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A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata

A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by... Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the posi- tive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis- epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demon- strated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5. Keywords: ABA, Citrus reticulata, fruit ripening, MYB transcription factor, NAC transcription factor, postharvest, synergistic transcriptional regulation. Introduction Fruit ripening is a complex process, and based on the patterns categories. Knocking-down 9-cis-epoxycarotenoid dioxygenases of respiration and ethylene biosynthesis during fruit ripening, (NCEDs) led to a significant down-regulation of the expres- fleshy fruits can be divided into two categories, namely, climac- sion of genes associated with cell wall catabolism (such as pectate teric and non-climacteric fruits (Giovannoni, 2004). Abscisic acid lyase and expansin) in tomato. Moreover, the application of ex- (ABA) is a vital hormone that affects the ripening process of both ogenous ABA or treatment with an inhibitor of its biosynthesis © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3614 | Zhu et al. can significantly promote or hinder the color change, softening, help researchers to overcome these limitations. In the past dec- and flavor component accumulation of tomato, peach, straw- ades, experiments with ABA-deficient natural mutants and berry, and grape fruits (Zhang et  al., 2009a,b; Jia et  al., 2011; ABA treatments have indicated that ABA is a vital regulator of Sun et  al., 2012; Leng et  al., 2014). Beside the effect on fruit color change and the metabolism of both sugars and organic ripening, exogenous ABA treatment can also induce the ex- acids (Rodrigo et al., 2003; Wu et al., 2014; Zhang et al., 2014; pression of genes associated with chlorophyll degradation (such Rehman et  al., 2018). Recently, we found a novel stay-green as stay-green and pheophytinase) and promote the senescence of natural mutant (MT) in Citrus reticulata cv. Suavissima, and used Arabidopsis (Yang et al., 2014). Owing to ABA’s vital role in fruit it to study the transcriptional regulation of CrMYB68 on ca- ripening, the pathway of its biosynthesis has been well character- rotenoid metabolism via effects on CrBCH2 and CrNCED5 ized: violaxanthin derived from carotenoid metabolism is trans- (Zhu et al., 2017). However, research concerning comprehen- formed by ABA4, NCEDs, and several other enzymes to yield sive effects of ABA on the ripening process (such as chlorophyll ABA (Finkelstein, 2013). As the rate-limiting enzymes of ABA degradation, chloroplast disassembly, and ripening-related gene biosynthesis, NCEDs have attracted considerable of attention expression) and synergistic transcriptional regulation of ABA and the transcriptional regulation of NCEDs is one of the hot biosynthesis in citrus is rare. In the present study, we compre- topics in ABA research. In tomato, banana, peach, and citrus fruit, hensively analysed the effect of ABA on citrus ripening and WRKYs, ERF3, and bHLH1 transcription factors can directly further demonstrated that a novel NAC transcription factor, bind to their promoters and regulate the expression of NCEDs CrNAC036, directly regulated the expression of CrNCED5 (Endo et  al., 2016; Luo et  al., 2017; Wang et  al., 2019b). In the and could additionally interact with CrMYB68 to synergis- well-known model of anthocyanin biosynthesis, the synergistic tically down-regulate the expression of CrNCED5, providing transcriptional regulation of the MBW complex (MYB–bHLH– mechanistic insight into the low ABA content in MT. WD40) can induce higher expression of anthocyanin-associated genes than the effect of the single transcription factors separately, which demonstrated that synergistic regulation of transcription Materials and methods factor complexes is more efficient in the fruit ripening process Plant materials (Schaart et al., 2013; Wang et al., 2019a). However, reports con- Fruit from Citrus reticulata cv. Suavissima (wild type, WT) and its spon- cerning the synergistic transcriptional regulation of multiple taneous stay-green mutant (mutant type, MT) were harvested from trees transcription factors on fruit NCEDs remain limited. in the same commercial orchard in Wenzhou (Zhejiang Province, P.R. The regulatory mechanism of fruit ripening originated China) at 120 (enlarged stage), 170 (mature green stage), 210 (commer- cially ripe stage), and 245 (fully ripe stage) days after flowering (DAF) in from those of carpel senescence. The carpel senescence- 2011 and 2012. Given that we only observed slight phenotypic differ- related NAC (NAM/ATAF/CUC) transcription factors ences between WT and MT at 120 DAF and intended to obtain more play vital roles in different fruit ripening processes (Lü et  al., information about the fruit at the breaker and postharvest stages, fruit 2018). SlNOR-like1 and SlNAC4 can act as positive regu- samples were collected at 170, 185 (the breaker stage), and 210 DAF and lators of tomato color formation and several NACs play im- 30 d after storage (DAS) in 2013. portant roles in apple and oil palm fruit development (Zhu et  al., 2014; Tranbarger et  al., 2017; Gao et  al., 2018; Zhang Treatments and sampling et  al., 2018). Moreover, NAC proteins can also directly con- Both MT and WT fruits were harvested at 170 DAF and randomly divided trol the monoterpene synthesis of kiwifruit and the lignifica- into two groups. MT and WT group I fruits were dipped into 100 μM tion of postharvest loquat fruit (Nieuwenhuizen et  al., 2015; ABA solution (132 mg ABA was first dissolved in 1 ml ethanol and then Xu et al., 2015). In order to enhance the regulatory effect on added to 5 liters of distilled water containing 0.1% Tween 80) for 2 min. MT and WT group II fruits were dipped into 5 liters of distilled water the downstream genes, NAC transcription factors often form (containing 1  ml ethanol and 0.1% Tween 80)  for 2  min. After drying homodimers or heterodimers with other proteins. In peach, at room temperature for 30 min, the fruits were transferred to a sealed the NAC transcription factor BL can interact with NAC1 chamber (temperature 20–25  °C and relative humidity 75–85%) and to amplify its regulatory effect on anthocyanin biosynthesis; sampled at 0 and 6 h after treatment. The concentration and the method and in banana fruit, NAC5 can interact with WRKY1/2 to of ABA treatment were according to former reports (Zhang et al., 2009b; Oh et al., 2018). All sampled tissues were immediately frozen in liquid enhance the induction of resistance genes (Zhou et  al., 2015; nitrogen, powdered, and stored at −80 °C until analysis. Shan et  al., 2016). Besides the positive effect of NAC regu- lators on ripening and senescence, overexpressing the NAC transcription factors JUNGBRUNNEN1 (JUB1, ANAC042) Fruit storage and sampling and VNI2 (ANAC083), and TaNAC-S significantly delayed The fruits harvested at 210 DAF in 2013 were used for a storage experi- the senescence of Arabidopsis and wheat seedlings (Triticum ment. They were packed in plastic bags and kept at the optimum cold aestivum L.), respectively (Yang et  al., 2011; Wu et  al., 2012; storage temperature (15–18 °C) and relative humidity (75–85%). At 30 DAS, the flavedo was sampled, frozen in liquid nitrogen, and immediately Zhao et  al., 2015). This fact notwithstanding, little is known stored at −80 °C. concerning the negative regulatory effects of NAC on fruit ripening. Citrus is one of the most important fleshy fruit crops in the Extraction and UPLC analysis of chlorophylls and chlorophyll world. However, research on this important crop is restricted derivatives by its long juvenile phase, high heterozygosity and the difficulty Chlorophylls and chlorophyll derivatives were extracted according to a method described by Minguez-Mosquera and Garrido-Fernandez in obtaining transgenic plants. Fortunately, natural mutants can Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3615 (1989). The separation and quantification of chlorophylls and deriva- RNA isolation and comparative quantitative real time PCR tives were carried out by a UPLC (Waters, H-Class) system using a C18 analysis column (BEH C18, 50×2.1 mm, i.d. 1.8 μm). Separation was performed Total RNA was extracted as described previously (Cao et  al., 2012). −1 using an elution gradient (0.4  ml min ) with the mobile phases (A: The integrity of the RNA preparations was evaluated by electrophor- water: ion pair reagent: methanol (1:1:8, v/v/v), B: methanol: acetone esis and then their concentrations, A /A ratios and A /A ratios 260 280 230 260 (1:1, v/v)) as described by Mínguez-Mosquera et al. (1991). The elution were determined using a Nanodrop spectrophotometer (Agilent 2100, gradient program was optimized as follows (time, A): 0 min, 75%; 3 min, USA). Two biological replicates of RNA from the flavedo of MT and 35%; 4 min, 35%; 5 min, 25%; 6 min, 16%; 8 min, 0%; 9 min, 75%; 0 min, WT (170DAF) treated with ABA were hybridized to GeneChip Citrus 75%. The on-line UV-visible spectra were recorded from 350 to 750 nm Genome Arrays (Affymetrix ; Santa Clara, CA, USA). The analyses of with a photodiode array detector (eλ PAD). Detection was at 654  nm the gene annotation and differentially expressed genes (DEGs) were for chlorophyll b and its derivatives and 664 nm for chlorophyll a and its performed as described previously and DEGs were detected with re- derivatives. Data were collected and processed with Epower 3 software. striction of P>0.01 and fold-change greater than 2 (see Supplementary Chlorophylls and their derivatives were identified by comparing their re- Table S1 at JXB online) (Ma et  al., 2014b). The transcriptome datasets tention time and spectral characteristics with those of authentic standards. generated using the GeneChip Citrus Genome Array platform can At least three independent extractions and detection were performed for be found in the Gene Expression Omnibus (GEO) with the accession each sample. number GSE113669. Genes and primers used for the quantitative reverse transcription-PCR analysis are listed in Supplementary Table S2. Analysis of soluble sugars and organic acids by gas chromatography Isolation and analysis of CrNAC036 sequence Contents of soluble sugars and organic acids were determined using gas The coding sequences of CrNAC036 were amplified from cDNA using chromatography. The samples were frozen with liquid nitrogen and pow- gene-specific primers (see Supplementary Table S2). The Clustal W pro- dered. A total of 1 g of frozen powder was analysed by gas chromatography gram and GeneDoc software were used to align and edit the different as described previously (Wu et al., 2014) with minor modification. The amino acid sequences. Using the neighbor-joining algorithm, a phylogen- powder was suspended in 8 ml pre-cooled 80% methanol and incubated etic tree was constructed with the amino acid sequence of CrNAC036 and in a 70 °C water bath for 30 min. After a 1.5 h ultrasonic extraction and those of 105 NAC transcription factors from Arabidopsis using MEGA centrifugation at 4000 g for 10 min, the supernatant was collected and 5.0 (Tamura et  al., 2011). Bootstrap analysis was performed using 1000 0.2 ml internal standard (2.5% w/v phenyl-β-D-glucopyranoside, 2.5% replicates in MEGA 5.0 to evaluate the reliability of the different phylo- w/v methyl-α-D-glucopyranoside) was added. The solution was made genetic group assignments. The respective names and TAIR ID numbers up to 50 ml with 80% methanol, and a 1 ml aliquot of this final super- of the 105 NAC sequences are presented in Supplementary Table S3. natant was vacuum-dried. The dried sample was re-dissolved in 800 μl 2% w/v hydroxylamine hydrochloride in pyridine at 70 °C for 1 h and then 400 μl hexamethyldisilazane and 200 μl trimethylchlorosilane were Subcellular localization of CrNAC036 added for incubation at 70 °C for 2 h; 0.5 μl of the supernatant was ana- The subcellular localization of the CrNAC036 protein was determined as lysed with an Agilent 6890 N device (Santa Clara, CA, USA) equipped described previously (Zhu et al., 2017). Protoplasts were co-transformed with a flame ionization detector. Sugars and organic acids were identified with 35S:CrNAC036-pM999-GFP and the nuclear marker vector through a comparison of retention times using standard compounds from 35S:OsGhd7-CFP. Fluorescence from green fluorescent protein (GFP) Sigma-Aldrich (St Louis, MO, USA). and cyan fluorescent protein (CFP) was observed using a confocal laser- scanning microscope (TCS SP2, Leica, Germany). The excitation and Transmission electron microscopy emission filters used to detect fluorescence from GFP were 488 nm and 500–530  nm, respectively. The excitation and emission filters used to The flavedo from the fruit harvested without damage at 170, 185 and detect signals from CFP were 430  nm and 470–510  nm, respectively. 210 DAF and 30 DAS was analysed using transmission electron micros- Chlorophyll autofluorescence was monitored using the excitation wave- copy as described previously (Cao et al., 2012) with minor modification. length of either 488 or 514 nm and the emission wavelengths from 650 The flavedos of MT and WT fruits were fixed with 2.5% glutaraldehyde to 750 nm. and 0.1 M phosphate buffer with 2% OsO4. The fixed samples were de- hydrated in epoxy resin and embedded in SPI-812. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl Protein preparation, identification, and electrophoretic mobility acetate and subsequently with lead citrate. The images were captured by shift assay a HITACHI H-7650 transmission electron microscope at 80 kV and a Gatan 832 CCD camera. pET15 (Novagen) was used to produce a recombinant CrNAC036 pro- tein with a 6×His tag fused to the N-terminus. Escherichia coli strain BL21 (DE3) was used to express the recombinant CrNAC036 protein. We puri- Western blot analysis fied and characterized the recombinant protein as previously described (Zhu et  al., 2017) with minor modification. The recombinant protein Total proteins were extracted as described previously (Cao et al., 2012), was analysed by matrix-assisted laser desorption/ionization time-of- and quantified using a RC DC protein assay kit (Bio-Rad, Hercules, flight tandem mass spectrometry (5800 MALDI-TOF/TOF, AB SCIEX) CA, USA). Then, 30  μg of total flavedo protein was resolved by SDS- with a mass spectrometer to acquire MALDI and MS/MS spectra after PAGE (12.5%) and transferred to polyvinylidene fluoride membranes tryptic digestion. The MS spectra were recorded in reflector mode with (Millipore, USA). The subsequent western blot analysis was conducted a mass range of 800–4000. In MS/MS positive ion mode, for one main as described previously (Cao et  al., 2012), using the following primary MS spectrum, 50 subspectra with 50 shots per subspectrum were ac- antibodies (1:3000, v/v): rabbit anti-Lhca1, anti-Lhca2, anti-Lhca3, anti- cumulated using a random search pattern. Collision energy was 2  kV Lhca4 and anti-Lhcb1 (Agrisera, Sweden); and the following secondary and the collision gas was air. The database search was performed using antibodies (1:15  000, v/v): peroxidase-conjugated immunopure goat the MASCOT search engine 2.2 (Matrix Science, Ltd) embedded into anti-rabbit or goat anti-mouse IgG [H+L] (Pierce, USA). Signals were GPS-Explorer Software 3.6 (Applied Biosystems) against non-redundant detected using a Clarity Western ECL Substrate (Bio-Rad) according to protein databases of Citrus clementina (https://phytozome.jgi.doe.gov/pz/ the manufacturer’s instructions. The chemiluminescence signal was im- portal.html#!info?alias=Org_Cclementina). Additionally, MS/MS frag- aged using a ChemiDoc XRS (Bio-Rad) and quantified using Quantity ment tolerance was set to 0.4 Da. A protein confidence index ≥95% was One software (Bio-Rad). The calculated intensity volumes were fitted used for further manual validation. with a variable slope dose–response relationship using ImageJ. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3616 | Zhu et al. An electrophoretic mobility shift assay (EMSA) was performed as index, softening, and rotting rate during storage in MT fruit previously described (Zong et  al., 2016; Zhu et  al., 2017). Briefly, the are in accordance with those displayed by other ABA-deficient His-tagged CrNAC036 protein and 5′-FAM-labeled oligonucleotide citrus mutants (Rodrigo et  al., 2003; Wu et  al., 2014; Zhang probes (synthesized by the Shanghai Sangon Company) were incu- et  al., 2014; Zhu et  al., 2017). To further analyse the effect of bated in a binding solution (0.1% Nonidet P-40, 1  mM benzamidine, −1 ABA on fruit ripening process, we systematically analysed other 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 50 μg ml BSA −1 and 100 ng μl poly (dI-dC)) at 4 °C for 45 min. For the competition ABA-associated phenotypes such as the levels of chlorophyll- assays, after the protein was incubated with non-labeled probe at 4  °C associated metabolites and photosynthesis-related protein, the −1 for 45 min, 1 µl of the 5′-FAM-labeled probe (10 µmol l ) was added subcellular morphology of the chloroplast–chromoplast con- to the mixture and incubated at 4 °C for 45 min. The binding reactions version, and the content of sugars and organic acids. were resolved using electrophoresis with 6% polyacrylamide gels at 4 °C The WT ripening process was characterized by a decrease in in 0.5×TBE (Tris-Borate-EDTA) in the dark for 1 h and imaged with an Amersham TM Imager 600 (GE Healthcare). the levels of chlorophyll-related metabolites, such as chlorophyll a, chlorophyll b, and chlorophyllide a (Fig.  1; Supplementary Fig. S1). At the same time, the thylakoid membranes were dis- Dual luciferase and bimolecular fluorescence assembled and the chloroplasts were gradually converted to complementation assays chromoplasts, which contained plastoglobules filled with ca- We used rice protoplasts for the dual luciferase transcriptional activity rotenoids. By contrast to WT flavedo, MT flavedo still retained assay as described previously (Zong et  al., 2016) because of the high stability, transformation efficiency, and short growth cycle of rice. The chloroplasts with intact thylakoid membranes at 210 DAF (see Dual-Luciferase Reporter Assay System (Promega) was used to measure Supplementary Fig. S2). Consistently, the MT flavedo con- the luciferase activity according to the manufacturer’s instructions. The tained higher levels of photosystem-associated proteins than relative luciferase activity was calculated as the ratio of firefly luciferase the WT flavedo across the ripening process (Supplementary (fLUC)/Renilla luciferase (rLUC). Fig. S3). Moreover, compared with the results of the previous To prevent chlorophyll fluorescence from interfering with the bi- molecular fluorescence complementation (BiFC) assay, we prepared work, similar increases in sugars and decreases in organic acids protoplasts from etiolated rice seedlings. The coding sequence from were also observed in the WT flavedo during fruit ripening CrNAC036 was inserted into a BiFC expression vector (pCL112) to in this study; however, the rates of these changes in the MT produce the nYFP vectors. CrMYB68 was inserted into a BiFC expres- flavedo were slower than those in the WT flavedo (Fig.  1). sion vector (pCL113) to produce cYFP vectors (Bhargava et  al., 2010). Furthermore, we observed the same trend of change in the Florescence was observed using a confocal laser-scanning microscope (TCS SP2, Leica, Germany). All the plasmids used in the dual luciferase levels of sugars and organic acids in the flesh of WT and MT transcriptional activity assay and the BiFC assay were purified using the fruit (Supplementary Fig. S1). QIAGEN Plasmid Midi Kit. To further explore the effect of ABA on ripening-related gene expression and the regulatory mechanism underlying the Yeast two-hybrid analysis ABA-deficient phenotype of MT, GeneChip Citrus Genome Arrays (Affymetrix, Santa Clara, CA, USA) were used to char- The CrNAC036 and CrMYB68 coding sequences were inserted into pGBKT7 and pGADT7 to generate pGBKT7-CrNAC036 and acterize change in the transcriptome of MT and WT fruits pGADT7-CrMYB68 vectors, respectively. A  yeast strain (AH109, (170 DAF) after exogenous ABA treatment. Combining the Clontech) was co-transformed with these two vectors and grown on a data obtained with former microarray and digital gene ex- selective SD/−Trp/−Leu medium. The interactions were evaluated on pression profiling experiments (Zhu et al., 2017), we analysed SD/−Trp/−Leu/−His/−Ade medium containing X-α-galactosidase. the DEGs after ABA treatment and between MT and WT at different fruit stages (Fig.  2A). These analyses indicated that Statistical analysis 21 genes were differentially expressed across all transcriptome The variance of the data was analysed using SPSS 16.0 (SPSS Inc. profiling experiments and the expression of 13 DEGs was Chicago, IL, USA). Multiple comparisons were performed by one-way consistently higher while one gene was consistently lower in ANOVA at the significance level of P<0.05 based on Duncan’s multiple ABA-treated fruits (in comparison with that of water-treated range test. Student’s paired t-test was performed to assess whether the dif- ferences between two genotypes were statistically significant. fruit) and WT (in comparison with that of MT at different fruit stage) (Table 1). Among these genes, CrNCED5 was in- duced in both WT and MT fruits after ABA treatment and Accession numbers also highly expressed in WT fruit during the fruit develop- CrNAC036 (coding sequence, MH339996), CrABA4 (promoter se- ment stages. Moreover, some genes involving in ABA-induced quence, MH339995), and CrNCED5 (promoter sequence, KY612516) ripening process, such as cell wall degradation, were induced are available at NCBI with the indicated accession numbers. The micro- array raw data are available at NCBI’s Gene Expression Omnibus with by ABA treatment and highly expressed in the WT fruit the accession code of GSE113669. (Table  1). Interestingly, there were two transcription factors that co-expressed with CrNCED5 and since NAC family pro- teins play important roles in both ABA biosynthesis and the Results fruit ripening process, we inferred that CrNAC036 may be an important regulator of CrNCED5 (Table 1). Differences in ABA-associated ripening phenotypes Owing to the slight differences between MT and WT fruit between WT and MT fruit at 120 DAF and in order to acquire more information about As described previously, the low ABA content and some late- the fruit at the breaker and postharvest stages, we analysed the ripening phenotypes such as the low color index, maturity expression of genes involved in ABA biosynthesis and related Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3617 Fig. 1. Chlorophyll a, chlorophyll b, chlorophyllide a, sugar, and organic acid levels in the flavedo of WT and MT. The values in each column are the means of three biological replicates. Error bars indicate SD. The asterisks indicate significant differences determined using Student’s t-test. *P<0.05; **P<0.01. DAF: days after flowering. ND: not detected. process (such as chlorophyll and cell wall degradation) using fusion protein and purified. We obtained a protein with a mo- qRT-PCR at 170, 185 and 210 DAF and 30 DAS (Fig.  2B). lecular mass of ca. 25 kDa (Fig. 3C). MS data indicated that the At 210 DAF, the expression levels of CrABA4, CrPPH band corresponded to the N-terminus of the CrNAC036 pro- (CrPheophytinase), CrSGR (CrStay Green), CrPEL (CrPectin tein and the a, c and d subdomains that are conserved among Lyase), and CrExpansin A8 were 4.02-, 1.15-, 1.48-, 27.04-, and NAC transcription factors could all be identified in the fu- 8.74-fold higher in WT fruit than in MT fruit, respectively. By sion protein (see Supplementary Table S4; Supplementary contrast, the expression level of CrNAC036 in MT fruit was Figs S5, S6). The c and d subdomains are responsible for the 16.13-fold higher than that in WT fruit. specific DNA binding activities of NAC transcription factors (Ooka et al., 2003). Therefore, the purified N-terminus of the CrNAC036 protein could be utilized for analysing the DNA Amino acid sequence alignment, subcellular binding activity of CrNAC036. localization, and prokaryotic expression of CrNAC036 To further analyse the potential function of CrNAC036, we CrNAC036 specifically repressed the expression of compared the amino acid sequence of CrNAC036 with that of CrNCED5 105 Arabidopsis NAC transcription factors. In the phylogen- etic analysis, CrNAC036 grouped with the clade containing Previous studies reported that the senescence-associated NAC At2G17040.1, At2G02450.1, At2G02450.2, At5G39820.1, transcription factor family can bind to the DNA sequences and At2G43000.1 (see Supplementary Fig. S4). Moreover, containing the conserved sequence motif CGT/ACG subcellular localization experiments demonstrated that the (Podzimska-Sroka et al., 2015). We found the CGT/ACG motif CrNAC036–GFP fusion protein was co-localized with a in the promoters of CrNCED5 and CrABA4 (Fig. 4A). T o test nuclear marker protein (OsGhd7–CFP), indicating that the whether CrNAC036 can bind to the promoters of CrNCED5 CrNAC036–GFP fusion protein was accumulated in the nu- and CrABA4, the purified 6×His–CrNAC036 fusion pro- cleus (Fig. 3A). tein was incubated with 20-bp probes containing the CGT/ To determine the DNA binding activity of the CrNAC036 ACG sequence from the promoter regions of CrNCED5 and protein, it was expressed in Escherichia coli as a 6×His–CrNAC036 CrABA4. It was found that the CrNAC036 protein did not Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3618 | Zhu et al. Fig. 2. Transcriptome analysis (A) and expression (B) of CrNAC036 and ABA-induced genes at different stages of ripening. (A) CK-MT-ABA and CK-WT- ABA indicate the DEGs of MT and WT between water treatment and ABA treatment, respectively. MT-170DAF-WT, MT-210DAF-WT, and MT-30DAS-WT indicate the DEGs at 170 DAF, 210 DAF, and 30 DAS between MT and WT, respectively. (B) The values in each column are the means of three biological replicates. Error bars indicate SD. The asterisks represent significant differences determined by Student’s t-test, **P<0.01. DAF: days after flowering; DAS: days after storage. CrPEL, CrPectate Lyase; CrPPH, CrPheophytinase; CrSGR, CrStay-Green. (This figure is available in color at JXB online.) bind to the two probes containing the CGT/ACG motifs from CrNAC036 protein, as indicated by the retardation of its mo- the CrABA4 promoter in the EMSA. However, the P1 probe bility in the EMSA. Furthermore, although the CrNAC036 from the CrNCED5 promoter was specifically bound by the protein bound the mutant probe derived from P1, the binding Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3619 Table 1. Consistently differentially expressed genes under ABA treatments and at different ripening stages Gene Probe set ID Transcriptome analysis (log (fold change)) Function Arabidopsis ortholog CK- CK- MT- MT- MT- WT- MT- 170DAF- 210DAF- 30DAS- ABA ABA WT WT WT Ciclev10014639m Cit.17235.1.S1_s_at −3.11 −1.07 −1.13 −1.71 −7.81 ABA biosynthesis AT1G30100 (AtNCED5) Ciclev10029007m Cit.31377.1.S1_at 1.88 1.98 1.12 3.32 5.50 Transcription factor AT2G17040 (AtNAC036) Ciclev10029283m Cit.10057.1.S1_s_at −1.63 −2.47 −1.52 −1.13 −2.68 Transcription factor AT2G28500 (AtLBD11) Ciclev10031429m Cit.35568.1.S1_s_at −1.25 −1.56 −1.74 −3.39 −4.91 Cell wall degradation AT1G67750 (AtPEL) Ciclev10032524m Cit.20839.1.S1_s_at −1.02 −1.18 −1.11 −1.81 −3.95 Cell wall degradation AT2G40610 (AtEXPA8) Ciclev10019301m Cit.2945.1.S1_s_at −1.59 −2.05 −1.09 −2.42 −4.74 Cell wall degradation AT1G64390 (AtGH9C2) Ciclev10012384m Cit.8763.1.S1_s_at −2.52 −3.60 −1.86 −2.59 −8.10 Water metabolism AT4G00430 (AtPIP1;4) Ciclev10004103m Cit.1002.1.S1_s_at −1.09 −0.96 −1.81 −1.08 −3.00 Glucosinolate me- AT4G31500 tabolism (AtCYP83B1) Ciclev10025900m Cit.15742.1.S1_at −1.72 −3.34 −1.43 −1.79 −2.16 Lipid metabolism AT1G75900 (AtEXL3) Ciclev10011714m Cit.1770.1.S1_at −1.80 −1.79 −1.88 −3.36 −7.47 Secondary metab- AT3G26040 olism Ciclev10001944m Cit.22427.1.S1_s_at −1.93 −2.49 −1.40 −1.19 −3.32 Development, un- AT4G15920 specified Ciclev10033996m Cit.26052.1.S1_s_at −1.06 −1.67 −2.05 −1.72 −4.77 Unknown AT2G39855 Ciclev10033283m Cit.21497.1.S1_at −1.87 −2.26 −1.72 −1.63 −4.12 Unknown AT2G38905 Ciclev10028078m Cit.10062.1.S1_at −1.88 −2.08 −1.14 −1.15 2.91 Amino acid metab- AT3G47340(AtASN1) olism Ciclev10008993m Cit.1718.1.S1_s_at 3.34 4.15 1.04 1.35 −1.25 Ethylene biosyn- AT2G19590 (AtACO1) thesis Ciclev10029695m Cit.9890.1.S1_s_at 1.80 1.01 −1.17 −1.53 −1.13 Gibberellin-regulated AT2G14900 family protein Ciclev10005627m Cit.28626.1.S1_s_at −1.29 −2.11 −1.83 −1.78 1.72 Cell wall modification AT1G65680(ATEXPB2) Ciclev10031099m Cit.4425.1.S1_at −1.32 2.21 1.54 −1.43 2.25 Cytochrome P450 AT5G05260 (AtCYP79A2) Ciclev10002768m Cit.12040.1.S1_s_at −1.36 −2.81 −2.02 −1.81 1.06 Metal handling AT4G08570 Ciclev10006006m Cit.165.1.S1_s_at −1.84 −2.18 −1.23 −3.96 1.76 Light signalling AT3G22840 (AtELIP1) Ciclev10017113m Cit.10672.1.S1_s_at −3.78 −3.28 1.17 −2.02 3.22 Light signalling AT3G26740 (AtCCL) CK-MT-ABA and CK-WT-ABA indicate the DEGs of MT and WT between water treatment and ABA treatment, respectively. MT-170DAF-WT, MT-210DAF-WT, and MT-30DAS-WT indicate the DEGs at 170 DAF, 210 DAF, and 30 DAS between MT and WT, respectively. activity was considerably lower than that observed for the et  al., 2017). In order to determine whether CrNAC036 can wild-type P1 probe (Fig. 4B). interact with CrMYB68 to synergistically regulate the expres- To further evaluate whether CrNAC036 affected the activ- sion of CrNCED5, we first tested whether CrMYB68 and ities of the CrNCED5 and CrABA4 promoters, we fused the CrNAC036 can interact in the yeast two-hybrid system and CrNCED5 and CrABA4 promoters including the core-binding BiFC assays. The yeast strains harboring both the pGADT7- motif of NAC family (i.e. the CGT/ACG sequence) to the CrMYB68 and the pGBKT7-CrNAC036 vectors could grow firefly luciferase reporter gene, and these were then transiently and exhibit blue color on a medium containing X-α-Gal co-expressed in protoplasts. Results indicated that CrNAC036 without leucine, tryptophan, histidine, and adenine. These re- significantly repressed the activity of the CrNCED5 promoter sults indicated that CrNAC036 can interact with CrMYB68 but did not affect that of the CrABA4 promoter (Fig.  4C). in the yeast two-hybrid system (Fig. 5A). The ability of these Based on the results of EMSA and dual-luciferase experiments, proteins to interact with each other was independently verified it can thus be inferred that CrNAC036 can specifically bind to by BiFC experiments. As shown in Fig. 5B, the interaction be- the promoter of CrNCED5 and represses its activity. tween CrNAC036 with the C-terminus and CrMYB68 with the N-terminus of yellow fluorescent protein (YFP) yielded a fluorescence signal in the nucleus (Fig. 5B). CrNAC036 interacted with CrMYB68 to synergistically Secondly, we perfor med a dual-luciferase exper iment by tran- down-regulate the expression of CrNCED5 siently co-expressing the two effector vectors and a luciferase NAC proteins usually enhance their own transcriptional ac- reporter gene in protoplasts. As a result, the combinations of tivities via interaction with other transcription factors (Olsen ‘None (empty vector)+CrNAC036-None’ and ‘None (empty et  al., 2005). Our previous study indicated that CrMYB68 vector)+CrMYB68-None’ significantly repressed the activity displays a similar expression pattern to CrNAC036 in the of the promoter from CrNCED5. However, the activity of the MT fruit and can also directly regulate CrNCED5 (Zhu CrNCED5 promoter was the most obviously reduced when Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3620 | Zhu et al. Fig. 3. Subcellular localization and prokaryotic expression of CrNAC036. (A) Subcellular localization of CrNAC036. 35S:CrNAC036-pM999-GFP and 35S:OsGhd7-CFP were co-transformed into protoplasts. OsGhd7–CFP was used as a nuclear marker. (i) CrNAC036–GFP, (ii) OsGhd7–CFP, (iii) bright field, (iv) merged image. (B) Subcellular localization of pM-999. (i) pM999–GFP, (ii) chlorophyll fluorescence, (iii) bright field, (iv) merged image. The scale bars in (A, B) indicate 10 μm. (C) Prokaryotic expression analysis of His-tagged CrNAC036 with a Coomassie blue-stained 12% SDS gel. Lane 1, His- tagged CrNAC036 protein; M, Marker. (This figure is available in color at JXB online.) ‘CrNAC036-None’ and ‘CrMYB68-None’ were co-expressed ripening-related process, such as chlorophyll degradation, ca- (Fig.  5E). These results indicated that the protein–protein rotenoid biosynthesis and softening, by regulating gene expres- interactions between CrNAC036 and CrMYB68 do synergis- sion (Zaharah et al., 2013; Gao et al., 2016; Rehman et al., 2018). tically affect the expression of CrNCED5. These effects of ABA could also be confirmed in MT fruit, with the expression of genes that contribute to chlorophyll degradation and cell wall modification (i.e. CrPEL, CrExpansin A8, CrPPH, and CrSGR) being consistently lower in MT fruit Discussion than in WT fruit during the ripening process and the dem- ABA is one of the key regulators of the fruit ripening process. onstration that the expression of CrPEL and CrExpansin A8 The transcriptional regulation of ABA biosynthetic genes has could be significantly induced by exogenous ABA treatment been well studied in fruits (Endo et al., 2016; Luo et al., 2017; (Fig.  2). Additionally, injection of ABA caused an increase in Wang et  al., 2019b). However, comprehensive details of the the glucose and fructose levels of citrus fruit and exogenous effect of ABA on citrus ripening and the regulation of ABA ABA treatment can accelerate fruit coloring of citrus (Kojima biosynthesis in citrus by a multi-transcription factor complex et  al., 1995; Wang et  al., 2016; Rehman et  al., 2018). Taken remain largely unknown. Here, we performed metabolic, cyto- together, all of the previously published work and our own logical, and transcriptome analysis of an ABA-deficient mu- findings indicate that ABA is an essential positive regulator of tant from Citrus reticulata cv. Suavissima. It was found that ABA ripening of citrus fruits. served as an important regulator of citrus ripening and its bio- synthesis was under the synergistic regulation of CrNAC036 CrNAC036 plays an important role in ABA-deficient and CrMYB68 by suppressing the expression of CrNCED5. phenotype of MT Our results from the transcriptome and qRT-PCR experi- ABA plays an important role in the ripening process of ments indicate that the expression of CrNCED5 and CrABA4 citrus fruit was robustly lower in MT than that in WT (Fig. 2; Table 1; Zhu In MT fruit, some ripening parameters (such as color index, et al., 2017). We also checked the expression of other genes in- maturity index, and softening) were significantly delayed, volved in ABA biosynthesis but found that their expression did which was in accordance with those displayed by other ABA- not vary robustly (Zhu et al., 2017). Thus, as NCED5 was the deficient citrus mutants (Rodrigo et al., 2003; Wu et al., 2014; dominant NCED for the ABA biosynthesis in C.  clementina Zhang et al., 2014; Zhu et al., 2017). Moreover, in this study, the flavedo compared with other NCEDs (Agustí et al., 2007), the delay of chlorophyll degradation, the chloroplast–chromoplast low expression of CrNCED5 and CrABA4 may lead to the transition, and sugar and organic acid metabolism in MT fur- low ABA level in MT. Moreover, we observed, in the tran- ther indicated that ABA can systematically affect the citrus fruit scriptome data, that the expression of CrNAC036 displayed ripening process (Fig. 1; Supplementary Fig. S1). Furthermore, a significantly negative correlation to that of CrNCED5 and many studies have reported that ABA can directly promote CrABA4 (Table  1; Fig.  2). Additionally, we found that the Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3621 Fig. 4. EMSA and dual luciferase assay. (A) Schematic diagram of the promoter model and sequences. The sequences used in the EMSA and dual luciferase assay are indicated on the left; the sequences used in the EMSA are indicated on the right. Black circle indicates the core-binding motif of NAC family. (B) Binding of CrNAC036 to the promoters of CrNCED5 and CrABA4. In EMSA, 10-, 20-, and 30-fold excess of non-labeled probes were used as competitors. (C) Diagram of the constructs used in the dual luciferase assays and the regulation of CrNAC036 on CrNCED5 and CrABA4. The fLUC/rLUC ratio represents the relative activity of the CrNCED5 and CrABA4 promoters. The values in each column are the means of three biological replicates. Error bars indicate the SD. The double asterisks represent statistically significant differences determined using Student’s t-test (**P<0.01). (This figure is available in color at JXB online.) amino acid sequence of CrNAC036 is similar to that of JUB1, CrABA4 promoter (Fig.  4). Based on these results, it can be which inhibits Arabidopsis senescence (see Supplementary concluded that CrNAC036 is an important regulator of ABA Fig. S4), and note that the ABA content of jub1-1 plants was biosynthesis in citrus fruit, a task it achieves by specific down- significantly increased compared with that of the wild type regulation of the expression of CrNCED5. (Wu et  al., 2012). Furthermore, DAP-seq analysis indicated Transcription factors and hormones such as ABA and that JUB1 can directly bind to the promoter of AtNCED5 ethylene substantially influence fruit ripening. Several tran- (O’Malley et al., 2016). When taken together these data suggest scription factor families have been proven to promote ripening that CrNAC036 may negatively regulate ABA biosynthesis by through affecting ABA (ABF2 in grape) or ethylene (RIN, down-regulating the expression of CrNCED5 or CrABA4. CNR in tomato and MdMYC2 in Malus domestica) metab- However, whilst conserved NAC-recognition sequences were olism and signaling (Klee and Giovannoni, 2011; Nicolas et al., recognized in the promoters of both CrNCED5 and CrABA4, 2014; Li et al., 2017b). However, until recently no transcription the data from the EMSA indicated that CrNAC036 can spe- factor family had been reported to regulate both fruit ABA and cifically bind to the promoter of CrNCED5 but not to that of ethylene metabolism. Recently, Lü et al. (2018) reported that CrABA4 (Fig. 4). Moreover, the data from the dual luciferase carpel senescence-related NAC transcription factors play vital assay indicated that CrNAC036 can repress the activity of the roles in the ripening process of both climacteric fruit without CrNCED5 promoter while not affecting the activity of the recent whole-genome duplication and non-climacteric fruit. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3622 | Zhu et al. Fig. 5. Interactions between CrNAC036 and CrMYB68 and their cooperative regulation of the CrNCED5 promoter. (A) Interactions between CrNAC036 and CrMYB68 in the yeast two-hybrid assays. Blue colonies growing on a synthetic drop-out medium lacking Trp, Leu, His, and Ade (SD/−Trp/−Leu/− His/−Ade) and containing X-α-galactosidase indicate protein–protein interactions. Co-transformation of pGBKT7-p53 and pGADT7-RecT was used as a positive control. (B–D) Nuclear interactions of CrNAC036 with CrMYB68. Confocal images of transiently transformed nYFP–CrNAC036 and cYFP– CrMYB68 generating YFP signal in nucleus (OsGhd7–CFP was used as a nuclear marker) (B). Interaction with empty vectors was not observed (C, D). Scale bars: 10 μm. (E) Dual luciferase assays in protoplasts co-expressing CrNAC036 and CrMYB68. The fLUC/rLUC ratio represents the relative activity of the CrNCED5 promoter (P2). The sequence of P2 is indicated in Fig. 4A. The values in each column are the means of three biological replicates. Error bars indicate SD. Different letters indicate significant differences according to Duncan’s test (P<0.05). (This figure is available in color at JXB online.) Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3623 In tomato, SlNAC19/48 can directly induce the expression regulatory network of transcription factor complexes in ABA of ACO1 and ACS2 and in Musa acuminata, MaNAC1/2 biosynthesis. can affect ethylene signaling by interacting with EIN3 (Shan et al., 2012; Ma et al., 2014a; Kou et al., 2016). In the present study, our findings concerning the transcriptional regulation of Supplementary data CrNAC036 greatly improves our understanding of the tran- Supplementary data are available at JXB online. scriptional regulation of ABA metabolism in fruit. They add- Fig. S1. Pheophorbide a and pheophytin a levels in the itionally enrich our knowledge concerning the importance of flavedo and sugar and organic acid levels in the flesh of WT regulators of the NAC transcription factor family on the bio- and MT. synthesis of two hormones known to be vital for fruit ripening. Fig. S2. Transmission electron microscopy analysis of the morphological changes in the flavedo plastids from WT CrNAC036 and CrMYB68 synergistically inhibit ABA and MT. biosynthesis by down-regulating the expression of Fig. S3. Levels of photosystem-associated proteins in the CrNCED5 flavedo at four developmental stages. Fig. S4. Phylogenetic analysis of CrNAC036 and NAC pro- Owing to their sessile nature, plants have evolved complex teins from Arabidopsis. mechanisms to activate or repress gene expression in order to Fig. S5. Peptides from CrNAC036 identified using adapt to their prevailing environments. As important regulators MALDI-TOF/TOF MS. of gene expression, besides working alone, different transcrip- Fig. S6. The MS/MS spectrum of the identified peptides of tion factors can form complexes and recognize cis-regulatory CrNAC036 protein. elements within a target gene promoter for synergistic or an- Table S1. The DEGs of MT and WT after ABA treatment tagonistic regulation of gene transcription. Given that ABA is Table S2. Primers used in this study. an important regulator of stress tolerance and the fruit ripening Table S3. Names and TAIR ID numbers of 105 NAC tran- process, the transcriptional regulation of NCEDs by a single scription factors from Arabidopsis. transcription factor has been reported in many crops and fruits Table S4. Peptides identified by 6×His–CrNAC036 protein (Jiang et al., 2012; Finkelstein, 2013; Jensen et al., 2013; Endo using the MALDI-TOF/TOF MS method. et al., 2016; Zong et al., 2016; Luo et al., 2017; Ma et al., 2018; Wang et  al., 2019b). However, to date research concerning synergistic regulation of fruit NCEDs by transcription factor Acknowledgements complexes remains rare. In previous publications, transcription factors of the NAC We thank Dr Masaru Ohme-Takagi (National Institute of Advanced Industrial Science and Technology, Japan) for providing the plasmids 35S′- family have been reported to interact with distinct types of GAL4-fLUC, Dr Shouyi Chen (Institute of Genetics and Developmental transcription factors including NAC family proteins, WRKY Biology, China) for providing the plasmids None and AtUbi3-rLUC, and family proteins, TCP family proteins, and zinc finger-containing Dr Wei Zong and Lizhong Xiong (Huazhong Agricultural University) transcription factors (Olsen et al., 2004; Tran et al., 2007; Wang for providing plasmids pM999 and 35S:OsGhd7-CFP. Moreover, we et al., 2015; Zhou et al., 2015; Li et al., 2017a). Moreover, our also thank the research associates at the Center for Protein Research earlier research demonstrated that CrMYB68 was highly ex- (CPR), Huazhong Agricultural University for technical support. This pressed in the MT fruit and the protein can directly repress work was supported by the National Key R&D Program of China the expression of CrNCED5 (Zhu et  al., 2017). In the pre- (2018YFD1000200); Huazhong Agricultural University Scientific & sent study, CrNAC036 was revealed to be highly co-expressed Technological Self–innovation Foundation; the National Natural Science with CrMYB68, and Y2H and BiFC results indicated that Foundation of China (No. 31772368, 31572176); the National Modern CrNAC036 physically interacted with CrMYB68. Given that Agricultural (Citrus) Technology Systems of China (CARS-27) and China Postdoctoral Science Foundation No. 2018M640713. Finally, we both CrNAC036 and CrMYB68 are repressors of CrNCED5, thank Prof. Zuoxiong Liu for English language editing. 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Plant Physiology 171, 2810–2825. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata

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Oxford University Press
Copyright
Copyright © 2022 Society for Experimental Biology
ISSN
0022-0957
eISSN
1460-2431
DOI
10.1093/jxb/eraa118
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Abstract

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the posi- tive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis- epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demon- strated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5. Keywords: ABA, Citrus reticulata, fruit ripening, MYB transcription factor, NAC transcription factor, postharvest, synergistic transcriptional regulation. Introduction Fruit ripening is a complex process, and based on the patterns categories. Knocking-down 9-cis-epoxycarotenoid dioxygenases of respiration and ethylene biosynthesis during fruit ripening, (NCEDs) led to a significant down-regulation of the expres- fleshy fruits can be divided into two categories, namely, climac- sion of genes associated with cell wall catabolism (such as pectate teric and non-climacteric fruits (Giovannoni, 2004). Abscisic acid lyase and expansin) in tomato. Moreover, the application of ex- (ABA) is a vital hormone that affects the ripening process of both ogenous ABA or treatment with an inhibitor of its biosynthesis © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3614 | Zhu et al. can significantly promote or hinder the color change, softening, help researchers to overcome these limitations. In the past dec- and flavor component accumulation of tomato, peach, straw- ades, experiments with ABA-deficient natural mutants and berry, and grape fruits (Zhang et  al., 2009a,b; Jia et  al., 2011; ABA treatments have indicated that ABA is a vital regulator of Sun et  al., 2012; Leng et  al., 2014). Beside the effect on fruit color change and the metabolism of both sugars and organic ripening, exogenous ABA treatment can also induce the ex- acids (Rodrigo et al., 2003; Wu et al., 2014; Zhang et al., 2014; pression of genes associated with chlorophyll degradation (such Rehman et  al., 2018). Recently, we found a novel stay-green as stay-green and pheophytinase) and promote the senescence of natural mutant (MT) in Citrus reticulata cv. Suavissima, and used Arabidopsis (Yang et al., 2014). Owing to ABA’s vital role in fruit it to study the transcriptional regulation of CrMYB68 on ca- ripening, the pathway of its biosynthesis has been well character- rotenoid metabolism via effects on CrBCH2 and CrNCED5 ized: violaxanthin derived from carotenoid metabolism is trans- (Zhu et al., 2017). However, research concerning comprehen- formed by ABA4, NCEDs, and several other enzymes to yield sive effects of ABA on the ripening process (such as chlorophyll ABA (Finkelstein, 2013). As the rate-limiting enzymes of ABA degradation, chloroplast disassembly, and ripening-related gene biosynthesis, NCEDs have attracted considerable of attention expression) and synergistic transcriptional regulation of ABA and the transcriptional regulation of NCEDs is one of the hot biosynthesis in citrus is rare. In the present study, we compre- topics in ABA research. In tomato, banana, peach, and citrus fruit, hensively analysed the effect of ABA on citrus ripening and WRKYs, ERF3, and bHLH1 transcription factors can directly further demonstrated that a novel NAC transcription factor, bind to their promoters and regulate the expression of NCEDs CrNAC036, directly regulated the expression of CrNCED5 (Endo et  al., 2016; Luo et  al., 2017; Wang et  al., 2019b). In the and could additionally interact with CrMYB68 to synergis- well-known model of anthocyanin biosynthesis, the synergistic tically down-regulate the expression of CrNCED5, providing transcriptional regulation of the MBW complex (MYB–bHLH– mechanistic insight into the low ABA content in MT. WD40) can induce higher expression of anthocyanin-associated genes than the effect of the single transcription factors separately, which demonstrated that synergistic regulation of transcription Materials and methods factor complexes is more efficient in the fruit ripening process Plant materials (Schaart et al., 2013; Wang et al., 2019a). However, reports con- Fruit from Citrus reticulata cv. Suavissima (wild type, WT) and its spon- cerning the synergistic transcriptional regulation of multiple taneous stay-green mutant (mutant type, MT) were harvested from trees transcription factors on fruit NCEDs remain limited. in the same commercial orchard in Wenzhou (Zhejiang Province, P.R. The regulatory mechanism of fruit ripening originated China) at 120 (enlarged stage), 170 (mature green stage), 210 (commer- cially ripe stage), and 245 (fully ripe stage) days after flowering (DAF) in from those of carpel senescence. The carpel senescence- 2011 and 2012. Given that we only observed slight phenotypic differ- related NAC (NAM/ATAF/CUC) transcription factors ences between WT and MT at 120 DAF and intended to obtain more play vital roles in different fruit ripening processes (Lü et  al., information about the fruit at the breaker and postharvest stages, fruit 2018). SlNOR-like1 and SlNAC4 can act as positive regu- samples were collected at 170, 185 (the breaker stage), and 210 DAF and lators of tomato color formation and several NACs play im- 30 d after storage (DAS) in 2013. portant roles in apple and oil palm fruit development (Zhu et  al., 2014; Tranbarger et  al., 2017; Gao et  al., 2018; Zhang Treatments and sampling et  al., 2018). Moreover, NAC proteins can also directly con- Both MT and WT fruits were harvested at 170 DAF and randomly divided trol the monoterpene synthesis of kiwifruit and the lignifica- into two groups. MT and WT group I fruits were dipped into 100 μM tion of postharvest loquat fruit (Nieuwenhuizen et  al., 2015; ABA solution (132 mg ABA was first dissolved in 1 ml ethanol and then Xu et al., 2015). In order to enhance the regulatory effect on added to 5 liters of distilled water containing 0.1% Tween 80) for 2 min. MT and WT group II fruits were dipped into 5 liters of distilled water the downstream genes, NAC transcription factors often form (containing 1  ml ethanol and 0.1% Tween 80)  for 2  min. After drying homodimers or heterodimers with other proteins. In peach, at room temperature for 30 min, the fruits were transferred to a sealed the NAC transcription factor BL can interact with NAC1 chamber (temperature 20–25  °C and relative humidity 75–85%) and to amplify its regulatory effect on anthocyanin biosynthesis; sampled at 0 and 6 h after treatment. The concentration and the method and in banana fruit, NAC5 can interact with WRKY1/2 to of ABA treatment were according to former reports (Zhang et al., 2009b; Oh et al., 2018). All sampled tissues were immediately frozen in liquid enhance the induction of resistance genes (Zhou et  al., 2015; nitrogen, powdered, and stored at −80 °C until analysis. Shan et  al., 2016). Besides the positive effect of NAC regu- lators on ripening and senescence, overexpressing the NAC transcription factors JUNGBRUNNEN1 (JUB1, ANAC042) Fruit storage and sampling and VNI2 (ANAC083), and TaNAC-S significantly delayed The fruits harvested at 210 DAF in 2013 were used for a storage experi- the senescence of Arabidopsis and wheat seedlings (Triticum ment. They were packed in plastic bags and kept at the optimum cold aestivum L.), respectively (Yang et  al., 2011; Wu et  al., 2012; storage temperature (15–18 °C) and relative humidity (75–85%). At 30 DAS, the flavedo was sampled, frozen in liquid nitrogen, and immediately Zhao et  al., 2015). This fact notwithstanding, little is known stored at −80 °C. concerning the negative regulatory effects of NAC on fruit ripening. Citrus is one of the most important fleshy fruit crops in the Extraction and UPLC analysis of chlorophylls and chlorophyll world. However, research on this important crop is restricted derivatives by its long juvenile phase, high heterozygosity and the difficulty Chlorophylls and chlorophyll derivatives were extracted according to a method described by Minguez-Mosquera and Garrido-Fernandez in obtaining transgenic plants. Fortunately, natural mutants can Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3615 (1989). The separation and quantification of chlorophylls and deriva- RNA isolation and comparative quantitative real time PCR tives were carried out by a UPLC (Waters, H-Class) system using a C18 analysis column (BEH C18, 50×2.1 mm, i.d. 1.8 μm). Separation was performed Total RNA was extracted as described previously (Cao et  al., 2012). −1 using an elution gradient (0.4  ml min ) with the mobile phases (A: The integrity of the RNA preparations was evaluated by electrophor- water: ion pair reagent: methanol (1:1:8, v/v/v), B: methanol: acetone esis and then their concentrations, A /A ratios and A /A ratios 260 280 230 260 (1:1, v/v)) as described by Mínguez-Mosquera et al. (1991). The elution were determined using a Nanodrop spectrophotometer (Agilent 2100, gradient program was optimized as follows (time, A): 0 min, 75%; 3 min, USA). Two biological replicates of RNA from the flavedo of MT and 35%; 4 min, 35%; 5 min, 25%; 6 min, 16%; 8 min, 0%; 9 min, 75%; 0 min, WT (170DAF) treated with ABA were hybridized to GeneChip Citrus 75%. The on-line UV-visible spectra were recorded from 350 to 750 nm Genome Arrays (Affymetrix ; Santa Clara, CA, USA). The analyses of with a photodiode array detector (eλ PAD). Detection was at 654  nm the gene annotation and differentially expressed genes (DEGs) were for chlorophyll b and its derivatives and 664 nm for chlorophyll a and its performed as described previously and DEGs were detected with re- derivatives. Data were collected and processed with Epower 3 software. striction of P>0.01 and fold-change greater than 2 (see Supplementary Chlorophylls and their derivatives were identified by comparing their re- Table S1 at JXB online) (Ma et  al., 2014b). The transcriptome datasets tention time and spectral characteristics with those of authentic standards. generated using the GeneChip Citrus Genome Array platform can At least three independent extractions and detection were performed for be found in the Gene Expression Omnibus (GEO) with the accession each sample. number GSE113669. Genes and primers used for the quantitative reverse transcription-PCR analysis are listed in Supplementary Table S2. Analysis of soluble sugars and organic acids by gas chromatography Isolation and analysis of CrNAC036 sequence Contents of soluble sugars and organic acids were determined using gas The coding sequences of CrNAC036 were amplified from cDNA using chromatography. The samples were frozen with liquid nitrogen and pow- gene-specific primers (see Supplementary Table S2). The Clustal W pro- dered. A total of 1 g of frozen powder was analysed by gas chromatography gram and GeneDoc software were used to align and edit the different as described previously (Wu et al., 2014) with minor modification. The amino acid sequences. Using the neighbor-joining algorithm, a phylogen- powder was suspended in 8 ml pre-cooled 80% methanol and incubated etic tree was constructed with the amino acid sequence of CrNAC036 and in a 70 °C water bath for 30 min. After a 1.5 h ultrasonic extraction and those of 105 NAC transcription factors from Arabidopsis using MEGA centrifugation at 4000 g for 10 min, the supernatant was collected and 5.0 (Tamura et  al., 2011). Bootstrap analysis was performed using 1000 0.2 ml internal standard (2.5% w/v phenyl-β-D-glucopyranoside, 2.5% replicates in MEGA 5.0 to evaluate the reliability of the different phylo- w/v methyl-α-D-glucopyranoside) was added. The solution was made genetic group assignments. The respective names and TAIR ID numbers up to 50 ml with 80% methanol, and a 1 ml aliquot of this final super- of the 105 NAC sequences are presented in Supplementary Table S3. natant was vacuum-dried. The dried sample was re-dissolved in 800 μl 2% w/v hydroxylamine hydrochloride in pyridine at 70 °C for 1 h and then 400 μl hexamethyldisilazane and 200 μl trimethylchlorosilane were Subcellular localization of CrNAC036 added for incubation at 70 °C for 2 h; 0.5 μl of the supernatant was ana- The subcellular localization of the CrNAC036 protein was determined as lysed with an Agilent 6890 N device (Santa Clara, CA, USA) equipped described previously (Zhu et al., 2017). Protoplasts were co-transformed with a flame ionization detector. Sugars and organic acids were identified with 35S:CrNAC036-pM999-GFP and the nuclear marker vector through a comparison of retention times using standard compounds from 35S:OsGhd7-CFP. Fluorescence from green fluorescent protein (GFP) Sigma-Aldrich (St Louis, MO, USA). and cyan fluorescent protein (CFP) was observed using a confocal laser- scanning microscope (TCS SP2, Leica, Germany). The excitation and Transmission electron microscopy emission filters used to detect fluorescence from GFP were 488 nm and 500–530  nm, respectively. The excitation and emission filters used to The flavedo from the fruit harvested without damage at 170, 185 and detect signals from CFP were 430  nm and 470–510  nm, respectively. 210 DAF and 30 DAS was analysed using transmission electron micros- Chlorophyll autofluorescence was monitored using the excitation wave- copy as described previously (Cao et al., 2012) with minor modification. length of either 488 or 514 nm and the emission wavelengths from 650 The flavedos of MT and WT fruits were fixed with 2.5% glutaraldehyde to 750 nm. and 0.1 M phosphate buffer with 2% OsO4. The fixed samples were de- hydrated in epoxy resin and embedded in SPI-812. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl Protein preparation, identification, and electrophoretic mobility acetate and subsequently with lead citrate. The images were captured by shift assay a HITACHI H-7650 transmission electron microscope at 80 kV and a Gatan 832 CCD camera. pET15 (Novagen) was used to produce a recombinant CrNAC036 pro- tein with a 6×His tag fused to the N-terminus. Escherichia coli strain BL21 (DE3) was used to express the recombinant CrNAC036 protein. We puri- Western blot analysis fied and characterized the recombinant protein as previously described (Zhu et  al., 2017) with minor modification. The recombinant protein Total proteins were extracted as described previously (Cao et al., 2012), was analysed by matrix-assisted laser desorption/ionization time-of- and quantified using a RC DC protein assay kit (Bio-Rad, Hercules, flight tandem mass spectrometry (5800 MALDI-TOF/TOF, AB SCIEX) CA, USA). Then, 30  μg of total flavedo protein was resolved by SDS- with a mass spectrometer to acquire MALDI and MS/MS spectra after PAGE (12.5%) and transferred to polyvinylidene fluoride membranes tryptic digestion. The MS spectra were recorded in reflector mode with (Millipore, USA). The subsequent western blot analysis was conducted a mass range of 800–4000. In MS/MS positive ion mode, for one main as described previously (Cao et  al., 2012), using the following primary MS spectrum, 50 subspectra with 50 shots per subspectrum were ac- antibodies (1:3000, v/v): rabbit anti-Lhca1, anti-Lhca2, anti-Lhca3, anti- cumulated using a random search pattern. Collision energy was 2  kV Lhca4 and anti-Lhcb1 (Agrisera, Sweden); and the following secondary and the collision gas was air. The database search was performed using antibodies (1:15  000, v/v): peroxidase-conjugated immunopure goat the MASCOT search engine 2.2 (Matrix Science, Ltd) embedded into anti-rabbit or goat anti-mouse IgG [H+L] (Pierce, USA). Signals were GPS-Explorer Software 3.6 (Applied Biosystems) against non-redundant detected using a Clarity Western ECL Substrate (Bio-Rad) according to protein databases of Citrus clementina (https://phytozome.jgi.doe.gov/pz/ the manufacturer’s instructions. The chemiluminescence signal was im- portal.html#!info?alias=Org_Cclementina). Additionally, MS/MS frag- aged using a ChemiDoc XRS (Bio-Rad) and quantified using Quantity ment tolerance was set to 0.4 Da. A protein confidence index ≥95% was One software (Bio-Rad). The calculated intensity volumes were fitted used for further manual validation. with a variable slope dose–response relationship using ImageJ. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3616 | Zhu et al. An electrophoretic mobility shift assay (EMSA) was performed as index, softening, and rotting rate during storage in MT fruit previously described (Zong et  al., 2016; Zhu et  al., 2017). Briefly, the are in accordance with those displayed by other ABA-deficient His-tagged CrNAC036 protein and 5′-FAM-labeled oligonucleotide citrus mutants (Rodrigo et  al., 2003; Wu et  al., 2014; Zhang probes (synthesized by the Shanghai Sangon Company) were incu- et  al., 2014; Zhu et  al., 2017). To further analyse the effect of bated in a binding solution (0.1% Nonidet P-40, 1  mM benzamidine, −1 ABA on fruit ripening process, we systematically analysed other 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 50 μg ml BSA −1 and 100 ng μl poly (dI-dC)) at 4 °C for 45 min. For the competition ABA-associated phenotypes such as the levels of chlorophyll- assays, after the protein was incubated with non-labeled probe at 4  °C associated metabolites and photosynthesis-related protein, the −1 for 45 min, 1 µl of the 5′-FAM-labeled probe (10 µmol l ) was added subcellular morphology of the chloroplast–chromoplast con- to the mixture and incubated at 4 °C for 45 min. The binding reactions version, and the content of sugars and organic acids. were resolved using electrophoresis with 6% polyacrylamide gels at 4 °C The WT ripening process was characterized by a decrease in in 0.5×TBE (Tris-Borate-EDTA) in the dark for 1 h and imaged with an Amersham TM Imager 600 (GE Healthcare). the levels of chlorophyll-related metabolites, such as chlorophyll a, chlorophyll b, and chlorophyllide a (Fig.  1; Supplementary Fig. S1). At the same time, the thylakoid membranes were dis- Dual luciferase and bimolecular fluorescence assembled and the chloroplasts were gradually converted to complementation assays chromoplasts, which contained plastoglobules filled with ca- We used rice protoplasts for the dual luciferase transcriptional activity rotenoids. By contrast to WT flavedo, MT flavedo still retained assay as described previously (Zong et  al., 2016) because of the high stability, transformation efficiency, and short growth cycle of rice. The chloroplasts with intact thylakoid membranes at 210 DAF (see Dual-Luciferase Reporter Assay System (Promega) was used to measure Supplementary Fig. S2). Consistently, the MT flavedo con- the luciferase activity according to the manufacturer’s instructions. The tained higher levels of photosystem-associated proteins than relative luciferase activity was calculated as the ratio of firefly luciferase the WT flavedo across the ripening process (Supplementary (fLUC)/Renilla luciferase (rLUC). Fig. S3). Moreover, compared with the results of the previous To prevent chlorophyll fluorescence from interfering with the bi- molecular fluorescence complementation (BiFC) assay, we prepared work, similar increases in sugars and decreases in organic acids protoplasts from etiolated rice seedlings. The coding sequence from were also observed in the WT flavedo during fruit ripening CrNAC036 was inserted into a BiFC expression vector (pCL112) to in this study; however, the rates of these changes in the MT produce the nYFP vectors. CrMYB68 was inserted into a BiFC expres- flavedo were slower than those in the WT flavedo (Fig.  1). sion vector (pCL113) to produce cYFP vectors (Bhargava et  al., 2010). Furthermore, we observed the same trend of change in the Florescence was observed using a confocal laser-scanning microscope (TCS SP2, Leica, Germany). All the plasmids used in the dual luciferase levels of sugars and organic acids in the flesh of WT and MT transcriptional activity assay and the BiFC assay were purified using the fruit (Supplementary Fig. S1). QIAGEN Plasmid Midi Kit. To further explore the effect of ABA on ripening-related gene expression and the regulatory mechanism underlying the Yeast two-hybrid analysis ABA-deficient phenotype of MT, GeneChip Citrus Genome Arrays (Affymetrix, Santa Clara, CA, USA) were used to char- The CrNAC036 and CrMYB68 coding sequences were inserted into pGBKT7 and pGADT7 to generate pGBKT7-CrNAC036 and acterize change in the transcriptome of MT and WT fruits pGADT7-CrMYB68 vectors, respectively. A  yeast strain (AH109, (170 DAF) after exogenous ABA treatment. Combining the Clontech) was co-transformed with these two vectors and grown on a data obtained with former microarray and digital gene ex- selective SD/−Trp/−Leu medium. The interactions were evaluated on pression profiling experiments (Zhu et al., 2017), we analysed SD/−Trp/−Leu/−His/−Ade medium containing X-α-galactosidase. the DEGs after ABA treatment and between MT and WT at different fruit stages (Fig.  2A). These analyses indicated that Statistical analysis 21 genes were differentially expressed across all transcriptome The variance of the data was analysed using SPSS 16.0 (SPSS Inc. profiling experiments and the expression of 13 DEGs was Chicago, IL, USA). Multiple comparisons were performed by one-way consistently higher while one gene was consistently lower in ANOVA at the significance level of P<0.05 based on Duncan’s multiple ABA-treated fruits (in comparison with that of water-treated range test. Student’s paired t-test was performed to assess whether the dif- ferences between two genotypes were statistically significant. fruit) and WT (in comparison with that of MT at different fruit stage) (Table 1). Among these genes, CrNCED5 was in- duced in both WT and MT fruits after ABA treatment and Accession numbers also highly expressed in WT fruit during the fruit develop- CrNAC036 (coding sequence, MH339996), CrABA4 (promoter se- ment stages. Moreover, some genes involving in ABA-induced quence, MH339995), and CrNCED5 (promoter sequence, KY612516) ripening process, such as cell wall degradation, were induced are available at NCBI with the indicated accession numbers. The micro- array raw data are available at NCBI’s Gene Expression Omnibus with by ABA treatment and highly expressed in the WT fruit the accession code of GSE113669. (Table  1). Interestingly, there were two transcription factors that co-expressed with CrNCED5 and since NAC family pro- teins play important roles in both ABA biosynthesis and the Results fruit ripening process, we inferred that CrNAC036 may be an important regulator of CrNCED5 (Table 1). Differences in ABA-associated ripening phenotypes Owing to the slight differences between MT and WT fruit between WT and MT fruit at 120 DAF and in order to acquire more information about As described previously, the low ABA content and some late- the fruit at the breaker and postharvest stages, we analysed the ripening phenotypes such as the low color index, maturity expression of genes involved in ABA biosynthesis and related Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3617 Fig. 1. Chlorophyll a, chlorophyll b, chlorophyllide a, sugar, and organic acid levels in the flavedo of WT and MT. The values in each column are the means of three biological replicates. Error bars indicate SD. The asterisks indicate significant differences determined using Student’s t-test. *P<0.05; **P<0.01. DAF: days after flowering. ND: not detected. process (such as chlorophyll and cell wall degradation) using fusion protein and purified. We obtained a protein with a mo- qRT-PCR at 170, 185 and 210 DAF and 30 DAS (Fig.  2B). lecular mass of ca. 25 kDa (Fig. 3C). MS data indicated that the At 210 DAF, the expression levels of CrABA4, CrPPH band corresponded to the N-terminus of the CrNAC036 pro- (CrPheophytinase), CrSGR (CrStay Green), CrPEL (CrPectin tein and the a, c and d subdomains that are conserved among Lyase), and CrExpansin A8 were 4.02-, 1.15-, 1.48-, 27.04-, and NAC transcription factors could all be identified in the fu- 8.74-fold higher in WT fruit than in MT fruit, respectively. By sion protein (see Supplementary Table S4; Supplementary contrast, the expression level of CrNAC036 in MT fruit was Figs S5, S6). The c and d subdomains are responsible for the 16.13-fold higher than that in WT fruit. specific DNA binding activities of NAC transcription factors (Ooka et al., 2003). Therefore, the purified N-terminus of the CrNAC036 protein could be utilized for analysing the DNA Amino acid sequence alignment, subcellular binding activity of CrNAC036. localization, and prokaryotic expression of CrNAC036 To further analyse the potential function of CrNAC036, we CrNAC036 specifically repressed the expression of compared the amino acid sequence of CrNAC036 with that of CrNCED5 105 Arabidopsis NAC transcription factors. In the phylogen- etic analysis, CrNAC036 grouped with the clade containing Previous studies reported that the senescence-associated NAC At2G17040.1, At2G02450.1, At2G02450.2, At5G39820.1, transcription factor family can bind to the DNA sequences and At2G43000.1 (see Supplementary Fig. S4). Moreover, containing the conserved sequence motif CGT/ACG subcellular localization experiments demonstrated that the (Podzimska-Sroka et al., 2015). We found the CGT/ACG motif CrNAC036–GFP fusion protein was co-localized with a in the promoters of CrNCED5 and CrABA4 (Fig. 4A). T o test nuclear marker protein (OsGhd7–CFP), indicating that the whether CrNAC036 can bind to the promoters of CrNCED5 CrNAC036–GFP fusion protein was accumulated in the nu- and CrABA4, the purified 6×His–CrNAC036 fusion pro- cleus (Fig. 3A). tein was incubated with 20-bp probes containing the CGT/ To determine the DNA binding activity of the CrNAC036 ACG sequence from the promoter regions of CrNCED5 and protein, it was expressed in Escherichia coli as a 6×His–CrNAC036 CrABA4. It was found that the CrNAC036 protein did not Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3618 | Zhu et al. Fig. 2. Transcriptome analysis (A) and expression (B) of CrNAC036 and ABA-induced genes at different stages of ripening. (A) CK-MT-ABA and CK-WT- ABA indicate the DEGs of MT and WT between water treatment and ABA treatment, respectively. MT-170DAF-WT, MT-210DAF-WT, and MT-30DAS-WT indicate the DEGs at 170 DAF, 210 DAF, and 30 DAS between MT and WT, respectively. (B) The values in each column are the means of three biological replicates. Error bars indicate SD. The asterisks represent significant differences determined by Student’s t-test, **P<0.01. DAF: days after flowering; DAS: days after storage. CrPEL, CrPectate Lyase; CrPPH, CrPheophytinase; CrSGR, CrStay-Green. (This figure is available in color at JXB online.) bind to the two probes containing the CGT/ACG motifs from CrNAC036 protein, as indicated by the retardation of its mo- the CrABA4 promoter in the EMSA. However, the P1 probe bility in the EMSA. Furthermore, although the CrNAC036 from the CrNCED5 promoter was specifically bound by the protein bound the mutant probe derived from P1, the binding Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3619 Table 1. Consistently differentially expressed genes under ABA treatments and at different ripening stages Gene Probe set ID Transcriptome analysis (log (fold change)) Function Arabidopsis ortholog CK- CK- MT- MT- MT- WT- MT- 170DAF- 210DAF- 30DAS- ABA ABA WT WT WT Ciclev10014639m Cit.17235.1.S1_s_at −3.11 −1.07 −1.13 −1.71 −7.81 ABA biosynthesis AT1G30100 (AtNCED5) Ciclev10029007m Cit.31377.1.S1_at 1.88 1.98 1.12 3.32 5.50 Transcription factor AT2G17040 (AtNAC036) Ciclev10029283m Cit.10057.1.S1_s_at −1.63 −2.47 −1.52 −1.13 −2.68 Transcription factor AT2G28500 (AtLBD11) Ciclev10031429m Cit.35568.1.S1_s_at −1.25 −1.56 −1.74 −3.39 −4.91 Cell wall degradation AT1G67750 (AtPEL) Ciclev10032524m Cit.20839.1.S1_s_at −1.02 −1.18 −1.11 −1.81 −3.95 Cell wall degradation AT2G40610 (AtEXPA8) Ciclev10019301m Cit.2945.1.S1_s_at −1.59 −2.05 −1.09 −2.42 −4.74 Cell wall degradation AT1G64390 (AtGH9C2) Ciclev10012384m Cit.8763.1.S1_s_at −2.52 −3.60 −1.86 −2.59 −8.10 Water metabolism AT4G00430 (AtPIP1;4) Ciclev10004103m Cit.1002.1.S1_s_at −1.09 −0.96 −1.81 −1.08 −3.00 Glucosinolate me- AT4G31500 tabolism (AtCYP83B1) Ciclev10025900m Cit.15742.1.S1_at −1.72 −3.34 −1.43 −1.79 −2.16 Lipid metabolism AT1G75900 (AtEXL3) Ciclev10011714m Cit.1770.1.S1_at −1.80 −1.79 −1.88 −3.36 −7.47 Secondary metab- AT3G26040 olism Ciclev10001944m Cit.22427.1.S1_s_at −1.93 −2.49 −1.40 −1.19 −3.32 Development, un- AT4G15920 specified Ciclev10033996m Cit.26052.1.S1_s_at −1.06 −1.67 −2.05 −1.72 −4.77 Unknown AT2G39855 Ciclev10033283m Cit.21497.1.S1_at −1.87 −2.26 −1.72 −1.63 −4.12 Unknown AT2G38905 Ciclev10028078m Cit.10062.1.S1_at −1.88 −2.08 −1.14 −1.15 2.91 Amino acid metab- AT3G47340(AtASN1) olism Ciclev10008993m Cit.1718.1.S1_s_at 3.34 4.15 1.04 1.35 −1.25 Ethylene biosyn- AT2G19590 (AtACO1) thesis Ciclev10029695m Cit.9890.1.S1_s_at 1.80 1.01 −1.17 −1.53 −1.13 Gibberellin-regulated AT2G14900 family protein Ciclev10005627m Cit.28626.1.S1_s_at −1.29 −2.11 −1.83 −1.78 1.72 Cell wall modification AT1G65680(ATEXPB2) Ciclev10031099m Cit.4425.1.S1_at −1.32 2.21 1.54 −1.43 2.25 Cytochrome P450 AT5G05260 (AtCYP79A2) Ciclev10002768m Cit.12040.1.S1_s_at −1.36 −2.81 −2.02 −1.81 1.06 Metal handling AT4G08570 Ciclev10006006m Cit.165.1.S1_s_at −1.84 −2.18 −1.23 −3.96 1.76 Light signalling AT3G22840 (AtELIP1) Ciclev10017113m Cit.10672.1.S1_s_at −3.78 −3.28 1.17 −2.02 3.22 Light signalling AT3G26740 (AtCCL) CK-MT-ABA and CK-WT-ABA indicate the DEGs of MT and WT between water treatment and ABA treatment, respectively. MT-170DAF-WT, MT-210DAF-WT, and MT-30DAS-WT indicate the DEGs at 170 DAF, 210 DAF, and 30 DAS between MT and WT, respectively. activity was considerably lower than that observed for the et  al., 2017). In order to determine whether CrNAC036 can wild-type P1 probe (Fig. 4B). interact with CrMYB68 to synergistically regulate the expres- To further evaluate whether CrNAC036 affected the activ- sion of CrNCED5, we first tested whether CrMYB68 and ities of the CrNCED5 and CrABA4 promoters, we fused the CrNAC036 can interact in the yeast two-hybrid system and CrNCED5 and CrABA4 promoters including the core-binding BiFC assays. The yeast strains harboring both the pGADT7- motif of NAC family (i.e. the CGT/ACG sequence) to the CrMYB68 and the pGBKT7-CrNAC036 vectors could grow firefly luciferase reporter gene, and these were then transiently and exhibit blue color on a medium containing X-α-Gal co-expressed in protoplasts. Results indicated that CrNAC036 without leucine, tryptophan, histidine, and adenine. These re- significantly repressed the activity of the CrNCED5 promoter sults indicated that CrNAC036 can interact with CrMYB68 but did not affect that of the CrABA4 promoter (Fig.  4C). in the yeast two-hybrid system (Fig. 5A). The ability of these Based on the results of EMSA and dual-luciferase experiments, proteins to interact with each other was independently verified it can thus be inferred that CrNAC036 can specifically bind to by BiFC experiments. As shown in Fig. 5B, the interaction be- the promoter of CrNCED5 and represses its activity. tween CrNAC036 with the C-terminus and CrMYB68 with the N-terminus of yellow fluorescent protein (YFP) yielded a fluorescence signal in the nucleus (Fig. 5B). CrNAC036 interacted with CrMYB68 to synergistically Secondly, we perfor med a dual-luciferase exper iment by tran- down-regulate the expression of CrNCED5 siently co-expressing the two effector vectors and a luciferase NAC proteins usually enhance their own transcriptional ac- reporter gene in protoplasts. As a result, the combinations of tivities via interaction with other transcription factors (Olsen ‘None (empty vector)+CrNAC036-None’ and ‘None (empty et  al., 2005). Our previous study indicated that CrMYB68 vector)+CrMYB68-None’ significantly repressed the activity displays a similar expression pattern to CrNAC036 in the of the promoter from CrNCED5. However, the activity of the MT fruit and can also directly regulate CrNCED5 (Zhu CrNCED5 promoter was the most obviously reduced when Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3620 | Zhu et al. Fig. 3. Subcellular localization and prokaryotic expression of CrNAC036. (A) Subcellular localization of CrNAC036. 35S:CrNAC036-pM999-GFP and 35S:OsGhd7-CFP were co-transformed into protoplasts. OsGhd7–CFP was used as a nuclear marker. (i) CrNAC036–GFP, (ii) OsGhd7–CFP, (iii) bright field, (iv) merged image. (B) Subcellular localization of pM-999. (i) pM999–GFP, (ii) chlorophyll fluorescence, (iii) bright field, (iv) merged image. The scale bars in (A, B) indicate 10 μm. (C) Prokaryotic expression analysis of His-tagged CrNAC036 with a Coomassie blue-stained 12% SDS gel. Lane 1, His- tagged CrNAC036 protein; M, Marker. (This figure is available in color at JXB online.) ‘CrNAC036-None’ and ‘CrMYB68-None’ were co-expressed ripening-related process, such as chlorophyll degradation, ca- (Fig.  5E). These results indicated that the protein–protein rotenoid biosynthesis and softening, by regulating gene expres- interactions between CrNAC036 and CrMYB68 do synergis- sion (Zaharah et al., 2013; Gao et al., 2016; Rehman et al., 2018). tically affect the expression of CrNCED5. These effects of ABA could also be confirmed in MT fruit, with the expression of genes that contribute to chlorophyll degradation and cell wall modification (i.e. CrPEL, CrExpansin A8, CrPPH, and CrSGR) being consistently lower in MT fruit Discussion than in WT fruit during the ripening process and the dem- ABA is one of the key regulators of the fruit ripening process. onstration that the expression of CrPEL and CrExpansin A8 The transcriptional regulation of ABA biosynthetic genes has could be significantly induced by exogenous ABA treatment been well studied in fruits (Endo et al., 2016; Luo et al., 2017; (Fig.  2). Additionally, injection of ABA caused an increase in Wang et  al., 2019b). However, comprehensive details of the the glucose and fructose levels of citrus fruit and exogenous effect of ABA on citrus ripening and the regulation of ABA ABA treatment can accelerate fruit coloring of citrus (Kojima biosynthesis in citrus by a multi-transcription factor complex et  al., 1995; Wang et  al., 2016; Rehman et  al., 2018). Taken remain largely unknown. Here, we performed metabolic, cyto- together, all of the previously published work and our own logical, and transcriptome analysis of an ABA-deficient mu- findings indicate that ABA is an essential positive regulator of tant from Citrus reticulata cv. Suavissima. It was found that ABA ripening of citrus fruits. served as an important regulator of citrus ripening and its bio- synthesis was under the synergistic regulation of CrNAC036 CrNAC036 plays an important role in ABA-deficient and CrMYB68 by suppressing the expression of CrNCED5. phenotype of MT Our results from the transcriptome and qRT-PCR experi- ABA plays an important role in the ripening process of ments indicate that the expression of CrNCED5 and CrABA4 citrus fruit was robustly lower in MT than that in WT (Fig. 2; Table 1; Zhu In MT fruit, some ripening parameters (such as color index, et al., 2017). We also checked the expression of other genes in- maturity index, and softening) were significantly delayed, volved in ABA biosynthesis but found that their expression did which was in accordance with those displayed by other ABA- not vary robustly (Zhu et al., 2017). Thus, as NCED5 was the deficient citrus mutants (Rodrigo et al., 2003; Wu et al., 2014; dominant NCED for the ABA biosynthesis in C.  clementina Zhang et al., 2014; Zhu et al., 2017). Moreover, in this study, the flavedo compared with other NCEDs (Agustí et al., 2007), the delay of chlorophyll degradation, the chloroplast–chromoplast low expression of CrNCED5 and CrABA4 may lead to the transition, and sugar and organic acid metabolism in MT fur- low ABA level in MT. Moreover, we observed, in the tran- ther indicated that ABA can systematically affect the citrus fruit scriptome data, that the expression of CrNAC036 displayed ripening process (Fig. 1; Supplementary Fig. S1). Furthermore, a significantly negative correlation to that of CrNCED5 and many studies have reported that ABA can directly promote CrABA4 (Table  1; Fig.  2). Additionally, we found that the Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3621 Fig. 4. EMSA and dual luciferase assay. (A) Schematic diagram of the promoter model and sequences. The sequences used in the EMSA and dual luciferase assay are indicated on the left; the sequences used in the EMSA are indicated on the right. Black circle indicates the core-binding motif of NAC family. (B) Binding of CrNAC036 to the promoters of CrNCED5 and CrABA4. In EMSA, 10-, 20-, and 30-fold excess of non-labeled probes were used as competitors. (C) Diagram of the constructs used in the dual luciferase assays and the regulation of CrNAC036 on CrNCED5 and CrABA4. The fLUC/rLUC ratio represents the relative activity of the CrNCED5 and CrABA4 promoters. The values in each column are the means of three biological replicates. Error bars indicate the SD. The double asterisks represent statistically significant differences determined using Student’s t-test (**P<0.01). (This figure is available in color at JXB online.) amino acid sequence of CrNAC036 is similar to that of JUB1, CrABA4 promoter (Fig.  4). Based on these results, it can be which inhibits Arabidopsis senescence (see Supplementary concluded that CrNAC036 is an important regulator of ABA Fig. S4), and note that the ABA content of jub1-1 plants was biosynthesis in citrus fruit, a task it achieves by specific down- significantly increased compared with that of the wild type regulation of the expression of CrNCED5. (Wu et  al., 2012). Furthermore, DAP-seq analysis indicated Transcription factors and hormones such as ABA and that JUB1 can directly bind to the promoter of AtNCED5 ethylene substantially influence fruit ripening. Several tran- (O’Malley et al., 2016). When taken together these data suggest scription factor families have been proven to promote ripening that CrNAC036 may negatively regulate ABA biosynthesis by through affecting ABA (ABF2 in grape) or ethylene (RIN, down-regulating the expression of CrNCED5 or CrABA4. CNR in tomato and MdMYC2 in Malus domestica) metab- However, whilst conserved NAC-recognition sequences were olism and signaling (Klee and Giovannoni, 2011; Nicolas et al., recognized in the promoters of both CrNCED5 and CrABA4, 2014; Li et al., 2017b). However, until recently no transcription the data from the EMSA indicated that CrNAC036 can spe- factor family had been reported to regulate both fruit ABA and cifically bind to the promoter of CrNCED5 but not to that of ethylene metabolism. Recently, Lü et al. (2018) reported that CrABA4 (Fig. 4). Moreover, the data from the dual luciferase carpel senescence-related NAC transcription factors play vital assay indicated that CrNAC036 can repress the activity of the roles in the ripening process of both climacteric fruit without CrNCED5 promoter while not affecting the activity of the recent whole-genome duplication and non-climacteric fruit. Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 3622 | Zhu et al. Fig. 5. Interactions between CrNAC036 and CrMYB68 and their cooperative regulation of the CrNCED5 promoter. (A) Interactions between CrNAC036 and CrMYB68 in the yeast two-hybrid assays. Blue colonies growing on a synthetic drop-out medium lacking Trp, Leu, His, and Ade (SD/−Trp/−Leu/− His/−Ade) and containing X-α-galactosidase indicate protein–protein interactions. Co-transformation of pGBKT7-p53 and pGADT7-RecT was used as a positive control. (B–D) Nuclear interactions of CrNAC036 with CrMYB68. Confocal images of transiently transformed nYFP–CrNAC036 and cYFP– CrMYB68 generating YFP signal in nucleus (OsGhd7–CFP was used as a nuclear marker) (B). Interaction with empty vectors was not observed (C, D). Scale bars: 10 μm. (E) Dual luciferase assays in protoplasts co-expressing CrNAC036 and CrMYB68. The fLUC/rLUC ratio represents the relative activity of the CrNCED5 promoter (P2). The sequence of P2 is indicated in Fig. 4A. The values in each column are the means of three biological replicates. Error bars indicate SD. Different letters indicate significant differences according to Duncan’s test (P<0.05). (This figure is available in color at JXB online.) Downloaded from https://academic.oup.com/jxb/article/71/12/3613/5849422 by DeepDyve user on 20 July 2022 Synergistic transcriptional regulation of ABA biosynthesis | 3623 In tomato, SlNAC19/48 can directly induce the expression regulatory network of transcription factor complexes in ABA of ACO1 and ACS2 and in Musa acuminata, MaNAC1/2 biosynthesis. can affect ethylene signaling by interacting with EIN3 (Shan et al., 2012; Ma et al., 2014a; Kou et al., 2016). In the present study, our findings concerning the transcriptional regulation of Supplementary data CrNAC036 greatly improves our understanding of the tran- Supplementary data are available at JXB online. scriptional regulation of ABA metabolism in fruit. They add- Fig. S1. Pheophorbide a and pheophytin a levels in the itionally enrich our knowledge concerning the importance of flavedo and sugar and organic acid levels in the flesh of WT regulators of the NAC transcription factor family on the bio- and MT. synthesis of two hormones known to be vital for fruit ripening. Fig. S2. Transmission electron microscopy analysis of the morphological changes in the flavedo plastids from WT CrNAC036 and CrMYB68 synergistically inhibit ABA and MT. biosynthesis by down-regulating the expression of Fig. S3. Levels of photosystem-associated proteins in the CrNCED5 flavedo at four developmental stages. Fig. S4. Phylogenetic analysis of CrNAC036 and NAC pro- Owing to their sessile nature, plants have evolved complex teins from Arabidopsis. mechanisms to activate or repress gene expression in order to Fig. S5. Peptides from CrNAC036 identified using adapt to their prevailing environments. As important regulators MALDI-TOF/TOF MS. of gene expression, besides working alone, different transcrip- Fig. S6. The MS/MS spectrum of the identified peptides of tion factors can form complexes and recognize cis-regulatory CrNAC036 protein. elements within a target gene promoter for synergistic or an- Table S1. The DEGs of MT and WT after ABA treatment tagonistic regulation of gene transcription. Given that ABA is Table S2. Primers used in this study. an important regulator of stress tolerance and the fruit ripening Table S3. Names and TAIR ID numbers of 105 NAC tran- process, the transcriptional regulation of NCEDs by a single scription factors from Arabidopsis. transcription factor has been reported in many crops and fruits Table S4. Peptides identified by 6×His–CrNAC036 protein (Jiang et al., 2012; Finkelstein, 2013; Jensen et al., 2013; Endo using the MALDI-TOF/TOF MS method. et al., 2016; Zong et al., 2016; Luo et al., 2017; Ma et al., 2018; Wang et  al., 2019b). However, to date research concerning synergistic regulation of fruit NCEDs by transcription factor Acknowledgements complexes remains rare. In previous publications, transcription factors of the NAC We thank Dr Masaru Ohme-Takagi (National Institute of Advanced Industrial Science and Technology, Japan) for providing the plasmids 35S′- family have been reported to interact with distinct types of GAL4-fLUC, Dr Shouyi Chen (Institute of Genetics and Developmental transcription factors including NAC family proteins, WRKY Biology, China) for providing the plasmids None and AtUbi3-rLUC, and family proteins, TCP family proteins, and zinc finger-containing Dr Wei Zong and Lizhong Xiong (Huazhong Agricultural University) transcription factors (Olsen et al., 2004; Tran et al., 2007; Wang for providing plasmids pM999 and 35S:OsGhd7-CFP. Moreover, we et al., 2015; Zhou et al., 2015; Li et al., 2017a). Moreover, our also thank the research associates at the Center for Protein Research earlier research demonstrated that CrMYB68 was highly ex- (CPR), Huazhong Agricultural University for technical support. This pressed in the MT fruit and the protein can directly repress work was supported by the National Key R&D Program of China the expression of CrNCED5 (Zhu et  al., 2017). In the pre- (2018YFD1000200); Huazhong Agricultural University Scientific & sent study, CrNAC036 was revealed to be highly co-expressed Technological Self–innovation Foundation; the National Natural Science with CrMYB68, and Y2H and BiFC results indicated that Foundation of China (No. 31772368, 31572176); the National Modern CrNAC036 physically interacted with CrMYB68. Given that Agricultural (Citrus) Technology Systems of China (CARS-27) and China Postdoctoral Science Foundation No. 2018M640713. Finally, we both CrNAC036 and CrMYB68 are repressors of CrNCED5, thank Prof. Zuoxiong Liu for English language editing. 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Published: Jun 22, 2020

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