Identification and functional characterization of a rice NAC gene involved in the regulation of leaf senescence
© Zhou et al.; licensee BioMed Central Ltd. 2013
Received: 29 April 2013
Accepted: 13 August 2013
Published: 12 September 2013
As the final stage of leaf development, leaf senescence may cause the decline of photosynthesis and gradual reduction of carbon assimilation, which makes it a possible limiting factor for crop yield. NACs are plant-specific transcription factors and some NACs have been confirmed to play important roles in regulating leaf senescence.
In this study, we reported a member of the NAC transcription factor family named OsNAP whose expression is associated with leaf senescence, and investigated its preliminary function during the process of leaf senescence. The results of qRT-PCR showed that the OsNAP transcripts were accumulated gradually in response to leaf senescence and treatment with methyl jasmonic acid (MeJA). A subcellular localization assay indicated that OsNAP is a nuclear-localized protein. Yeast one-hybrid experiments indicated that OsNAP can bind the NAC recognition site (NACRS)-like sequence. OsNAP-overexpressing transgenic plants displayed an accelerated leaf senescence phenotype at the grain-filling stage, which might be caused by the elevated JA levels and the increased expression of the JA biosynthesis-related genes LOX2 and AOC1, and showed enhanced tolerance ability to MeJA treatment at the seedling stage. Nevertheless, the leaf senescence process was delayed in OsNAP RNAi transgenic plants with a dramatic drop in JA levels and with decreased expression levels of the JA biosynthesis-related genes AOS2, AOC1 and OPR7.
These results suggest that OsNAP acts as a positive regulator of leaf senescence and this regulation may occur via the JA pathway.
KeywordsChlorophyll Leaf senescence NAC JA Rice (Oryza sativa L.)
Leaf senescence is complex in that it involves many highly organized molecular and cellular processes such as the disintegration of chloroplast, down-regulation of photosynthesis, degradation of nucleic acid, protein, and lipid and recycling of nutrients. And as the final stage of leaf development, leaf senescence eventually leads to leaf death, which is controlled by both internal and external factors. The internal factors include age, phytohormone levels and developmental processes, and the external factors mainly comprise environmental/biological stresses such as extreme temperature, shading, drought, wounding, nutrient limitation, pathogen attack and oxidative stress by UV-B irradiation and ozone [1–3].
NACs (NAM, ATAF and CUC) are plant-specific transcription factors and are widely found in plants. It has been reported that many NACs show enhanced expression during dark-induced and natural leaf senescence in Arabidopsis [4, 5], and may play a central role in mediating leaf senescence. The senescence-controlling NAC gene NAM-B1 was also reported to be associated with the contents of grain protein, zinc, and iron in wheat . ANAC092/AtNAC2/ORE1, whose expression correlates with senescence, positively regulates aging-induced cell death in Arabidopsis leaves . The oresara1 (ore1) mutant, which lacks the functional ANAC092/AtNAC2/ORE1 gene, displays a delayed leaf senescence phenotype . An ABA-responsive NAC transcription factor VND-INTERACTING2 (VNI2), whose expression shows a leaf aging or leaf longevity-dependent expression pattern, may mediate the crosstalk between the salt stress response and the leaf aging process . AtNAP is strongly up-regulated during leaf senescence in Arabidopsis, and atnap null mutants show a delayed leaf senescence phenotype, whereas the inducible overexpression of AtNAP causes precocious leaf senescence . Recent studies have shown that AtNAP can bind to the promoter region of SENESCENCE-ASSOCIATED GENE113 (SAG113) to form a ABA-AtNAP-SAG113 protein phosphastase 2C regulatory chain for controlling stomatal movement and water loss in senescing Arabidopsis leaves [11, 12]. Many NAC transcription factors can be induced by leaf senescence, but their particular roles in leaf senescence still remain largely unknown.
Many studies have revealed that JA and its derivatives play important roles in regulating the response to leaf senescence in plants. MeJA and its precursor JA were first isolated in oat and shown to promote senescence in detached oat leaves, suggesting that jasmonates might serve as powerful promoters to induce plant senescence . JA-induced leaf senescence is accompanied by the increased expression of several enzymes involved in JA biosynthesis and the decreased expression of the genes involved in photosynthesis [14–16]. In addition, JA can activate the expression of many senescence-regulated genes such as AtWRKY6, OsAkaGal and ESR/ESP, which play major roles in leaf senescence. Leaf senescence and MeJA can induce the expression of OsAkaGal, which encodes a chloroplast alkaline α-galactosidase involved in the degradation of digalactosyl diacylglycerol during leaf senescence in rice [18, 20]. Exogenous application of JA to attached and detached leaves promotes leaf senescence in Arabidopsis but does not induce leaf senescence in the JA-insensitive mutant coi1, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence . Furthermore, JA promotes H2O2 accumulation in the leaves of JA-sensitive cultivar TN1 seedlings to accelerate the process of senescence but not in the leaves of JA-insensitive cultivar TNG67 . Many JA and senescence-regulated genes have been identified, yet how the crosstalk between JA signaling and senescence occurs remains to be thoroughly understood.
In this study, we isolated and characterized the JA-induced senescence-associated gene OsNAP, which encodes a NAC transcription factor in rice. We generated the OsNAP overexpression and RNAi lines and analyzed the leaf senescence process of them. We also determined the endogenous JA levels and checked the expression of the genes encoding the enzymes of the JA biosynthetic pathway in the OsNAP transgenic lines and wild-type plants. Studies of these lines indicated that OsNAP acts as a positive regulator of the JA pathway to mediate the leaf senescence process in rice.
Characterization of OsNAPin rice
There are 75 predicted NAC proteins  and 140 putative NAC or NAC-like proteins (ONAC) in rice . Later computational analyses showed that there are at least 151 OsNAC genes in the rice genome . And phylogenetic analysis showed that there are 13 OsNACs which closely cluster with AtNAP: Os03g21060 (OsNAP), Os12g03040, Os11g03300 (OsNAC10), Os03g60080 (SNAC1/OsNAC9), Os01g60020 (OsNAC4), Os07g12340 (OsNAC3), Os11g08210 (OsNAC5), Os07g37920, Os05g34310, Os07g48450, Os01g01430, Os05g34830 and Os01g66120 (OsNAC6/SNAC2) (Additional file 1: Figure S1). Digital expression profile analysis showed that the expression levels of Os07g37920, Os05g34310, Os07g48450 and Os01g01430 are low and even undetectable in leaves. And other 9 genes are expressed in leaves, but only the transcripts of OsNAP, OsNAC5 and OsNAC6/SNAC2 in leaves are up-regulated during leaf senescence [25–27]. However, several studies have demonstrated that OsNAC5 and OsNAC6/SNAC2 are involved in stress tolerance in rice [28–30]. We therefore decided to examine the potential role of OsNAP in the leaf senescence of rice.
The full-length cDNA of OsNAP was isolated for further functional analysis. OsNAP cDNA (Accession number AK243514) encodes a protein with 392 amino acids. Sequence analysis suggested that OsNAP is identical to ONAC058, which belongs to the NAC family in rice . Twelve sequences were obtained from Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Gossypium hirsutum, Glycine max, Bambusa emeiensis, Brachypodium distachyon, Sorghum bicolor, Hordeum vulgare, and Zea mays by BLASTP search. The resulting phylogenetic relationships showed that each of them contained A-E subdomains (Additional file 1: Figure S2), which is consistent with the results previously reported [31, 32].
Subcellular localization of OsNAP
Biochemical function of OsNAP in yeast
Several NACs not only act on gene promoters but also have transactivation activity [30, 34, 36]. To determinate whether OsNAP has activation capacity, we tested the transactivation activity of OsNAP in yeast. The full-length and partial fragments of OsNAP were fused to the GAL4 DNA binding domain in the pGBKT7 vector and the resultant vectors were co-transformed with pGADT7 into the AH109 yeast strain (Figure 2C). The combinations of pHIS53/pGAD-Rec2-53 and pGBDK7/pGADT7 were used as the positive control and negative control, respectively. It was observed that all of the co-transformants grew well on the SD/Leu-/Trp- medium, but only the cells transformed with pHIS53/pGAD-Rec2-53 and pGBD-OsNAP-TR/pGADT7 could grow on the SD/Leu-/Trp-/His-/Ade- medium (Figure 2D), suggesting that the transactivation domain of OsNAP is located in the C-terminal transcriptional regulatory (TR) domain of the protein.
Expression pattern of OsNAP
In addition, qRT-PCR analysis showed that the expression of OsNAP was continuously increased during MeJA treatment (Figure 3C), indicating that the expression of OsNAP was induced by MeJA treatment.
Effect of the overexpression of OsNAPon leaf senescence at the grain-filling stage
Effect of knock-down of OsNAPon leaf senescence in rice
Relation between the senescence phenotype in OsNAPtransgenic plants and endogenous JA content
Important role of OsNAP in regulating leaf senescence in rice
NAC proteins are unique transcription factors involved in different developmental processes including senescence in plants . Approximately one-fifth of NACs (20/109) are present in the senescence ESTs (expressed sequence tags) in Arabidopsis . About 46% of the up-regulated genes in ANAC092/AtNAC2/ORE1-overexpressing transgenic plants are senescence-associated genes . These findings suggest that NAC proteins may play crucial roles in senescence.
In this study, we identified a member of the NAC transcription factors OsNAP, which plays an important role in regulating leaf senescence in rice. Firstly, the transcript of OsNAP was up-regulated during natural leaf senescence (Figure 3A, 3B, b-d). Secondly, OsNAP-overexpressing lines displayed an accelerated leaf senescence phenotype and showed a reduced net photosynthetic rate coupled with a decline in chlorophyll content during the reproductive growth stage (Figure 4). In addition, OsNAP RNAi lines showed a markedly delayed leaf senescence, which was confirmed by the higher expression of OsDOS (Figure 5). Finally, as the homolog of AtNAP in rice, OsNAP can restore the delayed leaf senescence phenotype in atnap mutant to a normal WT phenotype . These results suggest that OsNAP is associated with leaf senescence in rice.
The role of OsNAP in the crosstalk between senescence and the JA pathway
JA and its derivatives are known as endogenous modulators of many physiological processes in plants including senescence [14, 40, 41]. Our analysis revealed that the expression level of OsNAP was up-regulated with MeJA treatment (Figure 3C), suggesting that OsNAP may be involved in the JA pathway. There are two important processes in the JA pathway: JA biosynthesis and JA signal transduction [40, 42]. Accelerated or hindered JA biosynthesis can lead to more rapid or delayed senescence in plants. OsNAP overexpression resulted in accelerated senescence in transgenic rice and it was evident from the decreased chlorophyll content and net photosynthetic rate (Figure 4), which was correlated with accumulation of endogenous JA content and increased transcript levels of JA biosynthesis genes (Figure 7A, 7B). qRT-PCR results showed that the expression of the JA biosynthesis gene LOX2 was increased 2 to 4 fold in the OsNAP-overexpressing lines compared with in the WT plants (Figure 7B). JA levels were increased during senescence in OsNAP-overexpression transgenic lines and the WT plants, which is in accordance with previous conclusions [2, 4, 15]. On one hand, JA can induce the expression of the key enzymes of chlorophyll breakdown including chlorophyllase , promoting the loss of chlorophyll in leaves. On the other hand, JA can also induce the expression of many senescence-associated genes to promote senescence [14, 44]. Therefore, it is possible that the accelerated leaf senescence observed in OsNAP-overexpressing plants might have resulted from an increase in the endogenous JA levels. In addition, OsNAP RNAi transgenic plants displayed a marked delay of leaf senescence (Figure 5). JA in these lines declined dramatically to non-detectable levels (Figure 7A), and the transcripts of the genes encoding enzymes in the JA biosynthesis pathway, including AOS2, AOC and OPR7, dropped sharply (Figure 7C). The delayed yellowing during natural senescence in OsNAP RNAi transgenic plants at the grain-filling stage may be due to the reduced JA production. Hence, OsNAP may act as a positive regulator of leaf senescence by regulating the JA biosynthesis pathway.
Besides JA biosynthesis, a block in JA signal transduction can also alter the process of senescence. The mutant coi1, whose JA signaling has been impaired, exhibits delayed senescence phenotype . Rubisco activase (RCA) is a COI1-dependent JA-repressed protein and it has been shown that a loss-of-function mutation of RCA leads to typical signs of senescence . OsDOS is a nucleus-localized CCCH-type zinc finger protein in rice that acts as a negative regulator of the JA pathway and senescence process . The transcripts of OsDOS were significantly higher in OsNAP RNAi lines than in the WT plants (Figure 5E), suggesting that the JA signaling pathway was impaired. In addition, MeJA inhibition was significantly lower in the OsNAP high-expressing lines compared with in WT plants, suggesting that the overexpression of OsNAP enhances the resistance to exogenous MeJA in rice (Figure 6). These results indicate that JA signaling pathway is involved in the OsNAP-mediated senescence process.
The NAC transcription factors can bind to specific cis-elements of target gene promoters, and can thus regulate gene transcription. For example, the miR319-regulated TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factors regulate the JA biosynthesis gene LOX2, controlling JA content and affecting leaf senescence . And AtNAP can bind to the promoter region of SAG113, which negatively regulates the ABA signaling pathway that modulates stomatal movement and water loss in senescing leaves . In the present study, ABA did not have significant effects on OsNAP expression levels (data not shown), while MeJA gradually induced the expression of OsNAP (Figure 3C). In addition, OsNAP was able to bind to the NACRS-like sequence (Figure 2A, 2B), and the C-terminal region had transactivation activity in yeast (Figure 2C, 2D). It’s worth noting that there were a number of putative NAC recognition sequences (NACRS) and core DNA binding sequences (CDBS) in the 1 kb region upstream of the JA biosynthesis-related genes (Additional file 1: Table S2). For these reasons, it can be concluded that OsNAP may interact with JA biosynthesis genes directly or indirectly. Further experiments, particularly a screen for the interacting proteins, will be necessary to determine the specific roles of OsNAP in JA signaling and leaf senescence.
In this article, a NAC transcription factor OsNAP was isolated and characterized in rice, and OsNAP was identified as a key mediator between the JA pathway and leaf senescence. In addition, the expression of OsNAP was detected in the callus, sheath, mature seed, node and internode besides the leaves (Figure 3B), suggesting that OsNAP plays important roles in diverse biologic processes in rice. Nevertheless, further analyses are required to clarify the specific roles of OsNAP in rice.
Plant materials and growth conditions
Zhonghua 11 (Oryza Sativa L. ssp. Japonica cv. Zhonghua 11) was used for this study. Wild-type and OsNAP transgenic plants were planted in the field of Huazhong Agricultural University (Wuhan, China).
To check the OsNAP expression during MeJA treatment, Zhonghua 11 plants were grown in a greenhouse with a 14-h light/10-h dark cycle. MeJA treatment was conducted by spraying 0.2 mM MeJA on the leaves. Five plants were collected at each time point of 0, 1, 2, 3 and 12 h after treatment for RNA isolation.
For MeJA treatment at the seedling stage, T2 seeds of Zhonghua 11 and overexpression lines were sterilized and germinated on 1/2 MS medium with 16-h light/8-h dark cycle for 4 days, and then transferred to 1/2 MS medium containing 25 μM MeJA. The shoot and root lengths of the seedlings were measured after 10 days of growth.
Subcellular localization of OsNAP
The cDNA fragment of OsNAP was amplified using the full-length cDNA clone J100075D15 (http://cdna01.dna.affrc.go.jp/cDNA) as a template with the following primers: 5’- ATCggtaccATGGTTCTGTCGAACCCGGC-3’ and 5’-ATCggatccGTTCATCCCCATGTTAGAGT. The PCR products were sub-cloned into the pU1391a-GFP vector digested with Kpn I and BamH I to form pU1391a-OsNAP-GFP . The construct was transformed into onion (Allium cepa L.) epidermal cells using a Biolistic PDS-1000/He particle delivery system (Bio-Rad) according to a previously described method . After fixation in 4% PFA and staining with 10 μg/ml DAPI, transient expression of GFP fluorescence was observed using a Confocal Laser-scanning Microscope (TCS SP2, Leica, Germany).
A 1176-bp cDNA fragment of OsNAP was amplified using the following primers: 5’-gaattcATGGTTCTGTCGAACCCGGC-3’ and 5’-tctagaGTTCATCCCCATGTTAGAGT-3’ with a EcoR I site and a Xba I site. The PCR products were digested with EcoR I/Xba I and inserted into pM999-35S-EGFP. The OsNAP-EGFP construct and a positive control construct 35S::Ghd7::CFP containing a marker gene were co-introduced into Arabidopsis protoplasts as previously described . The GFP and CFP fluorescences were visualized using Confocal Laser-scanning Microscopy (TCS SP2, Leica, Germany).
Functional characterization of OsNAP in yeast
For the yeast one-hybrid assay, the cDNA fragment of OsNAP was amplified and cloned into the pGADT7 (Clontech, Palo Alto, CA, USA) vector to form pGAD-OsNAP. pGAD-OsNAP/pHIS2-cis, the positive control (pHIS53/pGAD-Rec2-53) and the negative control (pHIS2/pGAD-OsNAP) were co-transformed into the Y187 yeast strain for determination of the DNA-protein interactions according to a previous report . For the transactivation assay, the PCR product of the full-length and partial fragments of OsNAP were fused in frame to the GAL4 DNA binding domain in the pGBDK7 vector to form pGBD-OsNAP, pGBD-OsNAP-ABC, pGBD-OsNAP-DE and pGBD-OsNAP-TR. The combinations of pGBD-OsNAP/pGADT7, pGBD-OsNAP-ABC/pGADT7, pGBD-OsNAP-DE/pGADT7, pGBD-OsNAP-TR/pGADT7, the positive control (pHIS53/pGAD-Rec2-53) and the negative control (pGBDK7/pGADT7) were co-transformed into the AH109 yeast strain for the verification of transactivation activity.
Construction of expression plasmids and transformation into rice
To investigate the expression pattern of OsNAP, the promoter fragment of OsNAP was amplified using the following primers: 5’- ATCGaagcttCCGTTGCATTAGGAAACGTC-3’ and 5’ -ATCGtctagaACCCACACACAACACACACC-3’ and then cloned into the DX2181b vector , yielding the OsNAP promoter::GUS construct. To create the overexpression construct, the OsNAP genomic DNA sequence was amplified using the following primers: 5’- ATCGccatggTTCGCCATGTGCAATTATGT-3’ and 5’-ATCGggttaccCAGGGAGGTGTGTGTTGTGT-3’. The PCR fragments were digested with Nco I and BstE II and cloned into the pCAMBIA1301 expression vector. To suppress the OsNAP gene, a cDNA fragment of OsNAP was amplified using the following primers:5’- ATCggatccCCACCACCAACAACAACAAC-3’ and 5’-ATCggtaccCTCAGTCCCAGTGACGATCC-3’ and inserted into pMCG161 and pDS1301  to form pMCG161-OsNAP and pDS1301-OsNAP, respectively. The pMCG161-OsNAP plasmid was digested with Sac I/Spe I and inserted into the pDS1301-OsNAP to generate the RNAi construct. All the constructs were introduced into the EHA105 Agrobacterium tumefaciens strain and then introduced into the rice callus of Zhonghua 11 according to a previous method .
Molecular analysis of putative transgenic plants
The putative transgenic plants were identified by PCR according to a previously described method . The copy numbers of the transgenic plants were confirmed by Southern blot using a published protocol . The transcripts of OsNAP in single-copy transgenic lines were detected by Northern blot as described previously . The probes for Southern and Northern blot were amplified using PCR with gene-specific primers listed in Additional file 1: Table S1. The PCR program was as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and finally 72°C for 7 min.
GUS staining assay
GUS staining and observation were carried out as described previously . Various tissues and organs, including the callus, leaves, sheaths, nodes and mature seeds from P OsNAP ::GUS transgenic plants were immersed in GUS staining solution (0.1% Triton X-100, 1 mg/mL X-Gluc, 100 μg/ml chloramphenicol, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 10 mM Na2-EDTA, 20% methanol and 50 mM sodium phosphate, pH 7.0) at 37°C overnight. After staining, the samples were bleached with 75% (v/v) ethanol and photographed using a dissecting microscope (Leica, Germany).
RNA isolation and quantitative RT-PCR analysis
Total RNAs were extracted using Trizol reagent according to the manufacturer’s protocol (Invitrogen, USA). First-strand cDNAs were synthesized in a reaction volume of 20 μL containing 3 μg RNase-free DNase I treated total RNA, 0.5 mg Oligo(dT)15, 1 mM dNTPs, 10 mM dithiothreitol, and 200 units of SuperScript™ III reverse transcriptase (Invitrogen, USA). Quantitative RT-PCR (qRT-PCR) was carried out as described previously . Three replicates were performed for the analysis of each gene and for determining the relative expression levels between the repeats according to a previous report . Actin was used as an internal control for normalization. The primers are listed in Additional file 1: Table S1.
Measurement of chlorophyll content, net photosynthetic rate and endogenous JA levels
Chlorophyll content was determined as described previously . Net photosynthetic rate was measured from 9:00 am to 11:00 am or 3:00 pm to 5:00 pm using CIRAS-2 according to the manufacturer’s instructions (PP system, USA). The endogenous JA level was extracted and quantified using the previously described method .
Methyl jasmonate acid
Open reading frame
- TR domain:
Transcriptional regulatory (TR) domain
Day after heading.
This research was funded by the National High Technology Research and Development Program of China (863 Program), the National Natural Science Foundation of China and the National Program of Transgenic Variety Development of China. We are grateful Dr. Zuoxiong Liu for reading the manuscript. We also thank Huazhi Song for technical support in using confocal scanning laser microscopy (CSLM).
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