Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
IDENTIFICATION OF ADDITIONAL GENES REGULATED BY SULFUR SHORTAGE IN TOBACCO Małgorzata Lewandowska and Agnieszka Sirko1 Department of Plant Biochemistry, Institute of Biochemistry and Biophysics PAS, Pawinskiego 5A, 02-106 Warsaw, Poland Abstract To identify genes regulated by short-term sulfur starvation, the suppression subtractive hybridization (SSH) method was used. This method allows performing a global transcription responses analysis in plants with lack of a complete genomic sequence like Nicotiana tabacum. Results presented in this manuscript are continuation of our approach described previously (Wawrzyńska et al. 2005; Lewandowska et al. 2005). Shortly, the analysis was performed on two months-old plants of Nicotiana tabacum LA Burley 21 treated or untreated with short-term sulfur starvation. Two month-old plants were grown in hydroponic conditions in sulfur sufficient medium and then were divided into two groups. Ten plants from each group were transferred for two days either into sulfursufficient medium or sulfur-deficient medium. After 48 h the plants were harvested and fractionated into four parts, young leaves, mature leaves, stalks and roots. The fractionated parts of 10 plants were then pooled and used for further analysis. Because any visible symptoms of sulfur deficiency had appeared in the plants from S-starved group, molecular and biochemical analysis were performed. Both types of analysis confirmed influence of sulfur starvation on plants from S-starved group, and indicated young leaves as a most affected by sulfur limitation, comparing to other parts of plants (Wawrzyńska et al. 2005). For constructing of the SSH libraries the young leaves were chosen. Two sets of subtracted cDNAs cloned into the pGEM-T Easy vector were obtained, one containing fragments of cDNA of genes putatively up-regulated upon S Sulfur Metabolism in Higher Plants, pp. xx-xx Edited by A. Sirko et al. 2009 Backhuys Publishers, Leiden, The Netherlands starvation, and second containing fragments of cDNA of genes putatively downregulated upon S starvation. The differences between earlier described and here presented approach were: (i) nature of the spotted material, (ii) way of denaturation of the spotted material and (iii) semi-mechanical screening of the libraries. On the membrane were spotted not as in the first experiment plasmids isolated from bacteria, but overnight cultures of bacteria grown in 384 wall plates filled with LB medium with ampicillin. The bacteria’s cultures were spotted using BioGrid robotic system on the nylon membrane saturated with denaturating buffer. After neutralization step further procedure was similar to the procedure used in the first approach. Identification of the clones was a little bit difficult, probably because of the contamination from lysated bacteria’s cell, which was left on the membrane despite addition washing steps. After the primary screen, plasmids with cDNA representing the putatively regulated genes were isolated from the bacterial culture and re-tested in two rounds of hybridizations as described in Wawrzyńska et al. (2005). The positive verified plasmids were selected for sequencing. The entire procedure resulted in identification of cDNA fragments corresponding to 5 up-regulated and 17 down-regulated independent genes. Some of genes identified in this screen were also identified in earlier experiments (Wawrzyńska et al. 2005; Table 1.). Table 2 contains the list of the cDNAs corresponding to the genes identified solely in present experiment. Table 1. The up- and down regulated genes identified in earlier experiments. Clones identified GenBank In earlier accession no. experiments UP-REGULATED CLONES UP32 UP28 CO046514 UP35 U19 CO046507 UP36 DOWN-REGULATED CLONES D32 D-8a AY547456 D28 D29 D-10b AY547457 Clone D33 D38 D37 D42 D48 D25c CO046506 D7 D13 D21 AY547455 CO046499 CO046503 Homology Putative ID Histone H3 LHC-I chlorophyll a/b binding protein Subunit X (PsaK) of PSI H subunit of glycine decarboxylase multienzyme complex Subunit A of chloroplast glyceraldehyde-3phosphate dehydrogenase Aquaporin 1 NtpII, 10kDa peptide of PSII (PsbR) CAB36 gene for chlorophyll a/b binding protein a two clones isolated (D8, D11) clones isolated (D10, D24) cthree clones isolated (D16, D19, D25) btwo Regulation of clones was verified using the quantitative RT-PCR method in young leaves (selected clones, Fig.1A) and mature leaves (all clones despite UP37, Fig. 1B). We were not able to confirm the regulation of UP37 (not shown). In the case of D40 the regulation observed using qRT-PCR method was very slight, while D26 seemed to be down regulated only in young leaves. As a control were used: in young leaves UP9, which was found in our laboratory to be induced by sulfur starvation, in mature leaves up-regulated APR and also found in our laboratory down-regulated D21 corresponding to identified in this screen clone D48. Table 2. The up- and down-regulated genes identified present screen. Clone Homology/Putative ID UP-REGULATED CLONES UP33 phylloplanin UP34 extensin UP37 Oxygen-evolving enhancer protein 1, chloroplast precursor (OEE1) (33 kDa subunit of oxygen evolving system of photosystem II) DOWN-REGULATED CLONES D26 Chlorophyll a-b binding protein 7 D35 D30 D27 D31 D34 D41 D39 D40 D36 D45 D46 D47 D49 BLAST program * Matching sequence** (Probability) /plant N N N, X DQ451214.1 (0.0)/N. tabacum X71602.1 (0.0)/ N. tabacum AY220076.1 (0.0)/ N. tabacum Q40459 (2e-135)/N. tabacum N, X M21317(2e-176)/ Petunia x hybryda P10708 (2e-60)/ S. lycopersicum uroporphyrinogen decarboxylase plastidic aldolase NPALDP1 putative elongation factor 1gamma-like Elongation factor 1, gamma chain; Glutathione S-transferase, C-terminal; Thioredoxin-like fold chloroplast chlorophyll a-b binding protein N N N, X X82833.1 (7e-128)/ N.tabacum AB027001.1 (7e-179)/ N. paniculata DQ241837.1 (6e-151)/ S. tuberosum ABB87108.1 (2e-51)/ S. tuberosum ABE91935.1| (3e-49)/ M. truncatula N, X 60S ribosomal protein L22-2 (RPL22B) geranylgeranyl reductase aldose 1-epimerase family protein protochlorophyllide oxidoreductase chlorophyll a/b binding protein Cab-4 X AY554168 (2e-63)/ N. tabacum AAW31513 (3e-25)/ P. sativum NP_187207 (5e-28)/ A. thaliana N N N, X N AJ007789.1 (9e-117)/ N. tabacum NP_194104.1 (9e-62)/ A. thaliana AB074571.1 (4e-100)/ N. tabacum BAB93004.1| (3e-20)/ N. tabacum AY219853.1 (5e-155)/ N. tabacum M17558.1(1e-96)/ S. lycopersicum D50 D43 D44 *BLAST program used for homology search: N, blastn and X, blastx using nr databases; E, blastn using EST database at NCBI (http:://www.ncbi.nlm.nih.gov/BLAST/), T, blastn at TIGR using databases for the selected plants (http://tigrblast.tigr.org/tgi/) **GenBank/EMBL/DDBJ/TIGR accession number for known sequences according to the BLAST search A) young leaves Normalized Fold Expression 3 2,5 2 nS 1,5 2d-S 1 0,5 0 UP9 UP33 D26 D27 D31 D34 D40 B) mature leaves Normalized Fold Expression 3 2,5 2 1,5 nS 2d-S 1 0,5 AP R U P3 3 U P D 48 34 /D 21 D 26 D 27 D 31 D 34 D 39 D 40 D 45 D 46 D 47 D 49 D 50 0 Fig. 1. Regulation of identified cDNAs using RT-PCR in young leaves (A) and mature leaves (B). The expression level of particular cDNAs was normalized to expression of these genes under control condition (1). UBQ2 and Tac9 were used as reference genes. NS, plants grown at optimal condition, 2d-S, plants grown for 2 days in medium without sulfur. As in earlier approach, the identified cDNAs may be divided in several functional categories, such as: stress-related/pathogen related (UP33, UP34, D37/D7), photosynthesis (UP35/UP19, UP37, D26, D32/D8, D39, D42/D13, D48/D21, D49, D50), related to photosynthesis/chlorophyll synthesis (D27, D46, D49), energy metabolism (D29/D10, D31, D33/D25, D47), protein synthesis (D34, D40, D45), DNAbinding (UP32/UP28). We suppose that presence of both up-, and down-regulated cDNA in some categories might give plants possibility to control an optimal level of particular processes in the condition of sulfur limitation. Acknowledgements: To Victoria Nikiforova and Rainer Hoefgen from Max Planck Institute of Molecular Plant Physiology Golm for possibility to perform the semimechanical screening of the libraries. This work was supported by grant PBZ-KBN110/P04/2004 from MNiSW. References Lewandowska M., Wawrzynska A., Kaminska J., Liszewska F. and Sirko A. 2005. Identification of novel proteins of Nicotiana tabacum regulated by short term sulfur starvation. In: Saito K., De Kok L.J., Stuhlen I., Hawkesford M.J., Schnug E., Sirko A. and Rennenberg H. (eds.), Sulfur Transport and Assimilation in Plants in the Postgenomic Era, pp. 153-156. Backhuys Publishers, Leiden. Wawrzynska, A., Lewandowska, M., Hawkesford, M.J. and Sirko, A. 2005. Using a suppression subtractive library-based approach to identify tobacco genes regulated in response to shortterm sulphur deficit. J. Exp. Bot. 56: 1575-1590.