Plant Mol Biol DOI 10.1007/s11103-015-0288-z
A maize phytochrome‑interacting factor 3 improves drought and salt stress tolerance in rice Yong Gao · Wei Jiang · Yi Dai · Ning Xiao · Changquan Zhang · Hua Li · Yi Lu · Meiqin Wu · Xiaoyi Tao · Dexiang Deng · Jianmin Chen
Received: 10 October 2014 / Accepted: 23 January 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Phytochrome-interacting factor 3 (PIF3) activates light-responsive transcriptional network genes in coordination with the circadian clock and plant hormones to modulate plant growth and development. However, little is known of the roles PIF3 plays in the responses to abiotic stresses. In this study, the cloning and functional characterization of the ZmPIF3 gene encoding a maize PIF3 protein is reported. Subcellular localization revealed the presence of ZmPIF3 in the cell nucleus. Expression patterns revealed that ZmPIF3 is expressed strongly in leaves. This expression responds to polyethylene glycol, NaCl stress, and abscisic acid application, but not to cold stress. ZmPIF3 under the control of the ubiquitin promoter was introduced into rice. No difference in growth and development between ZmPIF3 transgenic and wild-type plants was observed under normal growth conditions. However, ZmPIF3 transgenic plants were more tolerant to dehydration and salt stresses. ZmPIF3 transgenic plants had Yong Gao and Wei Jiang have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0288-z) contains supplementary material, which is available to authorized users. Y. Gao · W. Jiang · Y. Dai · N. Xiao · H. Li · Y. Lu · M. Wu · X. Tao · J. Chen (*) College of Bioscience and Biotechnology, Yangzhou University, 88 South University Ave, Yangzhou 225009, Jiangsu, People’s Republic of China e-mail: [email protected] Y. Gao · W. Jiang · Y. Dai · N. Xiao · C. Zhang · H. Li · Y. Lu · M. Wu · X. Tao · D. Deng · J. Chen Jiangsu Key Laboratories of Crop Genetics and Physiology and Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, Jiangsu, People’s Republic of China
increased relative water content, chlorophyll content, and chlorophyll fluorescence, as well as significantly enhanced cell membrane stability under stress conditions. The overexpression of ZmPIF3 increased the expression of stressresponsive genes, such as Rab16D, DREB2A, OSE2, PP2C, Rab21, BZ8 and P5CS, as detected by real-time PCR analysis. Taken together, these results improve our understanding of the role ZmPIF3 plays in abiotic stresses signaling pathways; our findings also indicate that ZmPIF3 regulates the plant response to drought and salt stresses. Keywords Abiotic stress tolerance · Expression pattern · Morphological character · Physiological trait · Transcription factor · Zea mays
Introduction Plants are constantly confronted by environmental stressors, including drought, high salinity, and extreme temperatures. These stressors affect both biomass and grain yield in crops. Plants have evolved different adaptive strategies to alleviate the adverse effects of these abiotic stresses; these tolerance or resistance mechanisms are mostly controlled by networks regulated by transcription factors (Tian et al. 2013). Transcription factors specifically bind to the cisacting elements of genes to activate or repress the expression of downstream target genes (Li et al. 2013). There is compelling evidence that stress-responsive genes, such as transcription factors, function in multiple pathways and facilitate crosstalk between different stress signaling pathways (Ludwig et al. 2004; Campo et al. 2012). The phytochrome (phy) family of sensory photoreceptors regulates plant development in response to informational light signals (Schafer and Nagy 2006). Phy interacts
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with members of the basic helix-loop-helix (bHLH) family of phytochrome-interacting factors (PIFs) that are involved in the modulation of light-regulated genes with roles in photomorphogenesis. The PIF family comprises 15 members (Toledo-Ortiz et al. 2003); most of these proteins are involved in light signaling (de Lucas and Prat 2014). The PIFs previously examined (PIF1, PIF3, PIF4, PIF5 and PIF7) bind to a core DNA G-box motif (CACGTG) in a sequence-specific manner (Toledo-Ortiz et al. 2003; Leivar et al. 2008; de Lucas et al. 2008). This suggests the existence of a direct signaling pathway between photoactivated phy molecules and their target genes. Almost all of these PIFs contain a conserved N-terminal sequence known as the active phytochrome B (APB) motif. This motif is necessary and sufficient for phyB-specific binding (Khanna et al. 2004). PIF1 and PIF3 each contain an additional domain, called the active phytochrome A-binding (APA) region, that is necessary for phyA binding (Al-Sady et al. 2006; Shen et al. 2008). Current evidence indicates that the intra-nuclear binding of the Pfr form of phyA and/or phyB to PIF proteins induces the rapid phosphorylation and degradation of these PIF transcription factors (Fairchild et al. 2000; Khanna et al. 2004); these may be the primary molecular events in phy signaling. PIF3, the foundational PIF member, was initially identified in a yeast two-hybrid screen for phyB-interacting proteins (Ni et al. 1999; Shimizu-Sato et al. 2002). A mutation in this factor results in an impaired response to red (R) and far-red (FR) light. This result suggests that PIF3 plays a role in the early steps of light signal transduction (Kim et al. 2003; Monte et al. 2004). The data also demonstrate that phy induces the rapid phosphorylation of PIF3 as a result of this interaction (Al-Sady et al. 2006), resulting in ubiquitylation and degradation (Park et al. 2004; Al-Sady et al. 2006). Subsequent studies revealed that this degradation is initiated by the direct binding of either phyA or phyB to their respective APA or APB interaction sites on PIF3 in the nucleus (Al-Sady et al. 2006, 2008). Therefore, PIF3 might represent the primary biochemical mechanism of signal transfer from photoactivated phy molecules to signaling partners within the cell (Leivar and Quail 2011). A number of studies have shown that PIF family members play a role in plant growth and development (Leivar and Quail 2011). As an important phytochrome signaling pathway hub, PIF integrates multiple signals to orchestrate the regulation of the transcriptional network that drives multiple facets of downstream morphogenesis. The data indicate that PIFs are involved in repressing seed germination (Oh et al. 2009), regulating de-etiolation (Lorrain et al. 2009; Chen et al. 2013), promoting seedling skotomorphogenesis (Leivar et al. 2009) and shade-avoidance (Lorrain et al. 2008; Keller et al. 2011). These complex networks of regulators underscore the pivotal role of PIFs
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in plant growth and development. Moreover, an increasing number of studies in recent years have identified PIF family members as key components of signaling pathways in addition to the well-characterized photoregulated pathway. These pathways converge at the PIFs and their close relatives, which regulate an array of developmental responses; these include the circadian clock (Nozue and Maloof 2006), high temperature (Koini et al. 2009; Stavang et al. 2009; Franklin et al. 2011) and plant hormones. For example, PIF4 functions in high-temperatureinduced hypocotyl elongation in de-etiolated Arabidopsis seedlings (Koini et al. 2009). Over the last few years, PIF family members have also been shown to be involved in the biosynthesis and signaling pathways of hormones (de Lucas and Prat 2014) such as gibberellin (GA) (de Lucas et al. 2008; Feng et al. 2008), abscisic-acid (ABA) (Kim et al. 2008; Oh et al. 2009), brassinosteroids (BRs) (Bai et al. 2012; Jaedicke et al. 2012;de Lucas and Prat 2014) and auxin (Franklin et al. 2011; Hornitschek et al. 2012; Sun et al. 2012). A number of studies have demonstrated that DELLA proteins, a class of nuclear transcriptional regulators, repress GA signaling and restrict plant growth (Achard and Genschik 2009). The interaction of DELLAs with PIF3 and PIF4 provide evidence for a crucial role for PIFs in the integration of both GA and light signaling (de Lucas et al. 2008; Feng et al. 2008; Achard and Genschik 2009). Recent studies have also found a function for DELLAs under abiotic stress (Achard et al. 2006, 2008; Navarro et al. 2008). DELLA proteins restrict plant growth during salt and cold stress to improve plant survival (Achard et al. 2006, 2008; Achard and Genschik 2009). In addition, PIF1/ PIL5 plays a pivotal role in mediating this response by regulating the expression of ABA-related genes and promoting ABA biosynthesis (Kim et al. 2008; Oh et al. 2007, 2009). ABA can regulate the plant adaptation to environmental stressors, including cold, drought, and salt (Spartz and Gray 2008). PIFs play broader roles than previously appreciated, functioning as cellular signaling hubs that integrate multiple signals to regulate the transcriptional network that drives multiple facets of downstream morphogenesis. PIFs can also regulate DELLA proteins and endogenous ABA in response to abiotic stress. However, the detailed roles of PIFs in the abiotic stress response remain to be elucidated. Maize is one of the most widely grown crops in the world and important for food and edible oil. In this study, a PIF transcription factor member from maize known as ZmPIF3 was cloned. The expression of this gene in response to water deficiency, high salinity, and ABA was described. Transgenic experiments indicated that the over-expression of ZmPIF3 in rice enhanced tolerance to drought and salt stresses without growth retardation; these findings suggest that the gene could be used in crop plants to improve tolerance to drought and salt stress.
Plant Mol Biol
Materials and methods Plant materials and stress treatment Maize (Zea mays, hybrid line ‘Zhengdan 958’) seeds were sterilized and germinated on wet filter paper for 6 days with a 16/8 h light/dark cycle at 25 °C. The seedlings were transferred to hydroponic growth conditions. Plants were grown in troughs filled with aerated nutrient solution under controlled conditions (28/26 °C, 16 h photoperiod, 70 % relative humidity). The full-strength nutrient solution had the following concentrations: 2.0 mM Ca(NO3)2, 1.0 mM K2SO4, 0.2 mM KH2PO4, 0.5 mM MgSO4, 2.0 mM CaCl2, 5.0 μM H3BO3, 2.0 μM MnSO4, 0.5 μM ZnSO4, 0.3 μM CuSO4, 0.01 μM (NH4)6Mo7O24, 200 μM Fe–EDTA. In this study all the stress treatments were performed with four-leaf-stage maize seedlings. For PEG, high-salinity and abscisic acid (ABA) treatment, the seedlings were grown hydroponically in nutrient solution supplied with 20 % PEG (molecular weight 6,000), 200 mM NaCl or 100 μM ABA respectively, and then sampled at 0, 1, 3, 6, 12, 24 and 48 h. For cold stress, seedlings were transferred to a growth chamber at 4 °C. Leaves were sampled at 0, 1, 3, 6, 12, 24 and 48 h after treatment. Immediately after excision, all samples were frozen in liquid nitrogen and stored at −80 °C until use. Rice (Oryza sativa L. Wuyunjing) for transgenic analysis was grown in a controlled environment chamber at 28/25 °C with a 16/8 h photoperiod and 70 % relative humidity. Three T3 homozygous transgenic lines with higher expression levels of the target gene were selected for phenotypic assays, and all seeds used for phenotypic assays were from the same harvest and stored under the same conditions. Cloning the full‑length cDNA and sequence analysis of ZmPIF3 Using PIF3 gene in Arabidopsis as a querying probe, a novel cDNA sequence was obtained from maize EST database of GenBank by in silico cloning, which was named ZmPIF3 gene. To obtain full-length ZmPIF3 cDNA, a pair of primers (F 5′-ATGTCCGACAGCAGCGACTTCG-3′ and R: 5′-TCATGTTTCAGCCTCATTTCTTCC-3′) were designed based on the lateral flanking sequence of the open reading frame (ORF) of the putative sequence. The obtained sequence ZmPIF3 was analyzed using bioinformatic tools at the websites (http://www.expasy. org/ and http://www.ncbi.nlm.nih.gov/). The nucleotide sequence, deduced amino acid sequence and open reading frame (ORF) were analyzed, and the sequence comparison was conducted through database search using BLAST program (NCBI, National Center for Biotechnology Services,
http://www.ncbi.nlm.nih.gov, http://www.maizegdb.org/). The phylogenetic analysis of PIFs from Arabidopsis thaliana was aligned with DNAMAN (Lynnon Biosoft, Vaudreuil, QC, Canada) using default parameters. A phylogenetic tree was constructed using MEGA version 6. The neighbor-joining method was used to construct the tree. Subcellular localization of the ZmPIF3 protein The ZmPIF3 ORF was fused upstream of the green fluorescent protein gene (GFP) under the control of the CaMV 35S promoter and the NOS terminator in the p2GWF7 expression vector. Proper restriction sites were added to the 5′- and 3′-ends of the coding region using PCR. The primers used were as follows: F: 5′-AATAAAGCTTATGTCCGACAGCAGCGACTT-3′ (HindIII site in bold), and R: 5′-AATACTCGAGTGTTTCAGCCTCATTTCTTC-3′ (XhoI site in bold). The PCR product was digested with the relevant restriction endonucleases and ligated with the p2GWF7 plasmid cut with the corresponding enzyme to create a recombinant plasmid for fusion protein expression. Positive plasmids were confirmed by restriction analysis and sequenced. Recombinant constructs were transformed into living onion epidermal cells by biolistic bombardment with a GeneGun (Biorad Helios) according to the instruction manual (helium pressure, 150–300 psi). After incubation in MS medium at 28 °C for 36–48 h, the onion cells were visualized with a laser scanning confocal microscope (Leica TCS-NT). The subcellular location of ZmPIF3 was detected by monitoring the transient expression of GFP in the onion epidermal cells as described. Quantitative real‑time PCR (qRT‑PCR) Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used to determine the expression patterns of ZmPIF3 in maize. Reverse transcription reactions were performed using total RNA from maize tissues. First-strand cDNA was synthesized from DNase-treated total RNA with Transcriptor Reverse Transcriptase (Roche, Mannhein, IN, Germany) and oligo(dT)18 following the manufacturer’s instructions. Real-time PCR (qPCR) was performed using an ABI 7300 system with a SYBR Green Master Mix kit (Roche, Mannhein, IN, Germany) and specific primers (qPCR, F: 5′-C AATCCAGCCACCATTCCC-3′; R: 5′-CTGTTGCTCCTGCACCATG-3′). Three replicate reactions were routinely performed for each sample. Relative gene expression levels were detected using the 2−ΔΔCT method (Livak and Schmittgen 2001). Actin transcript levels were used to quantify the expression of ZmPIF3 in maize. Three independent biological replicates were analyzed. Controls for the qPCR reactions were performed without the addition of
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the reverse transcriptase enzyme; these were systematically included in our experiments. Transformation of ZmPIF3 in rice The full-length cDNA of ZmPIF3 was amplified from maize with the primers 5′-GAAGATCTATGTCCGACAGCAGCG-3′ (Bgl II site in bold) and 5′-CCGGAATTCTCATGTTTCAGCCTCATTTC-3′ (EcoR I site in bold). The product was ligated into the pMD-19T Easy vector and sequenced. The ZmPIF3 fragment from pMD-19T was digested and inserted into the multiple cloning site of the binary plasmid p1011. In this construct, ZmPIF3 was driven by an ubiquitin promoter. The p1011-ZmPIF3 construct was electroporated into the Agrobacterium tumefaciens strain EHA105 and introduced into rice embryonic calli by A. tumefaciens-meditated methods (Xu et al. 2005). ZmPIF3 transgenic rice plants were selected in 1/2 MS medium containing 50 mg l−1 hygromycin. Phenotypic analyses were performed using T3 homozygous lines. Features of ZmPIF3 transgenic seedlings under dehydration, salt and cold stress Transgenic plants were characterized for morphological changes under a 16/8 h photoperiod in a growth chamber with a constant temperature of 28/25 °C. For drought stress experiments, 2-week-old seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were exposed to 1/2 MS medium containing 20 % PEG for 4 days. Plants were then recovered under normal growth conditions for 10 days. For salt stress experiments, 2-weekold seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were submerged into 1/2 MS medium supplemented with 150 mM NaCl for 2 days. The plants were then transferred into an incubation solution without NaCl for an additional 7 days. For cold stress experiments, 2-week-old seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were subjected to treatment at 4 °C for 2 days. Plants were then transferred to a greenhouse and incubated at a temperature of 28/25 °C with a 16 h photoperiod for 7 days. Abiotic stress tolerance assays Wild-type, vector-only control and transgenic plants grown in soil were used to determine stress tolerance. For drought stress experiments, 40-day-old wild-type, vector-only control and transgenic plants were planted in rectangular pots filled with same soil mixture and well watered. Seedlings were cultured in a greenhouse (28/25 °C with a 16/8 h photoperiod and 70 % relative humidity) without watering for 7 days until phenotypic differences were evident between
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Plant Mol Biol
the transgenic plants and the two controls. The plants were then rewatered. For salt stress assays, rice seedlings were cultured as described above. Water was withheld for 40 days before irrigation was performed with a NaCl solution (150 mM). Irrigation occurred from the bottoms of the pots, and the soil was completely saturated with salt water. When phenotypic differences were evident between the transgenic plants and the two controls, the NaCl solution was removed and the plants were cultured normally. For cold stress experiments, 40-day-old seedlings were transferred into a low-temperature growth chamber (4 °C) for 2 days. Plants were then recovered in a normal growth chamber for an additional 7 days. Relative water content The relative water content (RWC) was measured using 20 plants each from the wild-type, vector-only control and ZmPIF3 transgenic genotypes. Two-week-old plants were detached from the roots, and the leaves of 20 plants were cut into pieces one centimeter in length. Mixed small pieces of fresh samples with a fresh weight (FW) of approximately 0.5 g were selected. Pieces were floated on distilled water in Petri dishes for 4 h to regain turgidity to a constant weight; the tissue was then re-weighed to obtain the turgor weight (TW). The samples were dried at 80 °C for 24 h to determine the dry weight (DW). The relative water contents (RWCs) were measured using the following formula (Schonfeld et al. 1988): RWC (%) = [(FM − DM)/(TM − DM)] × 100 %. These experiments were repeated three times. Chlorophyll content assays Chlorophyll content was measured with a chlorophyll meter (SPAD 502 Plus; Konica Minolta Sensing). Chlorophyll contents under stress conditions were measured after treatment with 20 % PEG for 2 days. One measurement of the fully expanded leaves was made for each plant; 20 plants from each line were used for the chlorophyll content assays. Chlorophyll fluorescence assays Chlorophyll florescence was measured using a portable photosynthesis system (FMS-2 Hansatech UK). The maximum efficiencies of photosystem II (PSII) photochemistry, Fv/Fm = (Fm–F0)/Fm, was employed to assess changes in the primary photochemical reactions of the photosynthetic potential. Chlorophyll florescence under stress conditions was measured after treatment with 20 % PEG6000 for 2 days. One measurement of the fully expanded leaves was made for each plant; 20 plants from each line were used for the chlorophyll florescence assays.
Plant Mol Biol
Cell membrane stability analysis
Results
Plant cell membrane stabilities (CMS) were determined with a conductivity meter (EL30 K; Mettler Toledo). The following formula was used: CMS (%) = (1−initial electrical conductivity/electrical conductivity after boiling) × 100. Two-week-old seedlings were transferred to 1/2 MS medium supplemented with 20 % PEG for 2 days. Seedlings were removed and immediately rinsed with double distilled water (ddH2O). Plants were then immersed in 20 ml ddH2O at room temperature. After 2 h, the initial conductivities of the solutions were recorded. Samples were boiled for 30 min and cooled to room temperature; the final conductivities were then measured.
Molecular characterization of ZmPIF3
Determination of ion concentrations For salt stress analysis, 2-week-old seedlings of the wildtype, the vector-only control and ZmPIF3 transgenic rice were submerged into 1/2 MS medium supplemented with 150 mM NaCl for 2 days. Leaves and roots were ashed for 5 h in an oven at 600 °C. The analysis of cations was performed using the ashes of 200 mg of dried plant materials. The ashes were dissolved in 2 ml concentrated nitric acid with gentle heating. This suspension was adjusted to a volume of 50 ml with distilled water and filtered through a paper filter. Thereafter, Na+ and K+ concentrations were analyzed in the filtrate by means of atomic absorption spectrometry (PE-2100; Perkin Elmer Corporation, USA) and an inductively coupled plasma spectrometer (Optima 7300 DV; Perkin Elmer Corporation, USA). Gene expression analysis Two-week-old seedlings grown on 1/2 MS medium were treated with 20 % PEG6000 or left untreated. Total RNA was extracted using Trizol reagent and treated with RNasefree DNase. For real-time quantitative PCR, total RNA reverse-transcribed with Transcriptor Reverse Transcriptase (Roche, Mannhein, IN, Germany). Quantitative expression assays were performed with the SYBR Green Master Mix kit (Roche, Mannhein, IN, Germany) and an ABI 7300 sequence detection system according to the manufacturer’s protocol (Applied Biosystem). The RT-PCR conditions were as follows: 5 min at 95 °C, and 40 cycles of 15 s at 95 °C, 20 s at 60 °C, and 31 s at 72 °C. Three replicate reactions were routinely performed for each sample. Relative gene expression levels were detected using the 2−ΔΔCT method. Actin transcripts were used to quantify the expression levels of the abiotic stress-response genes in transgenic rice. The primer pairs used for real-time PCR are listed in Supplementary Table S1, which is available at JXB online.
A full-length cDNA of the target gene was obtained by screening full-length maize cDNA libraries. The full-length cDNA sequence, designated as ZmPIF3, contained an open reading frame of 1,914 bp. This ORF encoded a putative 67-kDa polypeptide of 638 amino acids with an isoelectric point of 6.45. NCBI Blast indicated that the deduced amino acid sequence of ZmPIF3 shared a high similarity with the A. thaliana PIFs proteins PIF3 (AT1G09530) (22.87 %), PIF1 (AT2G20180) (21.16 %), PIF4 (AT2G43010) (20.06 %), PIF5 (AT3G59060) (19.44 %), PIF6 (AT3G62090) (15.83 %), PIF7 (AT5G61270) (17.24 %), PIF8 (AT4G00050) (18.25 %) and PIL1(AT2G46970) (16.61 %). Three characteristic domains, the bHLH domain, the APB motif and the APA motif, were found in the deduced ZmPIF3 amino acid sequence (Fig. 1a). The deduced amino acid sequence of ZmPIF3 was further compared with a set of highly homologous PIF family proteins by molecular phylogenetic tree analysis (Fig. 1b). Compared with the other PIF proteins, ZmPIF3 was more closely related to PIF3 in Arabidopsis, VvPIF3 in Vitis vinifera, GmPIF3 in Glycine max, MdPIF3 in Malus domestica, and OsPIL15 and OsPIL16 in rice. Subcellular localization of ZmPIF3 Transcription factors are typically localized to cell nuclei, where they bind DNA and are involved in transcriptional activation. To identify the cellular localization of ZmPIF3, the expression and distribution of ZmPIF3 were examined in transgenic onion epidermal cells. A fusion protein with GFP was expressed, and its localization was visualized using a laser scanning confocal microscope. As shown in Fig. 2, ZmPIF3–GFP was present in the cell nucleus. This result demonstrates that ZmPIF3 localizes to nuclei (Fig. 2). Expression patterns of ZmPIF3 in different tissues and under abiotic stress The expression patterns of ZmPIF3 in different tissues were examined using qRT-PCR analysis. As shown in Fig. 3a, ZmPIF3 was strongly expressed in leaves and pistils; less expression was observed in the roots, stems, stamen, and seed. Various expression patterns were observed under PEG, salt, low temperature, and ABA treatments (Fig. 3b). The expression levels of ZmPIF3 increased significantly under salt stress, PEG stress and ABA treatment conditions. However, ZmPIF3 expression was not significantly
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Fig. 1 Sequence alignment of ZmPIF3 with other Arabidopsis PIF family members. a The comparison of the putative amino acid sequence of ZmPIF3 with the A. thaliana PIF3 protein. The identical amino acids are shown in white with a black background, and the conserved amino acids are shown in white with a gray background. The specific sites of the APB motif, APA motif and bHLH domain are presented. The aligned PIF3 sequences are from A. thaliana (GenBank accession no. AT1G09530). b Phylogenetic tree analysis of ZmPIF3 with other PIF family members. The neighbor-joining method was used to construct the tree. The amino acids sequences used in phylogenetic tree analysis were from plants, including A. thaliana PIF1 (GenBank accession no. Q8GZM7.1); PIF3 (GenBank accession no. O80536.1); PIF4 (GenBank accession no. Q8W2F3.1); PIF5 (GenBank accession no. Q84LH8.1); PIF6 (GenBank accession no. Q8L5W7.1); PIF7 (GenBank accession no. Q570R7.2); PIF8 (GenBank accession no. Q8GZ38.1); PIL1 (GenBank accession no. Q8L5W8.1); HFR1 (GenBank accession no. Q9FE22.1); bHLH127 (GenBank accession no. Q7XHI7.1); bHLH23 (GenBank accession no. Q9SVU6.1); bHLH119 (GenBank accession no. Q8GT73.2); bHLH56 (GenBank accession no. Q9SVU7.2); SPT (GenBank accession no. Q9FUA4.1); ALC (GenBank accession no. Q9FHA2.1); O. sativa OsPIL11 (GenBank accession no. NP_001067246); OsPIL12 (GenBank accession no. NP_001173558); OsPIL13 (GenBank accession no. NP_001051465); OsPIL14 (GenBank accession no. NP_001058876); OsPIL15 (GenBank accession no. NP_001042775); OsPIL16 (GenBank accession no. NP_001054593); V. vinifera VvPIF3 (GenBank accession no. XP_002276198); G. max GmPIF3 (GenBank accession no. XP_006589101); and M. domestica MdPIF3 (GenBank accession no. AEX32796)
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Plant Mol Biol
Plant Mol Biol
Fig. 2 Subcellular localization of ZmPIF3. Full-length ZmPIF3 fused to GFP was transiently transformed into onion epidermal cells. The plant nuclei were stained with DAPI. Images were taken using confocal microscopy (GFP fluorescence, green; DAPI fluorescence,
blue; visible, visible light image; merged, merged images of above three images). Cells transformed with an empty vector (smGFP) are shown as a control. Arrows indicate ZmPIF3-localized nuclei. Bar 100 mm
regulated by cold treatment. The expression patterns and maximum expression levels differed for each stress. For PEG, NaCl and ABA treatments, expression levels peaked at 12 h. The exogenous application of PEG appeared to upregulate the expression of ZmPIF3. The expression level of ZmPIF3 was significantly higher after 12 h exposure to 20 % PEG 6000 compared with the control (P
A maize phytochrome‑interacting factor 3 improves drought and salt stress tolerance in rice Yong Gao · Wei Jiang · Yi Dai · Ning Xiao · Changquan Zhang · Hua Li · Yi Lu · Meiqin Wu · Xiaoyi Tao · Dexiang Deng · Jianmin Chen
Received: 10 October 2014 / Accepted: 23 January 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Phytochrome-interacting factor 3 (PIF3) activates light-responsive transcriptional network genes in coordination with the circadian clock and plant hormones to modulate plant growth and development. However, little is known of the roles PIF3 plays in the responses to abiotic stresses. In this study, the cloning and functional characterization of the ZmPIF3 gene encoding a maize PIF3 protein is reported. Subcellular localization revealed the presence of ZmPIF3 in the cell nucleus. Expression patterns revealed that ZmPIF3 is expressed strongly in leaves. This expression responds to polyethylene glycol, NaCl stress, and abscisic acid application, but not to cold stress. ZmPIF3 under the control of the ubiquitin promoter was introduced into rice. No difference in growth and development between ZmPIF3 transgenic and wild-type plants was observed under normal growth conditions. However, ZmPIF3 transgenic plants were more tolerant to dehydration and salt stresses. ZmPIF3 transgenic plants had Yong Gao and Wei Jiang have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0288-z) contains supplementary material, which is available to authorized users. Y. Gao · W. Jiang · Y. Dai · N. Xiao · H. Li · Y. Lu · M. Wu · X. Tao · J. Chen (*) College of Bioscience and Biotechnology, Yangzhou University, 88 South University Ave, Yangzhou 225009, Jiangsu, People’s Republic of China e-mail: [email protected] Y. Gao · W. Jiang · Y. Dai · N. Xiao · C. Zhang · H. Li · Y. Lu · M. Wu · X. Tao · D. Deng · J. Chen Jiangsu Key Laboratories of Crop Genetics and Physiology and Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, Jiangsu, People’s Republic of China
increased relative water content, chlorophyll content, and chlorophyll fluorescence, as well as significantly enhanced cell membrane stability under stress conditions. The overexpression of ZmPIF3 increased the expression of stressresponsive genes, such as Rab16D, DREB2A, OSE2, PP2C, Rab21, BZ8 and P5CS, as detected by real-time PCR analysis. Taken together, these results improve our understanding of the role ZmPIF3 plays in abiotic stresses signaling pathways; our findings also indicate that ZmPIF3 regulates the plant response to drought and salt stresses. Keywords Abiotic stress tolerance · Expression pattern · Morphological character · Physiological trait · Transcription factor · Zea mays
Introduction Plants are constantly confronted by environmental stressors, including drought, high salinity, and extreme temperatures. These stressors affect both biomass and grain yield in crops. Plants have evolved different adaptive strategies to alleviate the adverse effects of these abiotic stresses; these tolerance or resistance mechanisms are mostly controlled by networks regulated by transcription factors (Tian et al. 2013). Transcription factors specifically bind to the cisacting elements of genes to activate or repress the expression of downstream target genes (Li et al. 2013). There is compelling evidence that stress-responsive genes, such as transcription factors, function in multiple pathways and facilitate crosstalk between different stress signaling pathways (Ludwig et al. 2004; Campo et al. 2012). The phytochrome (phy) family of sensory photoreceptors regulates plant development in response to informational light signals (Schafer and Nagy 2006). Phy interacts
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with members of the basic helix-loop-helix (bHLH) family of phytochrome-interacting factors (PIFs) that are involved in the modulation of light-regulated genes with roles in photomorphogenesis. The PIF family comprises 15 members (Toledo-Ortiz et al. 2003); most of these proteins are involved in light signaling (de Lucas and Prat 2014). The PIFs previously examined (PIF1, PIF3, PIF4, PIF5 and PIF7) bind to a core DNA G-box motif (CACGTG) in a sequence-specific manner (Toledo-Ortiz et al. 2003; Leivar et al. 2008; de Lucas et al. 2008). This suggests the existence of a direct signaling pathway between photoactivated phy molecules and their target genes. Almost all of these PIFs contain a conserved N-terminal sequence known as the active phytochrome B (APB) motif. This motif is necessary and sufficient for phyB-specific binding (Khanna et al. 2004). PIF1 and PIF3 each contain an additional domain, called the active phytochrome A-binding (APA) region, that is necessary for phyA binding (Al-Sady et al. 2006; Shen et al. 2008). Current evidence indicates that the intra-nuclear binding of the Pfr form of phyA and/or phyB to PIF proteins induces the rapid phosphorylation and degradation of these PIF transcription factors (Fairchild et al. 2000; Khanna et al. 2004); these may be the primary molecular events in phy signaling. PIF3, the foundational PIF member, was initially identified in a yeast two-hybrid screen for phyB-interacting proteins (Ni et al. 1999; Shimizu-Sato et al. 2002). A mutation in this factor results in an impaired response to red (R) and far-red (FR) light. This result suggests that PIF3 plays a role in the early steps of light signal transduction (Kim et al. 2003; Monte et al. 2004). The data also demonstrate that phy induces the rapid phosphorylation of PIF3 as a result of this interaction (Al-Sady et al. 2006), resulting in ubiquitylation and degradation (Park et al. 2004; Al-Sady et al. 2006). Subsequent studies revealed that this degradation is initiated by the direct binding of either phyA or phyB to their respective APA or APB interaction sites on PIF3 in the nucleus (Al-Sady et al. 2006, 2008). Therefore, PIF3 might represent the primary biochemical mechanism of signal transfer from photoactivated phy molecules to signaling partners within the cell (Leivar and Quail 2011). A number of studies have shown that PIF family members play a role in plant growth and development (Leivar and Quail 2011). As an important phytochrome signaling pathway hub, PIF integrates multiple signals to orchestrate the regulation of the transcriptional network that drives multiple facets of downstream morphogenesis. The data indicate that PIFs are involved in repressing seed germination (Oh et al. 2009), regulating de-etiolation (Lorrain et al. 2009; Chen et al. 2013), promoting seedling skotomorphogenesis (Leivar et al. 2009) and shade-avoidance (Lorrain et al. 2008; Keller et al. 2011). These complex networks of regulators underscore the pivotal role of PIFs
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in plant growth and development. Moreover, an increasing number of studies in recent years have identified PIF family members as key components of signaling pathways in addition to the well-characterized photoregulated pathway. These pathways converge at the PIFs and their close relatives, which regulate an array of developmental responses; these include the circadian clock (Nozue and Maloof 2006), high temperature (Koini et al. 2009; Stavang et al. 2009; Franklin et al. 2011) and plant hormones. For example, PIF4 functions in high-temperatureinduced hypocotyl elongation in de-etiolated Arabidopsis seedlings (Koini et al. 2009). Over the last few years, PIF family members have also been shown to be involved in the biosynthesis and signaling pathways of hormones (de Lucas and Prat 2014) such as gibberellin (GA) (de Lucas et al. 2008; Feng et al. 2008), abscisic-acid (ABA) (Kim et al. 2008; Oh et al. 2009), brassinosteroids (BRs) (Bai et al. 2012; Jaedicke et al. 2012;de Lucas and Prat 2014) and auxin (Franklin et al. 2011; Hornitschek et al. 2012; Sun et al. 2012). A number of studies have demonstrated that DELLA proteins, a class of nuclear transcriptional regulators, repress GA signaling and restrict plant growth (Achard and Genschik 2009). The interaction of DELLAs with PIF3 and PIF4 provide evidence for a crucial role for PIFs in the integration of both GA and light signaling (de Lucas et al. 2008; Feng et al. 2008; Achard and Genschik 2009). Recent studies have also found a function for DELLAs under abiotic stress (Achard et al. 2006, 2008; Navarro et al. 2008). DELLA proteins restrict plant growth during salt and cold stress to improve plant survival (Achard et al. 2006, 2008; Achard and Genschik 2009). In addition, PIF1/ PIL5 plays a pivotal role in mediating this response by regulating the expression of ABA-related genes and promoting ABA biosynthesis (Kim et al. 2008; Oh et al. 2007, 2009). ABA can regulate the plant adaptation to environmental stressors, including cold, drought, and salt (Spartz and Gray 2008). PIFs play broader roles than previously appreciated, functioning as cellular signaling hubs that integrate multiple signals to regulate the transcriptional network that drives multiple facets of downstream morphogenesis. PIFs can also regulate DELLA proteins and endogenous ABA in response to abiotic stress. However, the detailed roles of PIFs in the abiotic stress response remain to be elucidated. Maize is one of the most widely grown crops in the world and important for food and edible oil. In this study, a PIF transcription factor member from maize known as ZmPIF3 was cloned. The expression of this gene in response to water deficiency, high salinity, and ABA was described. Transgenic experiments indicated that the over-expression of ZmPIF3 in rice enhanced tolerance to drought and salt stresses without growth retardation; these findings suggest that the gene could be used in crop plants to improve tolerance to drought and salt stress.
Plant Mol Biol
Materials and methods Plant materials and stress treatment Maize (Zea mays, hybrid line ‘Zhengdan 958’) seeds were sterilized and germinated on wet filter paper for 6 days with a 16/8 h light/dark cycle at 25 °C. The seedlings were transferred to hydroponic growth conditions. Plants were grown in troughs filled with aerated nutrient solution under controlled conditions (28/26 °C, 16 h photoperiod, 70 % relative humidity). The full-strength nutrient solution had the following concentrations: 2.0 mM Ca(NO3)2, 1.0 mM K2SO4, 0.2 mM KH2PO4, 0.5 mM MgSO4, 2.0 mM CaCl2, 5.0 μM H3BO3, 2.0 μM MnSO4, 0.5 μM ZnSO4, 0.3 μM CuSO4, 0.01 μM (NH4)6Mo7O24, 200 μM Fe–EDTA. In this study all the stress treatments were performed with four-leaf-stage maize seedlings. For PEG, high-salinity and abscisic acid (ABA) treatment, the seedlings were grown hydroponically in nutrient solution supplied with 20 % PEG (molecular weight 6,000), 200 mM NaCl or 100 μM ABA respectively, and then sampled at 0, 1, 3, 6, 12, 24 and 48 h. For cold stress, seedlings were transferred to a growth chamber at 4 °C. Leaves were sampled at 0, 1, 3, 6, 12, 24 and 48 h after treatment. Immediately after excision, all samples were frozen in liquid nitrogen and stored at −80 °C until use. Rice (Oryza sativa L. Wuyunjing) for transgenic analysis was grown in a controlled environment chamber at 28/25 °C with a 16/8 h photoperiod and 70 % relative humidity. Three T3 homozygous transgenic lines with higher expression levels of the target gene were selected for phenotypic assays, and all seeds used for phenotypic assays were from the same harvest and stored under the same conditions. Cloning the full‑length cDNA and sequence analysis of ZmPIF3 Using PIF3 gene in Arabidopsis as a querying probe, a novel cDNA sequence was obtained from maize EST database of GenBank by in silico cloning, which was named ZmPIF3 gene. To obtain full-length ZmPIF3 cDNA, a pair of primers (F 5′-ATGTCCGACAGCAGCGACTTCG-3′ and R: 5′-TCATGTTTCAGCCTCATTTCTTCC-3′) were designed based on the lateral flanking sequence of the open reading frame (ORF) of the putative sequence. The obtained sequence ZmPIF3 was analyzed using bioinformatic tools at the websites (http://www.expasy. org/ and http://www.ncbi.nlm.nih.gov/). The nucleotide sequence, deduced amino acid sequence and open reading frame (ORF) were analyzed, and the sequence comparison was conducted through database search using BLAST program (NCBI, National Center for Biotechnology Services,
http://www.ncbi.nlm.nih.gov, http://www.maizegdb.org/). The phylogenetic analysis of PIFs from Arabidopsis thaliana was aligned with DNAMAN (Lynnon Biosoft, Vaudreuil, QC, Canada) using default parameters. A phylogenetic tree was constructed using MEGA version 6. The neighbor-joining method was used to construct the tree. Subcellular localization of the ZmPIF3 protein The ZmPIF3 ORF was fused upstream of the green fluorescent protein gene (GFP) under the control of the CaMV 35S promoter and the NOS terminator in the p2GWF7 expression vector. Proper restriction sites were added to the 5′- and 3′-ends of the coding region using PCR. The primers used were as follows: F: 5′-AATAAAGCTTATGTCCGACAGCAGCGACTT-3′ (HindIII site in bold), and R: 5′-AATACTCGAGTGTTTCAGCCTCATTTCTTC-3′ (XhoI site in bold). The PCR product was digested with the relevant restriction endonucleases and ligated with the p2GWF7 plasmid cut with the corresponding enzyme to create a recombinant plasmid for fusion protein expression. Positive plasmids were confirmed by restriction analysis and sequenced. Recombinant constructs were transformed into living onion epidermal cells by biolistic bombardment with a GeneGun (Biorad Helios) according to the instruction manual (helium pressure, 150–300 psi). After incubation in MS medium at 28 °C for 36–48 h, the onion cells were visualized with a laser scanning confocal microscope (Leica TCS-NT). The subcellular location of ZmPIF3 was detected by monitoring the transient expression of GFP in the onion epidermal cells as described. Quantitative real‑time PCR (qRT‑PCR) Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used to determine the expression patterns of ZmPIF3 in maize. Reverse transcription reactions were performed using total RNA from maize tissues. First-strand cDNA was synthesized from DNase-treated total RNA with Transcriptor Reverse Transcriptase (Roche, Mannhein, IN, Germany) and oligo(dT)18 following the manufacturer’s instructions. Real-time PCR (qPCR) was performed using an ABI 7300 system with a SYBR Green Master Mix kit (Roche, Mannhein, IN, Germany) and specific primers (qPCR, F: 5′-C AATCCAGCCACCATTCCC-3′; R: 5′-CTGTTGCTCCTGCACCATG-3′). Three replicate reactions were routinely performed for each sample. Relative gene expression levels were detected using the 2−ΔΔCT method (Livak and Schmittgen 2001). Actin transcript levels were used to quantify the expression of ZmPIF3 in maize. Three independent biological replicates were analyzed. Controls for the qPCR reactions were performed without the addition of
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the reverse transcriptase enzyme; these were systematically included in our experiments. Transformation of ZmPIF3 in rice The full-length cDNA of ZmPIF3 was amplified from maize with the primers 5′-GAAGATCTATGTCCGACAGCAGCG-3′ (Bgl II site in bold) and 5′-CCGGAATTCTCATGTTTCAGCCTCATTTC-3′ (EcoR I site in bold). The product was ligated into the pMD-19T Easy vector and sequenced. The ZmPIF3 fragment from pMD-19T was digested and inserted into the multiple cloning site of the binary plasmid p1011. In this construct, ZmPIF3 was driven by an ubiquitin promoter. The p1011-ZmPIF3 construct was electroporated into the Agrobacterium tumefaciens strain EHA105 and introduced into rice embryonic calli by A. tumefaciens-meditated methods (Xu et al. 2005). ZmPIF3 transgenic rice plants were selected in 1/2 MS medium containing 50 mg l−1 hygromycin. Phenotypic analyses were performed using T3 homozygous lines. Features of ZmPIF3 transgenic seedlings under dehydration, salt and cold stress Transgenic plants were characterized for morphological changes under a 16/8 h photoperiod in a growth chamber with a constant temperature of 28/25 °C. For drought stress experiments, 2-week-old seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were exposed to 1/2 MS medium containing 20 % PEG for 4 days. Plants were then recovered under normal growth conditions for 10 days. For salt stress experiments, 2-weekold seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were submerged into 1/2 MS medium supplemented with 150 mM NaCl for 2 days. The plants were then transferred into an incubation solution without NaCl for an additional 7 days. For cold stress experiments, 2-week-old seedlings of the wild-type, the vector-only control and ZmPIF3 transgenic plants were subjected to treatment at 4 °C for 2 days. Plants were then transferred to a greenhouse and incubated at a temperature of 28/25 °C with a 16 h photoperiod for 7 days. Abiotic stress tolerance assays Wild-type, vector-only control and transgenic plants grown in soil were used to determine stress tolerance. For drought stress experiments, 40-day-old wild-type, vector-only control and transgenic plants were planted in rectangular pots filled with same soil mixture and well watered. Seedlings were cultured in a greenhouse (28/25 °C with a 16/8 h photoperiod and 70 % relative humidity) without watering for 7 days until phenotypic differences were evident between
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the transgenic plants and the two controls. The plants were then rewatered. For salt stress assays, rice seedlings were cultured as described above. Water was withheld for 40 days before irrigation was performed with a NaCl solution (150 mM). Irrigation occurred from the bottoms of the pots, and the soil was completely saturated with salt water. When phenotypic differences were evident between the transgenic plants and the two controls, the NaCl solution was removed and the plants were cultured normally. For cold stress experiments, 40-day-old seedlings were transferred into a low-temperature growth chamber (4 °C) for 2 days. Plants were then recovered in a normal growth chamber for an additional 7 days. Relative water content The relative water content (RWC) was measured using 20 plants each from the wild-type, vector-only control and ZmPIF3 transgenic genotypes. Two-week-old plants were detached from the roots, and the leaves of 20 plants were cut into pieces one centimeter in length. Mixed small pieces of fresh samples with a fresh weight (FW) of approximately 0.5 g were selected. Pieces were floated on distilled water in Petri dishes for 4 h to regain turgidity to a constant weight; the tissue was then re-weighed to obtain the turgor weight (TW). The samples were dried at 80 °C for 24 h to determine the dry weight (DW). The relative water contents (RWCs) were measured using the following formula (Schonfeld et al. 1988): RWC (%) = [(FM − DM)/(TM − DM)] × 100 %. These experiments were repeated three times. Chlorophyll content assays Chlorophyll content was measured with a chlorophyll meter (SPAD 502 Plus; Konica Minolta Sensing). Chlorophyll contents under stress conditions were measured after treatment with 20 % PEG for 2 days. One measurement of the fully expanded leaves was made for each plant; 20 plants from each line were used for the chlorophyll content assays. Chlorophyll fluorescence assays Chlorophyll florescence was measured using a portable photosynthesis system (FMS-2 Hansatech UK). The maximum efficiencies of photosystem II (PSII) photochemistry, Fv/Fm = (Fm–F0)/Fm, was employed to assess changes in the primary photochemical reactions of the photosynthetic potential. Chlorophyll florescence under stress conditions was measured after treatment with 20 % PEG6000 for 2 days. One measurement of the fully expanded leaves was made for each plant; 20 plants from each line were used for the chlorophyll florescence assays.
Plant Mol Biol
Cell membrane stability analysis
Results
Plant cell membrane stabilities (CMS) were determined with a conductivity meter (EL30 K; Mettler Toledo). The following formula was used: CMS (%) = (1−initial electrical conductivity/electrical conductivity after boiling) × 100. Two-week-old seedlings were transferred to 1/2 MS medium supplemented with 20 % PEG for 2 days. Seedlings were removed and immediately rinsed with double distilled water (ddH2O). Plants were then immersed in 20 ml ddH2O at room temperature. After 2 h, the initial conductivities of the solutions were recorded. Samples were boiled for 30 min and cooled to room temperature; the final conductivities were then measured.
Molecular characterization of ZmPIF3
Determination of ion concentrations For salt stress analysis, 2-week-old seedlings of the wildtype, the vector-only control and ZmPIF3 transgenic rice were submerged into 1/2 MS medium supplemented with 150 mM NaCl for 2 days. Leaves and roots were ashed for 5 h in an oven at 600 °C. The analysis of cations was performed using the ashes of 200 mg of dried plant materials. The ashes were dissolved in 2 ml concentrated nitric acid with gentle heating. This suspension was adjusted to a volume of 50 ml with distilled water and filtered through a paper filter. Thereafter, Na+ and K+ concentrations were analyzed in the filtrate by means of atomic absorption spectrometry (PE-2100; Perkin Elmer Corporation, USA) and an inductively coupled plasma spectrometer (Optima 7300 DV; Perkin Elmer Corporation, USA). Gene expression analysis Two-week-old seedlings grown on 1/2 MS medium were treated with 20 % PEG6000 or left untreated. Total RNA was extracted using Trizol reagent and treated with RNasefree DNase. For real-time quantitative PCR, total RNA reverse-transcribed with Transcriptor Reverse Transcriptase (Roche, Mannhein, IN, Germany). Quantitative expression assays were performed with the SYBR Green Master Mix kit (Roche, Mannhein, IN, Germany) and an ABI 7300 sequence detection system according to the manufacturer’s protocol (Applied Biosystem). The RT-PCR conditions were as follows: 5 min at 95 °C, and 40 cycles of 15 s at 95 °C, 20 s at 60 °C, and 31 s at 72 °C. Three replicate reactions were routinely performed for each sample. Relative gene expression levels were detected using the 2−ΔΔCT method. Actin transcripts were used to quantify the expression levels of the abiotic stress-response genes in transgenic rice. The primer pairs used for real-time PCR are listed in Supplementary Table S1, which is available at JXB online.
A full-length cDNA of the target gene was obtained by screening full-length maize cDNA libraries. The full-length cDNA sequence, designated as ZmPIF3, contained an open reading frame of 1,914 bp. This ORF encoded a putative 67-kDa polypeptide of 638 amino acids with an isoelectric point of 6.45. NCBI Blast indicated that the deduced amino acid sequence of ZmPIF3 shared a high similarity with the A. thaliana PIFs proteins PIF3 (AT1G09530) (22.87 %), PIF1 (AT2G20180) (21.16 %), PIF4 (AT2G43010) (20.06 %), PIF5 (AT3G59060) (19.44 %), PIF6 (AT3G62090) (15.83 %), PIF7 (AT5G61270) (17.24 %), PIF8 (AT4G00050) (18.25 %) and PIL1(AT2G46970) (16.61 %). Three characteristic domains, the bHLH domain, the APB motif and the APA motif, were found in the deduced ZmPIF3 amino acid sequence (Fig. 1a). The deduced amino acid sequence of ZmPIF3 was further compared with a set of highly homologous PIF family proteins by molecular phylogenetic tree analysis (Fig. 1b). Compared with the other PIF proteins, ZmPIF3 was more closely related to PIF3 in Arabidopsis, VvPIF3 in Vitis vinifera, GmPIF3 in Glycine max, MdPIF3 in Malus domestica, and OsPIL15 and OsPIL16 in rice. Subcellular localization of ZmPIF3 Transcription factors are typically localized to cell nuclei, where they bind DNA and are involved in transcriptional activation. To identify the cellular localization of ZmPIF3, the expression and distribution of ZmPIF3 were examined in transgenic onion epidermal cells. A fusion protein with GFP was expressed, and its localization was visualized using a laser scanning confocal microscope. As shown in Fig. 2, ZmPIF3–GFP was present in the cell nucleus. This result demonstrates that ZmPIF3 localizes to nuclei (Fig. 2). Expression patterns of ZmPIF3 in different tissues and under abiotic stress The expression patterns of ZmPIF3 in different tissues were examined using qRT-PCR analysis. As shown in Fig. 3a, ZmPIF3 was strongly expressed in leaves and pistils; less expression was observed in the roots, stems, stamen, and seed. Various expression patterns were observed under PEG, salt, low temperature, and ABA treatments (Fig. 3b). The expression levels of ZmPIF3 increased significantly under salt stress, PEG stress and ABA treatment conditions. However, ZmPIF3 expression was not significantly
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Fig. 1 Sequence alignment of ZmPIF3 with other Arabidopsis PIF family members. a The comparison of the putative amino acid sequence of ZmPIF3 with the A. thaliana PIF3 protein. The identical amino acids are shown in white with a black background, and the conserved amino acids are shown in white with a gray background. The specific sites of the APB motif, APA motif and bHLH domain are presented. The aligned PIF3 sequences are from A. thaliana (GenBank accession no. AT1G09530). b Phylogenetic tree analysis of ZmPIF3 with other PIF family members. The neighbor-joining method was used to construct the tree. The amino acids sequences used in phylogenetic tree analysis were from plants, including A. thaliana PIF1 (GenBank accession no. Q8GZM7.1); PIF3 (GenBank accession no. O80536.1); PIF4 (GenBank accession no. Q8W2F3.1); PIF5 (GenBank accession no. Q84LH8.1); PIF6 (GenBank accession no. Q8L5W7.1); PIF7 (GenBank accession no. Q570R7.2); PIF8 (GenBank accession no. Q8GZ38.1); PIL1 (GenBank accession no. Q8L5W8.1); HFR1 (GenBank accession no. Q9FE22.1); bHLH127 (GenBank accession no. Q7XHI7.1); bHLH23 (GenBank accession no. Q9SVU6.1); bHLH119 (GenBank accession no. Q8GT73.2); bHLH56 (GenBank accession no. Q9SVU7.2); SPT (GenBank accession no. Q9FUA4.1); ALC (GenBank accession no. Q9FHA2.1); O. sativa OsPIL11 (GenBank accession no. NP_001067246); OsPIL12 (GenBank accession no. NP_001173558); OsPIL13 (GenBank accession no. NP_001051465); OsPIL14 (GenBank accession no. NP_001058876); OsPIL15 (GenBank accession no. NP_001042775); OsPIL16 (GenBank accession no. NP_001054593); V. vinifera VvPIF3 (GenBank accession no. XP_002276198); G. max GmPIF3 (GenBank accession no. XP_006589101); and M. domestica MdPIF3 (GenBank accession no. AEX32796)
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Plant Mol Biol
Plant Mol Biol
Fig. 2 Subcellular localization of ZmPIF3. Full-length ZmPIF3 fused to GFP was transiently transformed into onion epidermal cells. The plant nuclei were stained with DAPI. Images were taken using confocal microscopy (GFP fluorescence, green; DAPI fluorescence,
blue; visible, visible light image; merged, merged images of above three images). Cells transformed with an empty vector (smGFP) are shown as a control. Arrows indicate ZmPIF3-localized nuclei. Bar 100 mm
regulated by cold treatment. The expression patterns and maximum expression levels differed for each stress. For PEG, NaCl and ABA treatments, expression levels peaked at 12 h. The exogenous application of PEG appeared to upregulate the expression of ZmPIF3. The expression level of ZmPIF3 was significantly higher after 12 h exposure to 20 % PEG 6000 compared with the control (P
