SUMMARY Chlorophyll biosynthesis:

Transcription

1 Summary

2 SUMMARY Plants are severely affected by various abiotic stresses due to their static nature. Abiotic stresses may be in the form of drought, salt, flooding, temperature, light or metal stress. Due to global warming many crops in various parts of the world are being subjected to drought stress and in the coming years this problem is going to become more severe. Plant growth and development is severely affected due to water stress. Photosynthesis is one of the most important factors determining the plant productivity under water limiting conditions. When plants germinate in the soil their seedlings remain in dark for a while. These seedlings do not synthesize chlorophyll and contain a special form of plastids called etioplasts. As seedlings come out of soil chlorophyll biosynthesis starts and etioplasts convert into chloroplasts. This process is called chloroplast biogenesis. Chloroplast biogenesis is a complex process and is regulated by light and other environmental factors like abiotic stress. Chloroplast biogenesis is intricately linked with photosynthesis, so the effect of drought stress on chloroplast biogenesis and photosynthesis was investigated. Because rice is a very important food crop in southern part of Asia including India, a drought-sensitive and high yielding cultivar of rice, Oryza sativa L cv Pusa Basmati 1, (PB1) was chosen for this study. Water stress was applied on six-day old etiolated seedlings by immersing their roots in 0 mm PEG 6000 or 30 mm PEG 6000 or 40 mm PEG 6000 or 50 mm PEG 6000 (half - strength Murashige & Skoog salt solution [vitamin and agar free] + 50 mm PEG 6000), 16 h prior to transfer to cool white fluorescent+incandescent light (100 µmol m -2 s -1 ), at 28 0 C. Protein contents of rice seedling declined in response to increasing water stress. Chlorophyll (Chl) and carotenoids contents were also reduced to various extents due to water stress. There was a minor decrease at 30mM PEG 6000 mediated water stress. But there was an increased Chl and carotenoids reduction in the seedlings as the PEG concentration was increased. Chl a/ Chl b ratio decreased in water-stressed seedlings. 50mM PEG 6000 concentration was chosen for further analysing the effect of water stress on rice seedlings. Chlorophyll biosynthesis: Chl biosynthetic intermediates glutamate-1-semialdehyde (GSA), 5 -aminolevulinic (ALA), Mg-protoporphyrin monomethyl (ester) (MP(E)) and protochlorophyllide (pchlide) were decreased in rice seedlings in response to water stress. After 72 h of greening, net accumulation of glutamate-1-semialdehyde, ALA, MP(E) and pchlide decreased by 55%, 80%, 53% and by 78% respectively in response to water stress.

3 Activity of chlorophyll biosynthesis pathway enzymes were decreased to various extents. Both, ALA dehydratase (ALAD) and Porphobilinogen deaminase (PBGD) activity decreased by almost 33 % after 72 h of greening. Coprogen oxidase, Protoprophyrinogen oxidase (protox) and Mg-chelatase activity decreased respectively by 33%, 38% and 50% in stressed seedlings after 24 h of greening. Relative Protochlorophyllide oxidoreductase (POR) activity, measured as percent phototransformation of pchlide, decreased by 62 % when measured in 72 h light-exposed stressed seedlings. GSAT and ChlI protein abundance increased due to water stress. UROD and Coprox decreased slightly but PPXI, POR and ChlP protein abundance was highly reduced in stressed seedlings. Gene expression was checked in water-stressed seedlings after 24 h and 72 h of greening. Except Gsat which was increased, gene expression of most of the enzymes of chlorophyll biosynthesis pathway was decreased to various extents. Coprox and HemA were decreased to lesser extent but Alad, Pbgd, Ppx1, Chlorina 1, Chlorina 9 and PorB decreased to high extents at h of greening. HemH and Mpec (Chl 27 homologue) were more decreased after 24 h of greening than at 72 h of greening. Shibata shift: POR-NADPH-Pchlide complex in etiolated seedlings has been studied by Shibata shift. Water stress caused a delayed Shibata shift in etiolated rice seedlings. Photosynthesis: Chl a fluorescence was used to measure photosynthetic parameters. F 0 declined after 72 h in response to water stress due to decrease in Chl contents. F v /F m declined by 30 % in 50 mm PEG induced water stress. Electron transport rate (etr), Photochemical quenching (qp), Quantum yield of photosystemii ( PSII) declined and Non-photochemical quenching (qn) increased in response to water stress. Whole chain activity decreased in response in response to water stress. PSI activity is partially affected in response to stress, however PSII activity was affected significantly.light saturation curve of PSII and PSI showed that both initial slope at low light intensity and saturated electron transport were affected in water-stressed seedlings due to damage in reaction centre core. Room temperature spectra showed a decrease in PSII fluorescence at 684 nm. To understand the structural organization of thylakoid membranes, low temperature (77K)

4 spectra were recorded in water-stressed seedlings after 72 h of greening. Increase in F686/F740 ratio in the presence of Mg 2+ was to the same extent in water-stressed seedlings as in control seedlings. Ultrastructure of chloroplast in stressed seedlings showed had swollen thylakoids and disintegration of granal-thylakoids. Immunoblotting of some photosynthetic proteins (Cyt b559, OEC16, 23, 33, Cyt b 6 /f sub IV, Cyt f, PSI-III, PSIV, PSV and PSVI) and antennae proteins Lhcb1, Lhcb2 and Lhca4 was performed to see the effect of water stress. Protein abundance of PSII proteins such as Cytb559, LHCPII, Lhcb1, Lhcb2, OEC16, OEC23 and OEC33 decreased in response to water stress. However there was a minor decrease in Cytb 6 /f components (Cytf, Cytb 6 /fsubunit IV). PSI components (PSI-III, PSI-V and PSI-VI) were decreased which showed that photosynthetic performance of PSI was regulated at protein level However PSI-IV subunit showed increased protein abundance. Transcript abundance of photosynthetic proteins PsbA, PsbD and PetD showed no significant changes due to water stress. PsbB transcript, which encodes for antenna protein CP47 was down-regulated, however Lhca4 encoding for PSI antenna protein was upregulated in rice seedlings under water stress conditions. Reactive oxygen species: In rice seedlings, Superoxide dismutase activity increased in response to water stress after h of greening. One Cu/Zn-SOD and one Fe-SOD isoform was increased in stressed rice seedlings. After 72 h of greening two-guaiacol peroxidase isoforms in stressedseedlings showed more activity than control. Catalase activity was decreased due to the reduction in the activity of a high molecular weight isoform in stressed-seedlings as seen in native gel assay. Gene expression of antioxidative enzymes such as Cu/Zn-SOD (SOD Cc2), Ascorbate peroxidase ( APx2) and Glutathione reductase ( GRase) increased in response to stress. Some of the genes encoding for known proteins involved in stress tolerance such as OsCIPK15, OsDREB2 and OsRAB7 were induced in response to water stress. MDA content increased in response to water stress after 72h of greening. After h of greening, H 2 O 2 production increased in rice seedlings due to water stress. Tocopherols contents were higher in stressed seedlings after 72 h of greening. Percentage of tocopherol in total tocopherol contents of rice seedlings increased due to

5 water stress which resulted in decrease of tocopherol ratio by half in stressed plants to that of control. Infra-Red Gas Analyser (IRGA) studies Nine day old green seedlings were treated with 40 mm PEG + nutrient soln. and IRGA data were taken after 24 h of stress. Light Saturation Curve of control seedlings showed saturation of Photosynthesis around 500 mol photons m -2 s -1 when measured at a Ca of 800 mol CO 2 mol -1 whereas water-stressed seedlings showed light saturation at same intensity but the photosynthesis was reduced by 60%. Highest photosynthesis rate in A-Ci curve reduced to 47% of control at maximum Ci (750 mol CO 2 mol -1 ) achieved in stressed seedlings at Ca of 1400 mol CO 2 mol -1. However extrapolation of fit curve showed saturation of photosynthesis at 10.5 mol CO 2 m -2 s -1 in stressed leaves, which was 62 % to that of control. Photosynthesis compensation point increased greatly in stressed seedlings from 39 mol CO 2 mol -1 in control seedlings to 190 mol CO 2 mol -1 in water-stressed seedlings. Proteomics: 2-DE gel electrophoresis of PEG fractionated soluble proteins showed that many proteins were differentially regulated in response to water stress. Proteins that were upregulated in 50 mm PEG 6000 induced water stressed rice seedlings after 72 h of greening could be clustered into various groups which included signaling (Receptor like kinase, RLK; putative receptor kinase; Phosphatidyl inositol 4-phosphate 5-kinase 4, PIP5K; Calcineurin like phosphoesterase), transcription (C 2 H 2 Zn-Finger; C3H Zn finger; RNA binding protein), retrotransposon (Ty-1 Copia type; Plant disease resistance protein; putative retrotransposon MLA-1), antioxidant defense mechanism (Ascorb ate peroxidase, APX and Dehydro ascorbate reductase, DHAR), cytoskeleton organization (Kinesin Heavy chain like), cell homeostasis (Cyt P450 and Glucosyl-galactosyl transferase), specific functions (SALT; IAA amino acid hydrolase and a protein similar to Atrophin-1) and uncharacterized proteins. Down-regulated proteins were grouped as photosynthesis light reaction (OEC23, OEC33), carbon fixation (Ribulose 1, 5 -bisphosphate carboxylase / oxygenase, Rubisco; Fructose 1, 6-bisphosphatase aldolase, FBP aldolase; phosphoglycerate kinase, PGK; triose phosphate isomerase, TPI), protein folding and degradation (Peptidyl prolyl isomerase,

6 PPIase and Lon protease), energy balance (putative ATP synthase delta chain; inorganic pyrophosphatase and mitochondrial formate dehydrogenase precursor, Epstein Barr Virus EBNA-1-like), transcription (RNA binding pherophorin like protein; mitochondrial transcription termination factor, mterf and cold shock domain containing protein), retrotransposon (NBS -LRR disease resistance protein and a putative retrotransposon), cytoskeleton organization (transducin /WD -40 domain containing), cell homeostasis (Cyt P450, CYP79A1; glycerol 3-phosphate dehydrogenase, GPDH; similar CRA_b having SAM synthase domain containing; TRX domain containing; cystein cynthase; aminotransferase like protein; malate dehydrogenase, MDH and anion transporting protein), protein synthesis (L18Ae ribosomal protein) and unknown/ uncharacterized proteins. Differential expression of proteins from so many categories suggests the complex pathways involved in water stress manifestation and adaptation in rice seedlings.