The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner

Plant Science 241 (2015) 199–210

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The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner Dong-Keun Lee a , Hyung Il Kim a , Geupil Jang b , Pil Joong Chung a , Jin Seo Jeong a , Youn Shic Kim a , Seung Woon Bang a , Harin Jung a , Yang Do Choi b , Ju-Kon Kim a,∗ a b

Crop Biotechnology Institute, Green Bio Science & Technology, Seoul National University, Gangwon-do 25354, South Korea Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea

a r t i c l e

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Article history: Received 14 August 2015 Received in revised form 7 October 2015 Accepted 11 October 2015 Available online 22 October 2015 Keywords: Nuclear factor (NF)-YA Transcription factor Abscisic acid Drought tolerance Rice

a b s t r a c t The mechanisms of plant response and adaptation to drought stress require the regulation of transcriptional networks via the induction of drought-responsive transcription factors. Nuclear Factor Y (NF-Y) transcription factors have aroused interest in roles of plant drought stress responses. However, the molecular mechanism of the NF-Y-induced drought tolerance is not well understood. Here, we functionally analyzed two rice NF-YA genes, OsNF-YA7 and OsNF-YA4. Expression of OsNF-YA7 was induced by drought stress and its overexpression in transgenic rice plants improved their drought tolerance. In contrast, OsNF-YA4 expression was not increased by drought stress and its overexpression in transgenic rice plants did not affect their sensitivity to drought stress. OsNF-YA4 expression was highly induced by the stress-related hormone abscisic acid (ABA), while OsNF-YA7 was not, indicating that OsNF-YA7 mediates drought tolerance in an ABA-independent manner. Analysis of the OsNF-YA7 promoter revealed three ABA-independent DRE/CTR elements and RNA-seq analysis identified 48 genes downstream of OsNFYA7 action putatively involved in the OsNF-YA7-mediated drought tolerance pathway. Taken together, our results suggest an important role for OsNF-YA7 in rice drought stress tolerance. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: ABA, abscisic acid; ABRE, ABA responsive elements; AP2/ERF, APETALA2/ERE binding factor; AREB/ABF, ABRE-binding proteins/ABRE-binding factors; BH, before heading; bZIP, basic leucine zipper transcription factor; DAPI, 4 ,6-diamidino-2-phenylindole; DRE/CRT, dehydration responsive element/Crepeat; DREB2, DRE/CRT-binding protein2; FPKM, reads per transcript kilobase per million fragments mapped reads; FR, filling rate; GFP, green fluorescent protein; HRD, HARDY; LEC1, LEAFY COTYLEDON1; LecRK, lectin receptor-like kinase; MS, murashige-skoog; MYB, myeloblastosis transcription factor; NAC, (NAM, ATAF1,2, CUC2) transcription factor; NF-Y, nuclear factor-Y; NP, number of panicles; NSP, number of total spikelets; NT, non-transgenic; PAM, pulse-amplitude modulation; PEG, polyethylene glycol; PGD1, PHOSPHOGLUCONATE DEHYDROGENASE1; SIK1, stress-induced protein kinase gene1; SnRK2, SNF1-related kinase; TGW, total grain weight. ∗ Corresponding author at: Crop Biotechnology Institute, Green Bio Science & Technology, Seoul National University, Gangwon-do 25354, South Korea. Fax: +82 33 339 5825. E-mail addresses: [email protected] (D.-K. Lee), [email protected] (H.I. Kim), [email protected] (G. Jang), [email protected] (P.J. Chung), [email protected] (J.S. Jeong), [email protected] (Y.S. Kim), [email protected] (S.W. Bang), [email protected] (H. Jung), [email protected] (Y.D. Choi), [email protected] (J.-K. Kim). http://dx.doi.org/10.1016/j.plantsci.2015.10.006 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.

Drought stress severely affects crop development and yield worldwide, and so the various mechanisms by which plants cope with insufficient water are of great interest. Plants have evolved numerous adaptive strategies, including developmental and physiological changes, to overcome drought stress [1–3], many of which involve the regulation of transcriptional networks by droughtresponsive transcription factors. Members of the AP2/ERF, bZIP, NAC and MYB transcription factor families have been associated with drought resistance mechanisms and their functions have been studied by gain-of-function approaches. For example, the expression levels of HRD (HARDY; an AP2/ERF transcription factor) or AtMYB96, from Arabidopsis thaliana, or OsAP37, OsNAC5, OsNAC9, OsNAC10, OsbZIP12, OsbZIP23, and OsMYB4 from rice (Oryza sativa) are up-regulated by drought stress, and overexpression of these genes promotes the expression of drought-responsive genes and enhances resistance to drought stress [4–12]. In addition, recent studies have suggested that Nuclear Factor Y (NF-Y) transcription factors have important functions in plant drought stress responses. The NF-Y transcription factor complex is composed of three subunits: NF-YA, NF-YB and NF-YC. There are

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10 NF-YA, 13 NF-YB and 13 NF-YC genes in A. thaliana, whereas 10 NF-YA, 11 NF-YB and 7 NF-YC genes have been identified in rice [13–14]. Overexpression of the drought-responsive genes AtNFYB1 from A. thaliana, or the maize (Zea mays) homolog, ZmNF-YB2, in transgenic plants was reported to increase resistant to drought stress [15]. The involvement of NF-YA in drought resistance mechanisms was further demonstrated by the characterization of the drought-responsive AtNF-YA5 gene during drought stress treatments. Transgenic A. thaliana plants constitutively over-expressing AtNF-YA5 showed improved resistance to drought, while nf-ya5 (NF-YA5 knockout mutant) lines were hypersensitive to drought stress compared to wild-type plants [16]. Finally, the GmNF-YA3, Cdt-NF-YC1 and ZmNF-YA14 genes have also been shown to be involved in drought resistance mechanisms [17–19]. However, despite increasing evidence that NF-Y genes promote drought tolerance, the underlying molecular mechanisms have not been yet been fully elucidated. NF-Y proteins are known to bind specifically to a highly conserved CCAAT sequence in the promoters of their target genes [20–22], and each NF-Y subunit contains conserved domains required for DNA binding and protein interactions [21,23]. NFYA proteins have two conserved domains, one in the N-terminus, which is required for the interaction with the NF-YB/NF-YC heterodimer, and one in the C-terminal region, which is involved in DNA-binding [24]. The N- and C-terminal domains are separated by a non-conserved spacer region of approximately 10 amino acids [25]. NF-YB and NF-YC proteins initially form a NF-YB/NFYC dimeric complex in the cytoplasm, which then traffics to the nucleus where it interacts with NF-YA, forming a heterotrimeric complex [21,23,26]. NF-Y transcription factors are present in all higher eukaryotes, including mammals and plants [27], and while they are encoded by a single gene in mammals they are present as gene families in plants [25,28–30]. The multiple copies of NF-Y genes in plants results in regulatory complexity as a consequence of interactions between different NF-Y subunit members, which then serve different roles [15]. In plants, the first reported function of NF-Y factors was in the regulation of plant development [31]. NF-Y genes such as LEAFY COTYLEDON1 (LEC1; AtNF-YB9), LEC1-LIKE (AtNF-YB6), AtNF-YA1, AtNF-YA5, AtNF-YA6, and AtNF-YA9 have been shown to be required for embryogenesis [32–34], while the OsHAP3 genes, which encode rice NF-YB subunits, regulate chloroplast biogenesis [35]. In addition, several NF-Y genes are important for flowering time regulation and root development in A. thaliana and rice [36–40]. Drought tolerance mechanisms are known to involve ABAdependent and ABA-independent pathways. Key components of ABA-dependent drought responses are ABA responsive elements (ABREs) and ABRE-binding proteins/ABRE-binding factors (AREB/ABF), which are members of the group-A class of bZIP transcription factors [41–44]. In contrast, the ABA-independent drought response involves dehydration responsive element/Crepeat (DRE/CRT) DNA motifs and DRE/CRT-binding protein 2 (DREB2), which belongs to the AP2/ERF transcription factor [45–46]. In spite of these distinct differences, some drought responsive genes are regulated by both pathways, and several crosstalk mechanisms between the two pathways have been identified. For example, the gene encoding the subclass III SNF1-related kinase (SnRK2), which activates AREB/ABF proteins associated with ABA-dependent gene expression, is induced by unknown ABAindependent regulators [47]. In addition, DREB2A, which operates in the ABA-independent pathway, is controlled by ABA-dependent regulators termed AREBs under drought stress, and interacts with AREB1, AREB2, and ABF3 in A. thaliana [48–49]. In this study, we undertook functional analyses of two rice NF-YA transcription factors, OsNF-YA4 and OsNF-YA7, investigating their putative role in drought resistance by evaluating the drought

tolerance of transgenic rice plants with increased expression of each of the genes. We also characterized the expression patterns of OsNF-YA4 and OsNF-YA7, and identified genes that are potentially related to the OsNF-YA7-mediated drought tolerance mechanism. 2. Materials and methods 2.1. Plasmid construction and transformation of rice protoplasts For the overexpression experiments, the full-length cDNAs of OsNF-YA4 (AK069854) and OsNF-YA7 (AK059903) were amplified from cDNA by PCR (Promega, Madison, WI) according to the manufacturer’s instruction. The primers used for cloning were forward 5 -ATGGAGTCGAGGCCGGGGGG-3 and reverse 5 -TCATGTTTCCTTCTGTAGGA-3 for OsNF-YA4 and forward 5 -ATGAAGCCAGATGGTGAAAC-3 and reverse 5 TCATACAACATCGGACGCAT-3 for OsNF-YA7. Each full-length cDNA was inserted into the p700 vector carrying the PGD1 promoter using the Gateway system (Invitrogen, Carlsbad, CA). PGD1::OsNF-YA7 and PGD1::OsNF-YF4 vectors were introduced into rice (O. sativa cv. Nackdong) using Agrobacterium tumefaciens (strain LBA4404)-mediated co-cultivation, as previously described [50]. For transient expression of OsNF-YA7-GFP and OsNF-YA4GFP in rice protoplasts, the predicted OsNF-YA7 and OsNF-YA4 coding regions without the stop codon were cloned into the pHBT vector (GenBank accession No. EF090408) between the 35S promoter and the GFP coding sequencing using the BamH1 and Stu1 restriction sites. The primers used for this cloning were forward 5 -ATGGATCCATGGAGTCGAGGCCGGGGGG-3 and reverse 5 -AGGCCTTGTTTCCTTCTGTAGGA-3 for OsNF-YA4 and forward 5 -ATGGATCCATGAAGCCAGATGGTGAAAC-3 and reverse 5 -AGGCCTTACAACATCGGACGCAT-3 for OsNF-YA7. The constructs, 35S::OsNF-YA7-GFP and 35S::OsNF-YA4-GFP were transformed into isolated rice protoplasts using PEG (polyethylene glycol)-mediated transformation. Isolation of protoplasts and PEG-mediated transformation were performed as previously described [51]. 2.2. Confocal microscopy The subcellular localization of the OsNF-YA7 and OsNF-YA4 proteins fused to the GFP reporter protein was analyzed using a Leica SP8 STED laser scanning confocal microscope (Leica, Solms, Germany). For DAPI (4 ,6-Diamidino-2-phenylindole) (Sigma, St. Louis, USA) staining, transformed protoplasts were incubated with 2 ␮g ml−1 DAPI for 2 min. GFP and chlorophyll were excited at 488 nm and then emitted light detected between 512 and 580 nm and between 700 and 790 nm, respectively. DAPI was simultaneously excited at 405 nm and detected between 430 and 450 nm. 2.3. Stress and ABA treatment for transcriptional expression analysis To analyze the expression patterns of the OsNF-YA7 and OsNFYA4 genes in response to abiotic stress, non-transgenic (NT) plants (O. sativa cv. Nackdong) were grown in soil for 4 weeks under standard greenhouse conditions (16 h light/8 h dark cycles at 28 ◦ C). Abiotic stress conditions treated as previous described [52]. Drought stress conditions consisted of air-drying whole plants by removing the plants from their pots for 3 h. For the salt and cold stress treatments, whole plants were transferred to distilled water containing 200 mM NaCl, or exposed to 4 ◦ C temperatures for 24 h, respectively. For the analysis of the OsNF-YA4 and OsNFYA7 expression patterns in response to ABA, NT plants were grown in Murashige–Skoog (MS) medium for 2 weeks in a growth chamber (16 h light/8 h dark cycles at 28 ◦ C). For a short-term treatment

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with ABA, ABA was introduced by transferring whole plants into distilled water containing 10 ␮M or 100 ␮M ABA for one day. For the long-term ABA treatment, NT seeds were planted and grown on MS medium supplemented with 0 ␮M, 0.5 ␮M, 1 ␮M or 3 ␮M of ABA for 2 weeks. 2.4. Real-time quantitative reverse transcription PCR Total RNA was extracted using a Qiagen RNeasy plant mini kit (Qiagen, Valencia, USA). One microgram of total RNA was used to synthesize first-strand cDNA in a 20 ␮l reaction using the Superscript II cDNA synthesis system (Invitrogen, Carlsbad, USA). To analyze gene expression levels, quantitative RT-PCR was carried out using an Mx3000p real-time PCR machine and the Platinum® SYBR® Green qPCR SuperMix-UDG system (Invitrogen, Carlsbad, USA). Rice Ubiquitin1 (AK121590) transcript abundance was used as the normalizing control and gene-specific primer sequences for the OsNF-YA4 and OsNF-YA7 were designed at the 3 UTR and 3 exon regions. Three biological and two technical replicates were analyzed for all quantitative experiments. Gene specific primers used for quantitative RT-PCR are listed in Table S1. 2.5. Phenotypic analysis associated with drought resistance Three single copy PGD1::OsNF-YA4 (#12, 13, and 17) T3 homozygous lines and five single copy PGD1::OsNF-YA7 (#1, 7, 9, 15, and 16) T3 homozygous lines isolated by genomic Southern blot analysis (Fig. S1A) were grown in soil for 4 weeks alongside NT plants, and drought stress was imposed by not watering. After 7 days of drought treatment, the plants were re-watered. Drought-induced visual symptoms were visualized by imaging PGD1::OsNF-YA4, PGD1::OsNF-YA7 and NT plants at the indicated time points using a NEX-5N camera (Sony, Tokyo, Japan). 2.6. PAM (pulse-amplitude modulation) test Each of three lines of PGD1::OsNF-YA4 and PGD1::OsNF-YA7 plants were grown in soil for 2 weeks in a greenhouse alongside NT plants (16 h light/8 h dark cycles at 28 ◦ C). Ten leaves of the 2-weekold rice plants were collected from each sample and then adapted to dark conditions for 10 min. For PAM analysis, drought conditions were imposed by air-drying for up to 3.5 h and data collected over a time-course by measuring every 0.5 h interval for 3.5 h at 28 ◦ C. At the indicated time, Fv /Fm values were measured by detecting chlorophyll fluorescence emission from the upper surface of the leaves with a pulse modulation fluorometer, Mini-PAM (Walz, Effeltrich, Germany). The dark-treated leaf was given a measuring light of 0.15 ␮mol photon m−2 s−1 for a minimal level of fluorescence, and then a 0.8 s actinic light of 10,000 ␮mol photon m−2 s−1 for a maximal level of fluorescence. The Fv /Fm values indicate the activity of photosystem II [53–54]. 2.7. Characterization of agronomic traits To characterize agronomic traits of the transgenic plants, three independent T3 homozygous lines of PGD1::OsNF-YA4 and PGD1::OsNF-YA7 together with NT and nullizygous plants were planted in a rice paddy field at Kyungpook National University, Gunwi (128:34E/36:15N), Korea. A randomized design was employed for three replicates using 3 different plots measuring 10 m2 each. Thirty seedlings of each individual line were transplanted into the plots at 25 days after sowing. Fertilizer was applied at 70N/40P/70K kg ha−1 after the last paddling. Growth and yield parameters were scored with 30 plants from each individual line of NT, nullizygotes, PGD1::OsNF-YA4 and PGD1::OsNF-YA7.

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2.8. RNA sequencing and transcriptome analysis Two-week-old NT, PGD1::OsNF-YA4 and PGD1::OsNF-YA7 plants were grown under normal conditions in a glasshouse (16-h light/8h dark) at 28 ◦ C. For the Illumina library preparation, total RNA was first extracted from whole NT, PGD1::OsNF-YA4 and PGD1::OsNFYA7 plants using the Plant RNeasy mini kit (Qiagen, Valencia, USA). Each RNA from three single copy homozygous PGD1::OsNF-YA4 and PGD1::OsNF-YA7 plants was equally combined into one total RNA of PGD1::OsNF-YA4 and PGD1::OsNF-YA7 for RNAseq, respectively. The quantity, quality, and purity of the total RNA was assessed using a Thermo Scientific Nanodrop 2000 and an Agilent Bioanalyzer 2100. RNA-seq libraries were prepared using the mRNA-seq 8 samples prep kit (Illumina, San Diego, USA) according to the manufacture’s protocol and sequenced (Macrogen, Seoul, Korea) using the Illumina HiSeq2000 (Illumina, San Diego, USA). The expression level of each transcript was expressed as the fragment per transcript kilobase per million fragments mapped reads (FPKM) value, which was calculated based on the number of mapped reads [55]. To make expression profiles for PGD1::OsNFYA4 or PGD1::OsNF-YA7, FPKM values for each transcript from PGD1::OsNF-YA4 or PGD1::OsNF-YA7 were normalized with FPKM values for each transcript from NT plants. We calculated the fold change FPKM values for each transcript and a cutoff fold change in gene expression level of at least three folds to identify up- and down-regulated genes. For identification of OsNF-YA7-mediated drought tolerance genes, the expression profile from PGD1::OsNFYA7 or PGD1::OsNF-YA4 was compared with drought and ABA high abundant gene profiles (expression fold change > 3) from previous microarray data [8].

3. Results 3.1. Expression patterns of OsNF-YA7 and OsNF-YA4 under abiotic stress conditions We previously reported gene expression profiles of 14-day old leaves subjected to drought, high salinity, low temperature and ABA treatments, based on a rice 3 -tiling microarray [8]. Of those abiotic stresses, drought stress in particular was observed to induce the expression of several transcription factors. Since Nuclear Factor (NF)-Y transcription factors have recently been identified as regulators of the drought tolerance [15], we focused on investigating the possible role of rice NF-YA genes in responses to drought stress. We first reanalyzed the microarray data and found that OsNFYA7 expression was strongly induced by abiotic stresses, including drought, high salinity, and low temperature (Fig. 1A). To validate this result, we performed an independent quantitative real-time PCR (q-RT PCR) analysis of gene expression in rice plants that had been exposed to drought, high salinity and low temperatures (Fig. 1B). As a control, we used OsNF-YA4 expression, since a phylogenetic analysis of NF-YA protein sequences indicated that OsNF-YA7 aligned within class 2 subclade together with OsNF-YA4 and OsNF-YA9 (Fig. 1C). We observed that the transcript levels of OsNF-YA7 increased in response to drought (4.4-fold), high salinity (2.9 fold) and low temperature treatments (2.7-fold) whereas, of these treatments, only low temperatures induced the expression of OsNF-YA4. NF-YA genes encode evolutionarily conserved transcription factors that have been identified in species ranging from yeast to plants and humans [29], and we similarly observed a cross kingdom conservation of the NA-YA transcription factors sequences (Fig. 1D). OsNF-YA proteins, including OsNF-YA7, have two conserved domains, the NF-YB/NF-YC interaction domain and the DNA binding domain, suggesting that they may share a molecular

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Fig. 1. Expression patterns of OsNF-YA7 and OsNF-YA4 in response to various abiotic stress conditions and multiple alignment of the NF-YA family. (A) Microarray analysis of 10 rice OsNF-YA genes in response to drought, salt and low temperature. (B) Quantitative RT-PCR analysis of OsNF-YA4 and OsNF-YA7 in response to abiotic stress. NT plants were grown in soil for 4 weeks and various abiotic stresses were introduced. Mock, drought, salt and low temperature represent non-treated control plants, air-dried for 3 h, 200 mM NaCl for 24 h, and 4 ◦ C for 24 h, respectively. Ubiquitin1 expression was used as an internal control. Data are shown as the mean ± SD of three biological and two technical replicates. Significant differences from WT plants are denoted by two asterisks corresponding to P < 0.01 and a single asterisk corresponding to P < 0.05 by the Student’s t-test. (C) Phylogenetic tree created using the neighbor-joining method in ClustalW using full-length amino acid sequences of rice NF-YA proteins (http:// www.genome.jp/tools/culstalw/). Bootstrap support (100 repetitions) is shown for each node. (D) Amino acid alignment of conserved regions in NF-YA proteins from various organisms (BOXSHADE of the Mobyle Web portal; http://mobyle.pasteur.fr/). Hs, Homo sapiens; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Os, Oryza sativa..

mechanism for regulating various developmental or environmental responses. However, despite the high degree of sequence similarity between OsNF-YA4 and OsNF-YA7, their expression patterns in response to abiotic stress were distinctly different. 3.2. Nuclear localization of OsNF-YA7 and OsNF-YA4 and the temporal expression patterns of their transcripts We next determined the temporal expression patterns of OsNFYA7 and OsNF-YA4 at various developmental stages of leaves, roots, and spikelets (Fig. 2A). OsNF-YA7 transcripts were detected at all developmental stages, but showed small differences in expression in different organs. Expression levels were higher in leaves of 7and 30-day old plants than in roots, while the opposite was true in 60-day old plants. OsNF-YA4 transcripts were also detected at all

developmental stages, but the pattern differed from that of OsNFYA7. The transcript levels of OsNF-YA4 were slightly higher in roots than in leaves of 7-day old seedlings, but the opposite was seen in 60-day old plants. An expression analysis of different reproductive stages showed that OsNF-YA4 transcripts were more abundant than those of OsNF-YA7 in the before heading (BH) stage of spikelet development. These distinct expression patterns suggested that OsNF-YA7 and OsNF-YA4 may serve different roles. The subcellular localization of OsNF-YA7 and OsNF-YA4 was investigated using a rice protoplast transient expression system (Fig. 2B). Chimeric genes encoding green fluorescent protein (GFP) fused to OsNF-YA7 or OsNF-YA4, were used to generate the constructs 35S::OsNF-YA7-GFP and 35S::OsNF-YA4-GFP, respectively, which were then transformed into rice protoplasts. GFP fluorescence was observed in the nuclei of protoplasts harboring either

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Fig. 2. Temporal expression of OsNF-YA7 and OsNF-YA4, and subcellular localization of OsNF-YA7-GFP and OsNF-YA4-GFP. (A) Quantitative RT-PCR of OsNF-YA4 and OsNF-YA7 in leaves and roots of NT plants at 7, 30 and 60 days after germination (DAG) and in spikelets of NT plants at two different stages of the reproductive development. The S1 spikelet stage represents less than 1.5 cm panicle length. BH spikelets refer to panicles before-heading. Ubiquitin1 expression was used as an internal control. Data are shown as the mean ± SD of three biological and two technical replicates. (B) Confocal images of OsNF-YA7-GFP and OsNF-YA4-GFP in protoplasts with or without simultaneous DAPI staining. Scale bars, 10 ␮m.

of the two constructs, as confirmed using colocalization of the GFP signal with the DAPI nuclear DNA stain (Fig. 2B). This indicated that OsNF-YA7 and OsNF-YA4 are nuclear localizing proteins, as would be expected for transcription factors. 3.3. Overexpression of OsNF-YA4 affects reproductive development To study the biological roles of OsNF-YA7 and OsNF-YA4 in rice development and in response to drought stress, we generated transgenic rice plants overexpressing OsNF-YA7 or OsNF-YA4 under the control of the PGD1 (PHOSPHOGLUCONATE DEHYDROGENASE1) promoter, which drives a constitutive and whole body expression of transgenes [56]. Initially, 25 and 47 individual lines of PGD1::OsNFYA7 and PGD1::OsNF-YA4, respectively, were generated, and from these, we selected single copy homozygous T2 lines. Finally, three single copy homozygous lines of PGD1::OsNF-YA7 (#7, 9, and 15) and PGD1::OsNF-YA4 (#12, 13, and 17) were selected for further studies. The expression levels of OsNF-YA7 in the PGD1::OsNF-YA7 lines was 25-36 fold higher than in non-transgenic (NT) lines, whereas the expression level of OsNF-YA4 in the PGD1::OsNF-YA4 lines showed a 5-fold higher expression than in the NT plants (Fig. S1B). Overexpression of OsNF-YA7 or OsNF-YA4 did not affect leaf or root development during vegetative growth, since the overexpressing transgenic plants showed similar shoot height and root

length to NT plants at 2-week old vegetative stages (Fig. 3A). We infer from these results that OsNF-YA7 and OsNF-YA4 may not play important biological roles under normal conditions. To take into account agronomic traits of PGD1::OsNF-YA4 and PGD1::OsNF-YA7 transgenic plants during the reproductive stage, we evaluated various aspects of their yield (yield components) when grown under field conditions. Three individual homozygous T3 lines of each genotype were transplanted in a paddy field and grown to maturity. Both NT and nullizygous plants from each of the heterozygous transgenic lines were grown together in the same field conditions and used as controls. We included the nullizygous plants since somatic variation in transgenic plants can generate phenotypes regardless of the transgene effect. Thus, yield component values of PGD1::OsNF-YA4 and PGD1::OsNF-YA7 transgenic plants were compared with those of the NT and nullizygous plants (Figs. 3B and S2). PGD1::OsNF-YA4 plants showed abnormal yield component values (Table S2-S4), such that grain filling rate (FR), number of total spikelets (NSP), number of panicles (NP), and total grain weight (TGW) were decreased compared to both controls. In contrast, PGD1::OsNF-YA7 transgenic plants showed no difference in yield components (Figs. 3B and S2 and Tables S2–S4). These results suggest that overexpression of OsNF-YA7 does not contribute to reproductive development, while overexpression of OsNF-YA4 is important, which is consistent with the high levels of expression of OsNF-YA4 in BH spikelets.

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Fig. 3. Morphological analysis of plants overexpressing OsNF-YA4 and OsNF-YA7. (A) The shoot height and root length of 2 -week-old PGD1::OsNF-YA7 and PGD1::OsNF-YA4. Three homozygous lines of PGD1::OsNF-YA7 (#7, 9, and 15) and three homozygous lines of PGD1::OsNF-YA4 (#12, 13, and 17) were measured. Data are shown as the mean ± SD (n > 10 for each line). (B) Agronomic traits of three independent T3 homozygous lines for each transgene together with the NT control. Each data point represents the percentage of the mean values (n = 30) with the NT plants assigned a reference value of 100%. CL, culm length; PL, panicle length; NP, number of panicles per hill; NSP, number of spikelets per panicle; TNS, total number of spikelets; FR, filling rate; NFG, number of filled grains; TGW, total grain weight; 1000 GW, 1000 grain weight.

3.4. Overexpression of OsNF-YA7 enhances drought resistance We next characterized the performance of PGD1::OsNF-YA7, PGD1::OsNF-YA4, and NT plants grown under drought conditions by monitoring visual symptoms induced by the drought stress (Fig. 4A). After 3 days of drought treatment, the NT plants started to display slight visual symptoms of drought-induced damage, such as leaf rolling and wilting. These symptoms became increasingly severe as the treatment continued, such that all leaves were dry until 7 days of drought treatment and re-watering did not result in recovery from the drought-induced damage (Fig. 4A). Similar to the NT plants, the PGD1::OsNF-YA4 transgenic plants did not exhibit drought tolerance and all the leaves of the transgenic plants were highly wilted and dry until 7 days of drought treatment (Fig. 4A), and also failed to recover following re-watering. In contrast, PGD1::OsNF-YA7 transgenic plants exhibited high resistance to the drought treatment (Fig. 4A). When exposed to drought for 3 days, PGD1::OsNF-YA7 transgenic plants were phenotypically similar to leaves of the untreated PGD1::OsNF-YA7 plants. Seven days of drought treatment resulted in damage to the PGD1::OsNF-YA7 transgenic plants, but the visual symptoms were considerably weaker than those of the NT plants. Re-watering resulted in recovery of the PGD1::OsNF-YA7 transgenic plants from the drought-induced damage. Taken together, the results suggest that the overexpression of the drought-responsive OsNF-YA7 gene in rice enhances drought resistance.

To independently confirm the resistance of PGD1::OsNF-YA7 and PGD1::OsNF-YA4 transgenic plants to drought stress, a PAM (pulseamplitude modulation) test was carried out (Fig. 4B), using the Fv /Fm values of the analysis to monitor the effect of abiotic stress [53–54]. We found that drought treatment substantially reduced the Fv /Fm values in the NT plants from the starting value of 0.8 as early as 1 h after the beginning of the treatment, and after 1.5 h the decrease was substantial. In contrast, the decrease in Fv /Fm values was delayed in the PGD1::OsNF-YA7 plants, such that all individual lines showed similar Fv /Fm values to those of the untreated control plants until 2.5 h after the drought treatment. Indeed, the Fv /Fm values only started to show a decrease after 3.0 h of treatment (Fig. 4B). Consequently, the Fv /Fm values of the PGD1::OsNF-YA7 transgenic plants were 2-fold higher than those of the NT plants at 2.5 h after drought treatment, whereas the PGD1::OsNF-YA4 plants showed no changes in the Fv /Fm values compared to the NT control (Fig. 4B). These results are consistent with the phenotypic observations and the conclusion that overexpression of OsNF-YA7 enhances resistance to drought stress. 3.5. OsNF-YA7 expression is not induced by ABA As previously mentioned above, molecular mechanisms for plant drought tolerance have been associated with ABA-dependent and -independent pathways [57]. To investigate the potential role of ABA in regulating OsNF-YA expression, we re-analyzed our previously published microarray data [8] and looked for evidence of

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Fig. 4. Drought stress resistance of PGD1::OsNF-YA7 and PGD1::OsNF-YA4 transgenic plants. (A) Phenotypes of four-week-old PGD1::OsNF-YA7, PGD1::OsNF-YA4 and NT were determined under drought treatment conditions. Drought stress was applied by no watering, and re-watering was performed after 7 days of drought treatment. Each number indicates individual lines of PGD1::OsNF-YA7 and PGD1::OsNF-YA4 and 10 plants from each individual line were used. (B) Pulse-Amplitude Modulation tests of PGD1::OsNF-YA7 and PGD1::OsNF-YA4 plants. Chlorophyll fluorescence (Fv /Fm ) of two-week-old PGD1::OsNF-YA7, PGD1::OsNF-YA4, and NT plants exposed to drought stress was measured in the dark using a pulse amplitude modulation fluorometer. Each data point represents the mean ± SD of triplicate experiments (n = 10).

responsiveness of the 10 OsNF-YA genes to ABA. Of the 10 OsNFYA genes, only OsNF-YA4 was induced by ABA (Fig. 5A), and this was confirmed using qRT-PCR to evaluate the effects of ABA treatment on OsNF-YA7 and OsNF-YA4 expression (Fig. 5B). The short term effects were quantified by applying 10 ␮M or 100 ␮M ABA to 14-day old rice seedlings for 24 h. This led to an approximately 30% decrease in OsNF-YA7 transcript levels, and a 4-fold increase in OsNF-YA4 transcript levels compared to levels in untreated control plants. The long term effects were tested by examining the expression of OsNF-YA4 and OsNF-YA7 in seedlings grown for 2 weeks on MS medium that included 0.5 ␮M, 1 ␮M or 3 ␮M ABA. OsNF-YA4 transcript levels gradually increased, depending on the ABA concentration, while OsNF-YA7 transcript levels were slightly reduced in all the ABA treated plants, indicating differential regulation of OsNF-YA7 and OsNF-YA4 by ABA. Based on the observed induction of OsNF-YA7 expression by drought but not by ABA, as well as the enhanced drought tolerance of the OsNF-YA7 overexpressing transgenic plants, we hypothesized that OsNF-YA7-mediated drought tolerance operates through an ABA-independent pathway. Typically, ABA-independent drought regulatory genes have a specific cis-element, known as the dehydration-responsive element/C-repeat (DRE/CRT), A/GCCGAC [45]. We therefore looked for the presence of DRE/CRT cis-elements in the promoters of OsNF-YA7 and OsNF-YA4, examining 2 kb upstream sequences from the transcription start sites. Interestingly, the promoter of OsNF-YA7 included 3 DRE/CRT elements, whereas the promoter of OsNF-YA4 had no DRE/CRT element (Fig. 5C). However, we identified an ABA-responsive (ABRE) cis-element in the OsNF-YA4 promoter, supporting the idea that OsNF-YA7-mediated drought tolerance is part of an ABAindependent pathway, while regulation of OsNF-YA4 expression by abiotic stresses, such as cold temperatures, may be part of an ABA-dependent pathway.

3.6. Identification of genes involved in the OsNF-YA7-mediated drought tolerance pathway To identify downstream genes that are regulated by OsNFYA7 and OsNF-YA4, we performed an RNA-sequencing (RNA-seq) transcriptome analysis of 2-week-old NT, PGD1::OsNF-YA7, and PGD1::OsNF-YA4 whole plants. We calculated the reads per transcript kilobase per million fragments mapped reads (FPKM) values for each sample with count data and a cutoff fold change in gene expression level of at least three fold to identify up- and down-regulated genes. We identified 771 up- and 1162 downregulated genes in the PGD1::OsNF-YA7 transgenic plants relative to NT, whereas PGD1::OsNF-YA4 transgenic plants showed 595 upregulated and 1777 down-regulated genes compared to the control (Fig. 6A and Table S5). Although there was an overlap of 212 upregulated and 664 down-regulated genes between PGD1::OsNF-YA4 and PGD1::OsNF-YA7, 1057 and 1496 genes were found to be independently regulated in the two transgenic lines, PGD1::OsNF-YA7 and PGD1::OsNF-YA4, respectively (Fig. 6B). From this we concluded that both OsNF-YA4 and OsNF-YA7 have both common and independent downstream gene targets. PGD1::OsNF-YA7 plants exhibited high resistance to drought treatments (Fig. 4), suggesting that an OsNF-YA7-mediated drought tolerance pathway was constitutively active in the overexpressing plants even when no drought stress is imposed. To identify key genes regulated by the OsNF-YA7-mediated drought tolerance pathway, the RNA-seq profiles were compared with previous microarray profiles (expression fold change > 3) regulated by drought and ABA [8]. The goal was to identify genes that were regulated by both drought- and OsNF-YA7 (Fig. 6C), and this analysis suggested 72 such genes (Fig. 6D and Table S6). The RNA-seq profile of OsNF-YA4 was also compared to the previously published microarray profiles, and since PGD1::OsNF-YA4 plants showed no

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Fig. 5. Expression of OsNF-YA7 and OsNF-YA4 in response to ABA treatment. (A) Microarray analysis of 10 rice OsNF-YA genes in response to ABA treatment. (B) Quantitative RT-PCR analysis of OsNF-YA7 and OsNF-YA4 expression in response to ABA treatment. The short-term ABA treatment consisted of 2-week old NT plants transiently exposed to 10 ␮M or 100 ␮M ABA for one day. The long-term ABA treatment consisted of 2-week-old NT plants that were germinated and grown on MS media supplemented with 0, 0.5, 1 or 3 ␮M ABA. Ubiquitin1 expression was used as an internal control. Data are shown as the mean ± SD of three biological and two technical replicates. (C) Promoter analysis of OsNF-YA7 and OsNF-YA4 for DRE/CRT and ABRE cis-elements.

drought tolerance phenotypes, ABA-independent genes common to both drought- and OsNF-YA4-regulated genes were considered to be non-specific background ‘noise’ (Fig. 6C). The non-specific background was 89 genes (Fig. 6D and Table S7), and we suggest that this was also the case for the 72 common genes regulated by both drought and OsNF-YA7. Thus, we compared the 72 genes to the 89 non-specific background genes, and then subtracted the common genes from the 72 OsNF-YA7-mediated drought regulated genes (Fig. 6C). The remaining 48 unique genes were then considered to be key genes involved in the ABA independent OsNF-YA7–mediated drought tolerance pathway (Fig. 6D). Of these 48 candidates, 26 genes were up- and 22 were downregulated (Tables 1 and 2) and the 26 up-regulated genes included several known drought-regulated genes, such as those encoding a Bowman–Birk trypsin inhibitor, a lectin-like receptor kinase, DIN1, an FtsH protease, a leucine-rich repeat receptor like kinase and a WRKY transcription factor [58–64]. Among the downregulated genes, DnaJ, ICE1-like, glutathione reductase, DERB1C, an ABC transporter, and dehydrin RAB 16C have all previously been identified as abiotic stress regulators [65–68]. We validated the expression patterns of 2 selected up- and 2 selected downregulated genes by qRT-PCR (Fig. 7), and found their expression patterns to be similar to the expression patterns derived from the

RNA-seq data. We therefore concluded that OsNF-YA7 does indeed regulate a unique set of drought-related genes, thereby contributing to enhanced drought tolerance.

4. Discussion The NF-Y complex consists of a set of evolutionarily conserved transcription factors that are present in a taxonomically broad range of organisms, including yeast, plants and humans [29]. They bind specifically to a CCAAT sequence in the promoters of their target genes, and this consensus sequence is present in approximately 30% of eukaryotic promoters [27]. Each subunit of the heterotrimeric NF-Y complex is encoded by a single gene in mammals, whereas plants have approximately 10 different genes that encode each subunit of the NF-Y transcription factor complex (e.g., 10 NF-YA, 13 NF-YB, and 13 NF-YC genes in A. thaliana; 7 NF-YA, 17 NF-YB and 12 NF-YC in Brachypodium distachyon; and 10 NFYA, 11 NF-YB and 7 NF-YC in rice) [13,14,26,29,69]. The presence of multiple genes for each subunit is thought to give rise to functional diversification through combinatorial interactions between different NF-Y subunit members [15]. OsNF-YA4 and OsNF-YA7 belong to the class 2 subclade (Fig. 1C) and have highly conserved NF-YA/B interaction and DNA bind-

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Fig. 6. Venn diagrams of OsNF-YA7- and OsNF-YA4-regulated genes. (A) Venn diagrams of up- (expression fold change > 3) and down-regulated genes (expression fold change < 3) in two-week-old PGD1::OsNF-YA7 and PGD1::OsNF-YA4 transgenic plants compared to NT plants. (B) Venn diagrams of gene expression common to PGD1::OsNFYA7 and PGD1::OsNF-YA4. (C) Strategies for isolation of ABA-independent and OsNF-YA7-mediated drought regulatory genes. (D) Venn diagrams of ABA-independent and OsNF-YA7-mediated drought regulatory genes.

ing domains (Fig. 1D). Nevertheless, their expression patterns in response to drought were distinct in that OsNF-YA4 was not upregulated by drought treatments while OsNF-YA7 expression was induced by drought treatments (Fig. 1B). OsNF-YA4 and OsNF-YA7 also differed in their temporal expression patterns throughout the rice life cycle (Fig. 2A). PGD1::OsNF-YA4 transgenic plants did not show any changes in sensitivity to drought stress, whereas, PGD1::OsNF-YA7 transgenic plants showed enhanced drought tolerance (Fig. 4), and the grain filling rate was affected only by overexpression of OsNF-YA4 (Fig. 3). Although the PGD1::OsNF-YA4 showed no sensitivity to drought stress, it is still possible that OsNFYA4 might be involved in a drought tolerance pathway. It is because PGD1::OsNF-YA4 transgenic plants showed mild overexpression of the OsNF-YA4 (about 5–7 folds higher than in NT) as compared to strong overexpression OsNF-YA7 (about 25–36 folds higher than in NT) of PGD1::OsNF-YA7 transgenic plants. However, these data indicate that OsNF-YA4 overexpression negatively regulates the grain filling process in rice spikelets. Interestingly, AtNF-YA1 and AtNFYA5 are known to be involved in A. thaliana seed development [34],

suggesting that OsNF-YA4 is involved in grain filling in rice spikelets and that OsNF-YA7 plays a role in rice drought tolerance. Recently studies have elucidated the roles of NF-Y transcription factors in abiotic stresses, such as drought. The expression levels of 15 of 36 A. thaliana genes encoding NF-Y subunits are responsive to drought stress, and overexpression of drought-responsive NFY subunits, such as AtNF-YA5 and AtNF-YB1, can enhance drought resistance [15,16,70]. Moreover, rice OsNF-YC1 and OsNF-YC6 are responsive to drought stress and overexpression of the Bermudagrass (Cynodon dactylon × Cynodon transvaalensis) Cdt-NF-YC1 gene in rice was reported to increase drought tolerance [17], indicating that the OsNF-Y complex is a component of a drought tolerance mechanism. In this study, we determined that among 10 OsNF-YA genes, OsNF-YA7 is the highest drought responsive member (Fig. 1). We therefore propose that OsNF-YA7 may bind OsNF-YC1 and/or OsNF-YC6, together with an uncharacterized OsNF-YB partner(s), to form a heterotrimeric complex, as part of an OsNF-Y-mediated drought tolerance mechanism. To determine the role of OsNFY-YA7 in drought resistance, we investigated whether the underlying mechanism was ABA-

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Table 1 List of genes up-regulated (> 3 fold) by OsNF-YA7 as part of ABA-independent drought regulation in rice. Gene ID

Annotation

Os01g0124000 Os01g0332900 Os01g0503400 Os01g0837900 Os01g0944100 Os03g0257900 Os03g0857500 Os04g0249600 Os04g0403400 Os04g0553000 Os05g0494600 Os05g0590000 Os06g0229000 Os06g0343000 Os06g0477600 Os06g0681200 Os07g0564500 Os08g0110600 Os08g0192200 Os08g0203400 Os08g0425800 Os09g0255400 Os09g0417800 Os11g0428500 Os12g0111700 Os12g0270900

Similar to Bowman Birk trypsin inhibitor Similar to immediate-early protein RSP40 Similar to hypothetical protein Similar to serine/threonine-protein kinase AFC1 like Similar to hypothetical protein Similar to lectin-like receptor kinase 7; 2 (receptor lectin like kinase) Similar to hypothetical protein Similar to senescence-associated protein DIN1 Tyrosyl-DNA phosphodiesterase family protein Similar to OSIGBa0143N19.6 protein (lipid transfer machine permease) Similar to Circadian clock coupling factor ZGT Peptidase A1 domain containing protein Similar to FtsH protease (VAR2) (zinc dependent protease) Similar to hypothetical protein Non-protein coding transcript Cupredoxin domain containing protein Pyridine nucleotide-disulphide oxidoreductase (NADH dehydrogenase-like) Similar to hypothetical protein Similar to hypothetical protein Protein kinase (Leucine-rich repeat receptor like kinase) Similar to hypothetical protein Similar to indole-3-glycerol phosphate synthase WRKY transcription factor 62 Similar to hypothetical protein Similar to hypothetical protein Sulfotransferase family protein

Table 2 List of genes down-regulated (<3 fold) by OsNF-YA7 as part of ABA-independent drought regulation in rice. ID

Annotation

Os01g0117600 Os01g0131900 Os01g0606900 Os01g0705700 Os02g0813500 Os03g0826800 Os03g0832800 Os04g0673700 Os05g0545300 Os05g0595100 Os06g0127100 Os06g0133100 Os07g0190600 Os07g0288700 Os07g0296000 Os08g0412800 Os08g0553800 Os09g0444900 Os11g0454000 Os11g0528200 Os12g0142900 Os12g0266000

Protein kinase, catalytic domain containing protein Similar to wound-induced protease inhibitor (WIP1) Heat shock protein DnaJ, N-terminal domain containing protein Similar to transcription factor ICE1 (inducer of CBF expression 1) Similar to glutathione reductase, cytosolic Similar to hypothetical protein Similar to glycerol-3-phosphate acyltransferase 1 Similar to hypothetical protein Serine/threonine protein kinase domain containing protein Similar to UDP-glucose-4-epimerase Dehydration-responsive element-binding protein 1C (DREB1C) Similar to hypothetical protein Similar to hypothetical protein Similar to white–brown-complex ABC transporter family Similar to hypothetical protein Similar to hypothetical protein NAD(P)-binding domain containing protein Similar to plant viral-response family protein Dehydrin RAB 16C Similar to NEF1 (no exine formation 1) Similar to H0321H01.8 protein Non-protein coding transcript

dependent or -independent pathway. Expression of OsNF-YA7 was not induced by an ABA treatment (Fig. 5B), and the promoter of OsNF-YA7 was shown to have 3 DRE/CRT elements (Fig. 5C), which are well known cis-elements that are regulated by ABAindependent, drought regulated OsDREB2 transcription factors. The rice genome contains 5 OsDREB2s, of which OsDREB2A and OsDREB2B are the member to be induced by drought stress [71]. Based on this information, we hypothesize that OsDREB2A and/or OsDREB2B bind to the DRE/CRT elements in the OsNF-YA7 promoter and directly regulates OsNF-YA7 expression in an ABA independent manner. ABA-independent and drought-induced OsNF-YA7 expression may regulate downstream genes to reduce damage as a consequence of severe drought conditions. We reasoned that the identification of genes that are downstream of OsNF-YA7 action would provide valuable mechanistic information and, RNA-

seq profiling analyses revealed 48 (26 up-regulated and 22 down-regulated genes) genes involved in the ABA independent OsNF-YA7-mediated drought pathway (Fig. 6). Interestingly, some of these 48 downstream target genes have already been reported to have a role in drought tolerance, or have been used as molecular markers for drought stress. For example, Bowman–Birk inhibitor transcripts were reported to accumulate at higher levels in a drought tolerant peanut cultivar than in a drought susceptible cultivar [60]. In addition, overexpression of receptor-like kinase OsSIK1 (stress-induced protein kinase1) and OsSIK2 was shown to improve drought stress tolerance in rice [58,62], and a mutation in AtLecRKb2 (At1g70130; a lectin receptor-like kinase) resulted in drought sensitive phenotypes [59]. These observations are congruent with a relationship between the activity of the genes downstream of OsNF-YA7 action contributing to the increased drought tolerance phenotype of PGD1::OsNF-YA7 plants. In conclusion, we propose

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[8]

[9]

[10]

[11]

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[13] [14]

[15]

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[17] Fig. 7. Validation of OsNF-YA7-mediated drought regulatory genes. Quantitative RT-PCR of OsNF-YA7-mediated drought regulatory genes in two-week-old NT and PGD1::OsNF-YA7 whole plants. Ubiquitin1 expression was used as an internal control. Data are shown as the mean ± SE of three biological and two technical replicates.

[18]

[19]

that OsNF-YA7 has an important role in regulating genes involved in drought tolerance in rice.

[20] [21]

Acknowledgements This research was supported by the Rural Development Administration under the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ906910), by the National Research Foundation of Korea under the MSIP grant (Project No. 2014051690), and by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2014R1A6A3A04053795 and NRF-2013R1A6A3A04060627).

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.10. 006.

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