Title page
Article title：
Overexpression of a phytochrome-regulated tandemzinc finger protein gene, OsTZF1, confers hypersensitivity to ABA and hyposensitivity to red light and far-red light in rice seedlings
Journal name:
Plant Cell Reports
Author names:
Cheng Zhang1, 2, Fang Zhang3, Jinjun Zhou1, 4, Zhongxue Fan1, 4, Fan Chen3, Huiquan Ma2, Xianzhi Xie1, 4*
Affiliations:
1 High-Tech Research Center, Shandong Academy of Agricultural Sciences，Jinan 250100, People’s Republic of China
2 College of Life Sciences, Shandong University of Technology, Zibo 255049, People’s Republic of China
3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
4 Laboratory of Crop Genetic Improvement and Biotechnology, Huanghuaihai, Ministry of Agriculture, Jinan 250100,People’s Republic of China
Corresponding author:
*Corresponding author. Tel.: 86-531-8317-8433; fax: 86-531-8317-8156. E-mail address: (X. Xie).
Supplementary Table 1 Fold changes of transcript levels of some differentially expressed genes in the WT relative to phyB mutants.
Accession number a / Annotation b / Fold change of WT relative to phyB cUp-regulated by phyB
AK059239 / Oryza sativa IAI2 mRNA for wound induced protein homolog, / 3.44±0.19
AK060082 / Unknown protein / 2.36±0.09
AK060202 / Arabidopsis thaliana sulfolipid synthase (SQD2) mRNA / 3.09±0.19
AK060800 / Oryza sativa chlorophyll a/b-binding protein precursor (Cab26) mRNA / 5.19±1.94
AK062101 / Unknown protein / 2.39±0.26
AK063600 / Unknown protein / 2.85±0.17
AK063903 / Unknown protein / 3.61±1.31
AK063904 / Oryza sativa IPS1 mRNA / 3.46±0.21
AK064148 / Arabidopsis thaliana mRNA for monogalactosyldiacylglycerol synthase / 2.68±0.19
AK065433 / N.tabacum mRNA for HSR201 protein. / 3.29±0.64
down-regulated by phyB
AK106392 / Arabidopsis thaliana putative CCCH-type zinc finger protein (At2g19810) mRNA / 0.48±0.02
AK107567 / Arabidopsis thaliana AT5g01210/F7J8_190 mRNA / 0.40±0.09
AK107691 / Unknown protein / 0.41±0.04
AK063186 / Unknown protein / 0.32±0.03
AK063845 / Unknown protein / 0.27±0.03
AK063896 / Unknown protein / 0.37±0.06
AK065150 / Oryza sativa (japonica cultivar-group) RAA1 (RAA1) mRNA / 0.13±0.01
AK065236 / Barley mRNA for NADPH-protochlorophyllide oxidoreductase (PCR / 0.22±0.06
AK069659 / Triticum aestivum mRNA for fatty acyl coA reductase (TAA1a gene). / 0.33±0.10
AK070510 / M.truncatula mRNA for MtN3 gene. / 0.50±0.17
Microarray analysis was performed with WTs and phyB mutants (phyB1, phyB2 and phyB3).
a Accession numbers according to KOME. (
b Annotation and description as shown in KOME (
c Average fold changes are means SE from three independent experiments (phyB1, phyB2 and phyB3 relative to WTs, respectively). For each experiment, genes with fold changes of more than 2.0 or less than 0.5 and a log ratio with a p-value < 0.01 indicated differentially expressed genes.
Supplementary Figure 1. R-induced expression of Lhcb genes in theOsTZF1-overexpression lines and WT seedlings.
WT and the OsTZF1-overexpression lines (#2 and #8) were grown under D or R for 7 days. Transcript levels of two Lhcb genes (Os03g0592500 and Os09g0346500) were analyzed by qRT-PCR (a) and RT-PCR (b). Ubiquitin (UBQ) was used as an internal control
Supplementary Methods
Microarray analysis
The aerial parts of the WTs (Nipponbare and Norin8 cultivars) and phyB1, phyB2, and phyB3 plants at the six-leaf stage were harvested. Total RNA was isolated from each pool using an RNeasy plant mini kit (QIAGEN GmbH, Hilden, Germany). RNA (400 ng aliquots) was labeled with a Low RNA Input Linear Amplification/Labeling Kit (Agilent Technologies) following the manufacturer’s instructions. Aliquots of Cy3-labeled cRNA (0.4 g) from phyB1, phyB2 or phyB3 samples and Cy5-labeled cRNA (0.4 g) from WT samples were used for hybridization to a rice oligo microarray (22K, custom-made; Agilent Technologies) for 16 h at 60 °C, respectively. Microarray background was subtracted and normalized using Feature Extraction software (version A7.1.1; Agilent Technologies) to the median of each experiment and transformed to log ratio using the default linear and lowess methods (Yang et al. 2002). A gene with a fold change of signal intensity of WT relative to phyB greater than 2 or less than 0.5 and a log ratio with a p-value < 0.01 indicated differential gene expression in each experiment (Agilent Reference Guide). The average fold changes of signal intensity for each target gene were calculated based on the fold change of three individual experiments, WT/phyB1, WT/phyB2 and WT/phyB3.
RNA analysis of light-regulatedgenes
The OsTZF1-overexpression lines (#2 and #8) and WT seedlings were grown either in continuous darkness or under R (23 mol photons m−2 s−1) for 6 d. The above-ground parts were harvested.Total RNAs were extracted from the 7-day old seedlings grown in continuous darkness or R by using RNAiso (TaKaRa, Dalian, China)according to the manufacturer’s instructions.First-strand cDNAs were synthesized from DNase I-treated total RNA using PrimeScriptRT Enzyme Mix I (TakaRa) according to the manufacturer’s instructions. Quantitative RT-PCR was performed on the ABI 7500 Fast Real-time PCR system (Applied Biosystems) using SYBR Premix Ex Taq™ (TaKaRa). Each reaction contained 10 µL of 2× SYBR Premix Ex Taq™ (TaKaRa), 2.0 µL of cDNA, and 0.2 µM gene-specific primer pairs in a final volume of 20 µL. The PCR thermal cycle used was as follows: denaturation at 95°C for 30 s and 40 cycles at 95°C for 5 s and at 60°C for 31 s. For Os03g0592500, the primer pair was F1 (5′ –TGAGCACAACGACACGAT- 3′) / R1 (5′ –TCTCCTCGATCGATCACA- 3′). For Os09g0346500, the primer pair was F1 (5′ –GTAGCTTAGCAGTGGTTAATTGT- 3′)/ R1 (5′ –TCTTCATCTTCTTAGTGTACACAAC- 3′). As an internal control, the rice ubiquitin gene (OsUBQ, Os03g13170) was used to quantify the relative level of each target gene(Table 1). Three replicate biological experiments were performed.
For RT-PCR, the cDNA and primer pairs same as listed above were used to analyze the expression of Lhcb genes. The PCR conditions were 94°C for 3 min and 20 cycles of 94°C for 1 min, 50°C, 45°C, or 55°C for 1 min for Os03g0592500,Os09g0346500, or OsUBQ, respectively, and 72°C for 1 min. PCR reagents were obtained from the TaKaRa Taq system(TaKaRa, Dalian, China).
Supplementary Reference:
Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15