seedlings with their root–shoot transition zones exposed to red light. Article 14 Photosynthesis Research 81, 31-40. 15 Solymosi K., Bóka K. & Böddi B. (2006). Transient etiolation: protochlorophyll(ide) and 16 chlorophyll forms in differentiating plastids of closed and breaking leaf buds of horse 17 chestnut ( Aesculus hippocastanum). Tree physiology 26, 1087-1096. 18 Stenbaek A., Hansson A., Wulff R.P., Hansson M., Dietz K.J. & Jensen P.E. (2008) NADPH- 19 dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection 20 of Mgprotoporphyrin monomethyl ester cyclase. FEBS Letters 582, 2773–2778. 21 Tanaka R., Yoshida K., Nakayashiki T., Masuda T., Tsuji H., Inokuchi H. & Tanaka A. (1996) 22 Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase 23 proteins in greening cucumber seedlings. Plant Physiology 110, 1223–1230. 24 Tanaka R. & Tanaka A. (2007) Tetrapyrrole biosynthesis in higher plants. Annual Review of 25 Plant Biology 58, 321-46. 26 Tewari A.K. & Tripathy B.C. (1998) Temperature-stress-induced impairment of chlorophyll 27 biosynthetic reactions in cucumber and wheat. Plant Physiology 117, 851-858. 28 Tewari A.K. & Tripathy B.C. (1999) Acclimation of chlorophyll biosynthetic reactions to 29 temperature stress in cucumber (Cucumis sativus L.). Planta 208, 431-437.

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© 2012 Blackwell Publishing Ltd 1 Tezara W., Mitchell V.J., Driscoll S.D. & Lawlor D.W. (1999) Water stress inhibits plant 2 photosynthesis by decreasing coupling factor and ATP. Nature 401, 914-917. 3 Tottey S., Block M.A., Allen M., Westergren T., Albrieux C., Scheller H.V., Merchant S. & 4 Jensen P.E. (2003). Arabidopsis CHL27, located in both envelope and thylakoid 5 membranes, is required for the synthesis of protochlorophyllide. Proceedings of the 6 National Academy of Sciences of the United States of America 100, 16119-16124. 7 Tripathy B.C. & Rebeiz C.A. (1986) Chloroplast biogenesis: Demonstration of the monovinyl 8 and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants. Journal 9 of Biological Chemistry 261, 13556-64. 10 Tripathy B.C. & Rebeiz C.A. (1987) Non-equivalence of glutamic and 5-aminolevulinic acids as 11 substrates for protochlorophyllide and chlorophyll biosynthesis in darkness. In Progress 12 in Photosynthesis Research (eds J. Biggins), pp. 439-443. Vol 4. Nijhoff Publishers, 13 Boston, MA, USA. Article 14 Tripathy B.C. & Rebeiz C.A. (1988) Chloroplast Biogenesis 60: Conversion of divinyl 15 protochlorophyllide to monovinyl protochlorophyllide in green(ing) barley, a dark 16 monovinyl/light divinyl plant species. Plant Physiology 87, 89-94. 17 Virgin H.I. (1965) Chlorophyll formation and water deficit. Physiol Plant 18, 994–1000. 18 Vothknecht U.C., Kannangara C.G. & von Wettstein D. (1998) Barley glutamyl tRNAGlu 19 reductase: mutations affecting haem inhibition and enzyme activity. Phytochemistry 47, 20 513-519. 21 Wang P., Gao J., Wan C., Zhang F., Xu Z., Huang X., Sun X. & Deng X. (2010) Divinyl 22 Chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl 23 reductase in rice. Plant Physiology 153, 994-1003. 24 Waters M. & Pyke K. (2005) Plastid development and differentiation. In: Plastids (eds S.G. 25 Moller), pp. 30-59. Blackwell, Oxford,OX, USA.

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© 2012 Blackwell Publishing Ltd 1 Welburn A.R. & Lichtenthaler H. (1984) Formula and program to determine total carotenoids 2 and Chla and b of leaf extracts in different solvents. In Advances in photosynthesis 3 research, vol II. (eds C. Sybesma), pp. 9-12. Martinus Nijoff/Dr. W. Junk Publishers, The 4 Hague, USA. 5 Wiktorsson B., Engdahl S., Zhong L.B., Böddi B., Ryberg M. & Sundqvist C. (1993) The effect 6 of cross-linking of the subunits of NADPH-protochlorophyllide oxidoreductase of the 7 aggregational state of protocholorophyllide. Photosynthetica 29, 205-218. 8 Willows R.D. & Beale S.I. (1998) Heterologous expression of the Rhodobacter capsulatus Bch - 9 I, -D, and -H genes that encode magnesium chelatase subunits and characterization of the 10 reconstituted enzyme. Journal of Biological Chemistry 273, 34206–34213. 11 Wu Z., Zhang X., He B., et al. (2007) A chlorophyll-deficient rice mutant with impaired 12 chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiology 145, 29-40. 13 Zhang H., Li J., Yoo J.H., Yoo S.C., Cho S.H., Koh H.J., Seo H.K. & Paek N.C. (2006) Rice Article 14 Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key 15 enzyme for chlorophyll synthesis and chloroplast development. Plant Molecular Biology 16 62, 325-327. 17

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Water-stress Salt-stress Chill-stress Heat-stress GluTR Protein − − Transcript − + − − GSA-AT Protein + + + + Transcript + + − + ALAD Enzyme Activity − − − − Transcript − − − − PBGD Enzyme Activity − − − − Transcript − − − 0 UROD Enzyme Activity − + Article Protein − − − + Transcript − − − + CPO Enzyme Activity − − − 0 Protein − 0 0 Transcript − − 0 0 Protox Enzyme Activity − − − 0 Protein − − Transcript − − Mg-chelatase Enzyme Activity − − − − Protein 0 − Transcript − − − − MPE cyclase Enzyme Activity − − Transcript − − − − POR Enzyme Activity − − 0 − Protein − 0 0 Transcript − CAO Transcript − ChlP Protein − − Transcript − −

Accepted 39

© 2012 Blackwell Publishing Ltd 1 Data compiled from the present study, Tewari & Tripathy 1998; Mohanty et al. 2006; Mohanty 2 & Tripathy 2011; Satpal & Tripathy unpublished. The 0, + and  denote no change, up- 3 regulation and down-regulation, respectively. Article

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2 Fig 1. Control and water-stressed (50 mM PEG) rice seedlings after 24 h (A and B) and 72 h (C 3 and D) of greening respectively. (E) Shoot and root length of control (black) and water-stressed 4 (grey, patterned) seedlings after 72 h of greening. Six-day old etiolated seedlings were treated 5 with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to 6 cool white fluorescent + incandescent light (100 moles photons m-2 s-1), at 28 0C. To see the 7 difference in greening, of control and stressed samples, rolled leaves from stressed seedlings 8 were spread open and put between two slides to take pictures (B & D).

9 Fig 2. Dry weight, DW (closed symbols) and protein content (open symbols) of control (circle) 10 and water-stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) 11 or 50 mM PEG 6000 (square) dissolved in half strength MS nutrient soln., 16 h prior to the 12 transfer to cool white fluorescent + incandescent light (100 moles photons m-2 s-1), at 28 0C.

13 SeedlingsArticle were harvested at 24 h, 48 h and 72 h of greening and their DW and protein contents 14 were measured. Each data point is the average of three replicates. The error bars represent SD.

15 Fig 3. Total Chl (closed symbols) and carotenoids (open symbols) content of control (circle) and 16 water-stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) or 17 50 mM (square) PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer 18 to cool white fluorescent + incandescent light (100 moles photons m-2 s-1), at 28 0C. Seedlings 19 were harvested at desired time points (24 h, 48 h and 72 h) and their Chl and carotenoids 20 contents were measured. Each data point is the average of three replicates. The error bars 21 represent SD.

22 Fig 4. Chl biosynthesis intermediates after 72 h of greening. Net synthesis of Glutamate 1- 23 semialdehyde (GSA), δ-amino levulinic acid (ALA), MP(E) and Pchlide in control (black) and 24 water-stressed (grey, patterned) seedlings. Six-day old etiolated seedlings were treated with 50 25 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool 26 white fluorescent + incandescent light (100 moles photons m-2 s-1), at 28 0C. The GSA and 27 ALA content was estimated after 4 h of illumination in presence of gabaculine or levulinic acid 28 respectively. Each data point is the average of three replicates and the error bars represent SD.

Accepted 41

© 2012 Blackwell Publishing Ltd 1 Fig 5. Activities of enzymes involved in Chl biosynthesis, control (black) vs 50 mM PEG (grey, 2 patterned) treated seedlings. Six-day old etiolated seedlings were treated with 50 mM PEG 6000 3 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + 4 incandescent light (100 moles photons m-2 s-1), at 28 0C. (A) ALA-dehydratase, (B) 5 Porphobilinogen deaminase (C) Coproporphyrinogen oxidase, (D) Protoporphyrinogen oxidase 6 (E) Mg-chelatase and (F) Protochlorophyllide oxidoreductase activities were measured after 24 h 7 or 72 h of greening as described in materials and methods. Each data point is the average of three 8 replicates. The error bars represent SD.

9 Fig 6. Low temperature (77K) fluorescence emission spectrum (E440) of (A) control and (B) 10 water-stressed (16 h) etiolated seedlings showing Shibata-shift.

11 Fig 7. Gene expression of chlorophyll biosynthetic enzymes. Six-day old etiolated seedlings 12 were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the

13 transferArticle to cool white fluorescent + incandescent light (100 moles photons m-2 s-1) at 28 0C for 14 24 h or 72 h. Total RNA was isolated from control and water-stressed leaves of rice seedlings 15 after 24 h and 72 h of greening and (A) semiquantitative RT-PCR was performed with cDNA 16 made from 2 µg of RNA. (B) Percent intensity of gene expression. Black bars represent control 17 samples after 24 h of greening; white, coarse patterned bars represent water-stressed samples 18 after 24 h of greening; grey, fine patterned and white, crossed bars represent control and water- 19 stressed samples respectively after 72 h of greening.

20 Fig 8. Immnoblot analysis of Chl biosynthetic enzymes. Six-day old etiolated seedlings were 21 treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the 22 transfer to cool white fluorescent + incandescent light (100 moles photons m-2 s-1) at 28 0C. 23 Plastids were isolated from control and water-stressed seedlings after 24 and 72 h of greening. 24 (A) Equal amount of (30 µg) of thylakoid proteins were separated by SDS-PAGE (12.5%). (B) 25 Western blot was performed with different antibodies as described in material and methods. (C) 26 Percent intensity of protein expression. Black bars represent control samples after 24 h of 27 greening; white, coarse patterned bars represent water-stressed samples after 24 h of greening; 28 grey, fine patterned and white, crossed bars represent control and water-stressed samples 29 respectively after 72 h of greening.

Accepted 42

2 Supporting Information 3 4 Additional Supporting Information may be found in the online version of this article: 5 6 Table S1. Gene specific primers used in this study 7 Table S2. Antibodies used in this study 8

11 Article

Accepted 43

14 E ing nn

10 Article

After 72 h of greening 72h of gree rr C 50 mM PEG Control 8 50 mM PEG Control D 6 gth (cm) afte nn 4

2 oot /Root le hh

S 0 Shoot Root

Fig 1. Control and water-stressed (50 mM PEG) rice seedlings after 24 h (A and B) and 72 h (C and D) of greening respectively. E, Shoot and root length of control (black) and water-stressed (grey, patterned) seedlings after 72 h of greening. Six-day old etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. To see the difference Accepted in greening, of control and stressed samples, rolled leaves from stressed seedlings were spread open and put between two slides to take pictures (B & D). 40

6 30 -1 -1

5 Article

, mg shoot 20 in, mg gFW ee WW 4 D Prot

3 10

2 24 h 48 h 72 h Greening period

Fig 2. Dry weight, DW (closed symbols) and protein content (open symbols) of control (circle) and water- stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) or 50 mM PEG 6000

(square) dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. Seedlings were harvested at 24 h, 48 h and 72 h of greening and their DW and protein contents were measured. Each data point is the average of three replicates.

The error bars represent SD. Accepted Accepted 1.0 0.20 080.8 -1

-1 0.6 0.15 Article gFW 0.4 gFW , mg gg ss

0.2 Chl , m 0.10

0.0 Carotenoid

-0.2 0.05

-0.4 24 h 48 h 72 h Greening period

Fig 3. Total Chl (closed symbols) and carotenoids (open symbols) content of control (circle) and water-stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) or 50 mM (square) PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. Seedlings were harvested at desired time points (24 h, 48 h and 72 h) and their Chl and carotenoids Accepted contents were measured. Each data point is the average of three replicates. The error bars represent SD. 300 A 10 BC4 18 D 16 250 -1

8 -1

-1 14 -1 3 200 12 6 10 olsFW g Article ols g FWols olsFW g olsFW g 150 2 mm mm mm mm 8

μ 4 100 6

GSA, GSA, 1 ALA, ALA, n 4 2 MP(E), n 50 Pchlide, n 2

0 0 0 0

Fig 4. Chl biosynthesis intermediates after 72 h of greening. Net synthesis of Glutamate 1-semialdehyde (GSA), δ-amino levulinic acid (ALA), MP(E) and Pchlide in control (black) and water-stressed (grey, patterned) seedlings. Six-day old etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. The GSA and ALA content was estimated after 4 h of illumination in presence of gabaculine or levulinic acid respectively. Each data point is the average of three replicates and the error bars represent SD. Accepted Accepted opoiioe emns C Copropor (C) deaminase Porphobilinogen h rnfrt olwiefurset+icnecn ih (100 light incandescent + fluorescent white cool to transfer the Six 5. Fig uts ciiiswr esrdatr2 r7 fgenn sdsrbdi aeil and materials in described as greening of h 72 thre or of average h the 24 is point after data measured Each methods. were activities oxidoreductase Protochlorophyllide - day D A Proto IX, µmols 100 mg protein-1 h-1 ciiiso nye novdi h isnhss oto ( control biosynthesis, Chl in involved enzymes of Activities PBG, nmols mg protein-1 h-1 0.0 0.5 1.0 151 2.0 2.5 old 10 20 30 40 . 5

Accepted Article0 24 hof greening etiolated 72 hof greening seedlings were B E MP(E) nmols 100 mg protein-1 h-1 treated Porphyrins, nmols mg protein-1 h-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 5 6 72 h of greening hrngnoiae D Protoporphyr (D) oxidase, phyrinogen 24h of greening with elcts h ro asrpeetSD. represent bars error The replicates. e 50 mM PEG 6000 C

oe htn m photons μmoles POR activity, relative percent F -1 -1 phototransformation Proto IX, nmols 100 mg protein h 100 120 10 20 30 40 50 60 70 lc)v 0m E ge,ptend rae seedlings. treated patterned) (grey, PEG mM 50 vs black) 20 40 60 80 dissolved 0 0 24 h of greening 24 hof 72 h of h 72 greening in half ngnoiae()M-hlts n (F) and Mg-Chelatase (E) oxidase inogen -2 s strength -1 ,a 28 at ), MS 0 .()AAdhdaae (B) ALA-dehydratase, (A) C. nutrient soln ., 16 h prior to A

Fig 6. Low temperature (77K) fluorescence emission spectra (E440) of (A) control and (B)

Article water-stressed (16 h) etiolated

seedlings shihowing Shibata-shifhift. B Accepted Accepted Con, 24h WS, 24hCon, 72h WS, 72h Fig 7. Gene expression of chlorophyll A biosynthetic enzymes. Six-day old etiolated HemA1 seedlings were treated with 50 mM PEG 6000 gsa dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + Alad incandescent light (100 μmoles photons m-2 s-1), pbgd at 280C for 24 h or 72 h. Total RNA was isolated from control and water-stressed leaves UroD of rice seedlings after 24 h and 72 h of greening and (A) semiquantitative RT-PCR was CPO performed with cDNA made from 2µg of RNA. (B) Percent intensity of gene expression. Black PPX‐I Article bars represent control samples after 24 h of Chlorina 1(ChlD)greening; white, coarse patterned bars represent water-stressed samples after 24 h of greening; Chlorina 9(Chl I) grey, fine patterned and white crossed bars represent control and water-stressed samples HemH2 respectively after 72 h of greening. Xantha‐l homolog (Chl27) Por B Rac2

B 250 Control, 24 h of greening 200 W ater stress, 24 h of greening Control, 72 h of greening 150 W ater stress, 72 h of greening y Units rr 100

50 Arbitra

Accepted Accepted HemA1 gsa Alad Pbgd UroD CPO PPX-1 Chlorina1Chlorina9 HemH2 Xantha-l Por B Con, 24h WS, 24h Con, 72h WS, 72h B A 97kD

66kD GSA-AT

43kD UroD

29kD CPO PPOX I 20kD Chl I POR 14kD ChlP

C 200 Control, 24 h of greening Water stress, 24 h of greening 150 Control, 72 h of greening 100 Water stress, 72 h of greening

Arbitrary Units Arbitrary 0 GSA-AT UroD CPO PPOX-1 Chl l POR Chl P

Fig 8. Immnoblot analysis of Chl biosynthetic enzymes. Six-day old etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. Plastids were isolated from control and water-stressed seedlings after 24 and 72 h of greening. (A) Equal amount of (30µg) of plastid proteins were separated by SDS-PAGE (12.5%). (B) Western blot was performed with different antibodies as described in material and methods. (C) Percent intensity of protein expression. Black bars represent control samples after 24 h of greening; white, coarse patterned bars represent water-stressed samples after 24 h of greening; grey, fine patterned and white crossed bars represent control and water-stressed samples respectively after 72 h of greening. Article Accepted