Download Abstract - Plant Sulfur Research

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.