Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus

ORIGINAL RESEARCH published: 08 February 2017 doi: 10.3389/fpls.2017.00126 Frontiers in Plant Science | www.frontiersin.org 1February 2017 | Volume 8 | Article 126 Edited by: Rüdiger Simon, University of Düsseldorf, Germany Reviewed by: Margret M. Sauter, University of Kiel, Germany Sarah Liljegren, University of Mississippi, USA *Correspondence: Francisco R. Tadeo [email protected] †Present Address: Paz Merelo, European Molecular Biology Laboratory, Developmental Biology Unit, Heidelberg, Germany; Javier Agustí, Departamento de Desarrollo y Acción Hormonal en Plantas, Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain; Vicent Arbona, Departament de Cièncias Agràries i del Medi Natural, Universitat Jaume I, Castelló de la Plana, Spain; Leandro H. Estornell, Department of Plant Biology, Swedish University of Agricultural Science, Uppsala BioCentre, Uppsala, Sweden Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science Received: 17 November 2016 Accepted: 20 January 2017 Published: 08 February 2017 Citation: Merelo P, Agustí J, Arbona V, Costa ML, Estornell LH, Gómez-Cadenas A, Coimbra S, Gómez MD, Pérez-Amador MA, Domingo C, Talón M and Tadeo FR (2017) Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus. Front. Plant Sci. 8:126. doi: 10.3389/fpls.2017.00126 Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus Paz Merelo1 †, Javier Agustí1 †, Vicent Arbona1 †, Mário L. Costa2, Leandro H. Estornell1 †, Aurelio Gómez-Cadenas3, Silvia Coimbra2, María D. Gómez4, Miguel A. Pérez-Amador4, Concha Domingo1, Manuel Talón1and Francisco R. Tadeo1* 1Centre de Genòmica, Institut Valencià d’ Agràries, València, Spain, 2Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal, 3Departament de Ciències Agràries i del Medi Natural, Universitat Jaume I, Castelló de la Plana, Spain, 4Departamento de Desarrollo y Acción Hormonal en Plantas, Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield. Keywords: calyx abscission zone, cell wall modification, citrus fruit abscission, ethylene, lignin biosynthesis, phylogeny, transcriptomics INTRODUCTION Abscission is a cell separation process by which plants can shed their aerial organs. It takes place in groups of functionally specialized cells known as abscission zones (AZs), which are located at specific sites of organ detachment in the plant (Roberts et al., 2002; Estornell et al., 2013; Tucker and Kim, 2015). Abscission is a fundamental process in plant biology that represented a highly beneficial evolutionary adaptation for plants: abscission allows for discarding senescent or physiologically damaged organs and for highly efficient seed dispersal. However, from an agricultural point of view, abscission has a tremendous impact on yield, leading to high yield losses in key crops like brassica Merelo et al. Cell Wall Remodeling during Abscission or citrus. In this way, understanding abscission at the molecular level is of top relevance not only to understand a fundamental process for plant physiology but also to generate new, improved, highly productive crops. Abscission related traits (i.e., reduced abscission of fruits or seeds) are among the main agronomic traits selected along plant domestication (Konishi et al., 2006; Pickersgill, 2007). A current example is the expansion of late-season varieties of sweet orange in the citrus market. In such varieties, the decline in the fruit retention force is delayed during the maturing period in comparison with early and mid-season varieties (Gallasch, 1996) that usually undergo pre-harvest fruit abscission (SpiegelRoy and Goldschmidt, 1996). Thus, late-season varieties of sweet orange extend the fruit harvesting season benefiting growers and food industry. Control of abscission is also relevant to facilitate mechanical harvesting, thus reducing collection costs. Mechanical fruit harvesting systems have been developed although they are still inefficient and cause tree damages (Li et al., 2011). On the other hand, several abscission-triggering compounds have been used to improve mechanical harvesting. In citrus, treatments with CMNP (5-chloro-3-methyl-4-nitro-1H-pyrazole) are used to promote fruit loosening and to facilitate and coordinate mechanical harvesting of fruits (Burns, 2002). In this regard, understanding the mechanisms underlying abscission is essential to control abscission and improve harvesting practices and productivity. Studies on floral organ abscission in the model system Arabidopsis thaliana have provided a wealth of valuable information. However, the current information about the molecular mechanisms underlying abscission in crop species is rather scarce. Most of the molecular studies of abscission in crops have mainly been focused on the characterization of individual or few genes. However, high-throughput approaches have recently been applied in AZ-containing tissues of tomato flowers (Meir et al., 2010) and apple (Zhu et al., 2011), mature olive (GilAmado and Gomez-Jimenez, 2013; Parra et al., 2013), melon (Corbacho et al., 2013), litchi (Li et al., 2015), and orange fruits (Cheng et al., 2015). In our previous studies (Agustí et al., 2008, 2009, 2012), global expression analyses provided a wide set of genes potentially involved in citrus leaf abscission. These datasets included a number of cell wall modification related genes as well as genes involved in signaling, transcription control, protein synthesis and degradation and vesicle transport. Our current challenge is to identify key regulatory genes of citrus fruit abscission which is, indeed, an economically important process. In citrus, maturing fruits are shed through the abscission zone C (AZ-C), located at the boundary between the calyx button and the fruit rind (FR). In this region, different tissues converge and the isolation of exclusive AZ-C cells for molecular studies without any contamination of other celltypes is extremely complicated. In this study, we have taken advantage of the optimization of laser microdissection (LM) in citrus tissues (Agustí et al., 2009; Matas et al., 2010; Caruso et al., 2012) for the accurate sampling of fruit AZ-C cells. This strategy has allowed the precise quantification of the timing and magnitude of gene expression and associate metabolites involved in the process of ethylene-promoted abscission in the specific cells of the AZ-C. Moreover, phylogenetic analyses of the most representative gene families during abscission in citrus and different plant species have revealed a link between phylogenetic proximity and expression pattern during this process suggesting highly conserved functions for specific members of these families in abscission. Overall, this study, through the identification of potential abscission-related genes and the detailed spatiotemporal analysis of the anatomical and histochemical changes in the activated AZ-C, provides crucial information for future biotechnological approaches aimed at improving citrus yield. MATERIALS AND METHODS Plant Material and In vitro Treatments We used fruits from two Citrus sinensis cultivars: a midseason orange cultivar (cv. Washington Navel) that usually undergoes pre-harvest abscission and a late-season orange cultivar (cv. Ricalate Navel) with delayed abscission. Maturing fruits were harvested after color change from adult trees grown in a homogeneous experimental orchard under normal cultural practices at the Institut Valencià d’Investigacions Agràries (IVIA). Fruits were separated from the tree leaving 2 cm peduncles to isolate the AZ-C for further analyses. For abscission kinetics studies and tissue collection, Washington Navel fruits were incubated for 0, 24, 48, and 96 h in the presence or absence of ethylene (10 µL/L) in sealed 10 l containers at 22◦C with a 16 h light period under fluorescent lighting. Ricalate Navel fruits were incubated for 0, 24, 48, 96, and 192 h in the presence of 1aminocyclopropane-l-carboxylic acid (ACC; 0.1 mM) or water under the same temperature and light conditions. In this case, a 3 mL Pasteur pipette containing the ACC solution or water was fitted to the fruit peduncles. Phloroglucinol Staining Phloroglucinol staining for lignin in fresh cut tissue portions (0.5 cm3) containing the AZ-C after 0, 24, and 48 h of ethylene or ACC treatment was performed according to Tadeo and PrimoMillo (1990). Samples were cut longitudinally to allow AZ-C staining and for further image acquisition. A saturated solution of phloroglucinol (Sigma-Aldrich) in 20% HCl was directly applied to samples. Observation was carried out with an Olympus SZ61 stereomicroscope (Olympus GmbH). Cryoscanning Electron Microscopy (cryo-SEM) Longitudinal sections as well as the proximal (peduncle) and distal (fruit) fracture plane of the ethylene-promoted AZ-C were observed using cryo-SEM. To examine longitudinal sections of the AZ-C, 1 cm portions of tissue were manually dissected with a razor blade. In the second case, the peduncle was forcibly separated from the fruit. Specimen mounting and AZC observation were carried out as previously described in Agustí et al. (2009). At least three samples containing the AZ-C after 24, 48, and 96 h of ethylene treatment were observed. Frontiers in Plant Science | www.frontiersin.org 2February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission Periodic Acid-Schiff (PAS) Staining Tissue containing the AZ-C after 0, 24, and 48 h of ACC treatment was manually dissected using a razor blade in 0.5 cm3portions. These samples were fixed overnight at 4◦C in a 4% (w/v) paraformaldehyde-PBS solution. After fixation, samples were washed with PBS, dehydrated in a graded ethanol series and embedded in LR White (Electron Microscopy Sciences). Longitudinal sections of the calyx button area (1 µm thickness) were cut with a Leica RM2165 microtome and placed on glass slides. Slides were further stained with PAS (Sigma-Aldrich) and mounted with DPX Mountant (Fluka). Observations were performed on a Leica DMLA microscope (Leica Microsystems) and images were processed with Leica ASMLD Version 4.0 software. Preparation of Tissue Containing the AZ-C for LM Portions of tissue containing the AZ-C (0.5 cm3) were dissected from fruits after 0, 12, and 24 h of ethylene treatment for the transcriptomics assay, and after 0, 12, 24, and 36 h of ACC treatment for lignin intermediates quantification. Preparation of cryosections and microdissection were performed as previously described in Agustí et al. (2009). Cells from the AZ-C and the adjacent FR were selected from 30 to 40 cryosections and collected separately. Phloroglucinol Staining of Cryosections Cryosections of 14 µm of tissue containing the AZ-C after 48 h of ethylene treatment were processed as described in Agustí et al. (2009) and mounted on CryoJane R adhesive coated slides (Instrumedics) following the manufacturer’s instructions. Slides were stored at −80◦C until phloroglucinol staining. Staining for lignin was performed using a saturated solution of phloroglucinol (Sigma-Aldrich) in 20% HCl. Observation was carried out with an Olympus SZ61 stereomicroscope (Olympus GmbH). RNA Isolation, Sample Labeling, and Microarray Hybridization Three independent biological replicates were collected for each cell type at 0, 12, and 24 h after ethylene treatment. For each independent sample, total RNA from ∼40,000 pooled cells was extracted using the RNeasy Micro Kit (Qiagen) following the manufacturer’s instructions. The RNA purity was assessed by measurements of OD260/OD280. Two RNA amplification rounds were performed utilizing the TargetAmpTM 2-Round Aminoallyl-aRNA Amplification Kit (EPICENTER) according to the manufacturer’s instructions to synthesize the antisense cRNA. The quality of the amplified RNA was evaluated by OD260/OD280 measurements and agarose gel electrophoresis. Each sample was labeled with Cy5 and cohybridized with Cy3-labeled antisense cRNA from a reference sample containing a mixture of equal amounts of RNA from all experimental samples (0, 6, 12, 24, 48, and 96 h of ethylene/air treatment). RNA labeling, microarray hybridization, and slide washes were performed as previously described in Cercos et al. (2006). Hybridized microarrays scanning, hybridization data acquisition, and microarray normalization and analysis were carried out as described in Agustí et al. (2009). A cDNA microarray including 21.081 putative genes of citrus was utilized (Martinez-Godoy et al., 2008). Gene expression differences were considered significant under a P-value lower than 0.05 and an M contrast cutoff value of +0.5 or −0.5. In this work, a time course experiment was designed for each cell type (AZ-C and FR), therefore, the expression level corresponds to M=log2[AZ-Ct/AZ-C0] or M =log2[FRt/FR0]. The raw microarray data as well as the protocols used to produce the data and the normalized data were deposited in the ArrayExpress database under the accession number E-MTAB-4538. Functional classification of the selected genes was performed using MIPS (Munich Information Center for Protein Sequences, http://www.helmholtzmuenchen.de/en/mips/) categorization. Amplified RNA was used for the validation of microarray hybridization data by semi-quantitative RT-PCR (sqRT-PCR) analysis (Figure S1). sqRT-PCR Analysis sqRT-PCR analysis was carried out using the SuperScript II Reverse Transcriptase kit (Invitrogen, Carlsbad) following the manufacturer’s instructions. After first-strand cDNA synthesis, PCR reactions were performed using the Biotools Taq DNA Polymerase (BIOTOOLS, B&M Labs). Size and intensity of expected bands were checked by 1% agarose gel electrophoresis. Citrus UBC gene (Ubiquitin-conjugating enzyme) was used as a reference to evaluate the amounts of mRNA in each sample. Primer sequences are available in Table S1. In situ Hybridization RNA in situ hybridization with digoxigenin-labeled probes was performed as described (Gomez et al., 2011). Portions of tissue containing the AZ-C (0.5 cm3) were dissected from fruits after 24 h of ethylene treatment and immediately fixed at 4◦C overnight in FAE (25% formaldehyde, 5% acetic acid, 50% ethanol), dehydrated, embedded in paraffin wax and sectioned to 8 µm. For CitCEL6 and CitPG20, RNA antisense and sense probes were generated with SP6 and T7 RNA polymerases, using as substrate a 1518 bp fragment of the CitCEL6 cDNA (1–1518 from ATG) or a 1110 bp fragment of the CitPG20 cDNA (217– 1326 from ATG), amplified by PCR and cloned into the pGEM-T Easy vector (Promega). Lignin Intermediates Quantification Coumaric acid, caffeic acid, and ferulic acid were analyzed by UPLC coupled to tandem mass spectrometry (UPLC-MS/MS) as described by Argamasilla et al. (2014). Three independent samples containing ∼40,000 pooled AZ-C cells were isolated by LM for each time point of ACC treatment (0, 12, 24, and 36 h) and collected in 60 µL of water. Sequence Identification, Alignment, and Phylogenetic Analysis Members of the different gene families associated with cell wall remodeling in citrus (based on CAZy classification; Carbohydrate-Active Enzymes; Cantarel et al., 2009; http://www.cazy.org/) were identified by TBLASTN search Frontiers in Plant Science | www.frontiersin.org 3February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission in the Citrus clementina haploid genome (version 0.9) database web browser (http://www.phytozome.net/search.php) using the consensus sequence for the catalytic domain of each family. Prediction of characteristic domains and conserved motifs was carried out through SMART (http://smart.embl-heidelberg.de/; Schultz et al., 1998; Letunic et al., 2009), PSORT (http://psort.hgc.jp/form.html), InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) and big-PI Plant Predictor (http://mendel.imp.ac.at/gpi/plant_server.html) servers. Phylogenetic trees are based on multiple alignments using the profile alignment function of ClustalW (www.ch.embnet.org/software/ClustalW-XXL.htm) and were generated with MEGA7 (Kumar et al., 2016) using the neighborjoining algorithm with 1000 bootstrap replicates. Poisson correction for multiple substitutions was used and only values higher than 50% were considered. Immunolocalization of Pectic Polyssacharides The primary monoclonal antibodies (mAbs) used in this study and provided by Prof. Paul Knox (Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, UK) were LM5 [anti-(1,4)-β-D-galactan; Jones et al., 1997], LM6 [anti-(1,5)-α-L-arabinan; Willats et al., 1998] and JIM5 [anti-partially methylesterified/de-esterified homogalacturonan; (Knox et al., 1990)]. The secondary antibody was fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Sigma-Aldrich). Immunolocalization of pectic polysaccharides was performed on semi-thin sections of tissue containing the AZ-C (0.5 cm3) from fruits after 0, 24, and 48 h of ACC treatment. Immunolocalization of pectic polyssacharides, light microscopy and image acquisition were perfomed as described in Coimbra et al. (2007). RESULTS AND DISCUSSION Ethylene Accelerates Citrus Fruit Abscission We performed a kinetics assay of citrus fruit abscission in response to abscission-accelerating treatments to determine the optimal sampling for the transcriptomic analysis. To that end, we carried out a comparison between orange (C. sinensis) fruits incubated with ethylene or its immediate metabolic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and fruits incubated with air or water (controls). We used maturing fruits from a mid-season orange cultivar (cv. Washington Navel) that usually undergoes pre-harvest abscission and from a late-season orange cultivar (cv. Ricalate Navel) with delayed abscission. We observed a faster decrease of fruit detachment force (FDF) in both Washington Navel and Ricalate Navel fruits treated with ethylene/ACC in comparison to air-/watertreated control fruits (Figure 1A). At 48 h after treatment, the FDF in fruits treated with ethylene or ACC was around 4 kgf. However, control fruits of Washington Navel and Ricalate Navel only showed values of FDF around 4 kgf at 96 and 192 h, respectively, a response that matches their pre-harvest abscission behavior. Thus, ethylene and ACC accelerated the abscission FIGURE 1 | Effect of ethylene and 1-aminocyclopropane1-carboxylic acid (ACC) on citrus fruit abscission. (A) Abscission kinetics of Washington Navel fruits non-treated or treated with ethylene and Ricalate Navel fruits non-treated or treated with ACC. The results are means of 10 fruits ±SE. (B,C) Phloroglucinol staining for lignin in the AZ-C of Washington Navel fruits after ethylene treatment (B) and Ricalate Navel fruits after ACC treatment (C). Dashed line, abscission zone C; , lignin deposition (phloroglucinol); FR, fruit rind; FD, floral disc; SP, sepals; VB, vascular bundles; P, parenchyma. process in both varieties tested. This result strongly suggests that the natural delay in the schedule of FDF decline in fruits of the late-season variety Ricalate Navel in comparison with those of the mid-season variety Washington Navel was not related to any impairment in the response of well-developed tissues to ethylene (Zacarias et al., 1993). Based on these findings, we used induced AZ-C samples from both varieties for further analyses. Phloroglucinol Staining Reveals a Positive Correlation between Abscission and Lignin Deposition Phloroglucinol staining in receptacles of both Washington Navel and Ricalate Navel fruits revealed lignin deposition at the central core of the AZ-C, between the axial vascular bundles, 24 h after ethylene and ACC treatments (Figures 1B,C). Forty-eight hours after the treatments, lignin deposition spread out along the AZ-C, Frontiers in Plant Science | www.frontiersin.org 4February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission FIGURE 2 | Cellular morphology of the AZ-C. (A–E) Scanning electron micrographs of longitudinal sections (A,B,D) and the proximal (C; calyx button side) and distal (E; fruit rind side) fracture planes of the AZ-C from Washington Navel fruits after 48 h (A,B) and 96 h (C–E) of ethylene treatment. High magnification pictures show cells of separation layers. AZ-C, abscission zone C; , separation line inside the AZ-C; CB, calyx button; FD, floral disc; FR, fruit rind; SP, sepal; VB, vascular bundles. Scale bars: 1 mm (A–E), 500 µm(A–C), 200 µm(E), 100 µm(C). Frontiers in Plant Science | www.frontiersin.org 5February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission FIGURE 3 | Anatomy of the AZ-C. Periodic acid-Schiff reactive (PAS) staining for insoluble carbohydrates of longitudinal sections of the AZ-C from Ricalate Navel fruits non-treated (A,D) and treated for 24 h (B,E) and 48 h (C,F) with ACC. Squares in (A–C) show the area magnified with the 40X objective. AZ-C, abscission zone C; FR, fruit rind; DCA, divided cells area; SA, starch-rich area; , recently divided cell; , cell containing amyloplasts. Scale bars: 500 µm and 50 µm. perfectly drawing the separation line between the calyx button and the FR. Accordingly, timing for lignin deposition positively correlated with abscission kinetics. Morphological Changes in Activated AZ-C Cells We used scanning electron microscopy (SEM) to examine changes in the cellular morphology of the AZ-C from Washington Navel fruits treated with ethylene (Figure 2). We observed the first cellular signs of activation of abscission by ethylene in the central core of the AZ-C at 48 h of treatment (Figures 2A,B). At that time, AZ-C samples could be split into two groups, one showing early stages of cell separation and the other one showing late events of cell separation. In the former group, the AZ-C was clearly distinguishable (Figure 2A), with accumulation of an amorphous material probably derived from the partial dissolution of the middle lamella and cell wall of the AZ-C cells. In the latter group of samples, cell separation was observed in the central core of the AZ-C (Figure 2B). A greater accumulation of amorphous material was observed, suggesting that cell wall and middle lamella degradation was complete after 48 h at the central region. At 96 h after ethylene treatment, cell separation extended from the central core to the periphery of the AZ-C (Figure 2D) and differential cell expansion was observed at proximal (calyx button) and distal (fruit) sides (Figures 2C,E). At the proximal side, parenchymatic pith cells underwent expansion (Figure 2C) while, at the distal side, expansion occurred in the cells of the axial vascular bundles (Figure 2E). Two Different Cell Areas Form the AZ-C Periodic acid-Schiff (PAS) staining was used to characterize anatomically the AZ-C after ACC treatment (Figure 3). This method detects insoluble carbohydrates and was used to distinguish the cells belonging to the AZ-C since these accumulate starch grains (Wilson and Hendershott, 1968; Huberman et al., 1983; Shiraishi and Yanagisawa, 1988; Goren, 1993). In addition to the starch-rich cell area (SA) previously identified by Wilson and Hendershott (1968) at the distal side of the AZ-C (FR side), we identified another cell area located at the proximal side of the AZ-C (pith side) and composed by recently divided cells based on the observation of thinner cell walls formed between cells (Divided Cells Area, DCA; Figure 3D). Then, the AZ-C was constituted by 10–15 cell layers distributed in cellular areas with two different cell morphologies and organellar composition (i.e., cells from the SA contain amyloplasts). The analysis of the AZ-C after 48 h of treatment suggests that cell wall degradation and cell degeneration occurred mainly at the layers of the SA in the fracture plane, adjacent to the mesocarp cells of the FR known as the albedo (see Figure 9). However, cell expansion occurred at the DCA (Figure 3F). These results together with kinetics (Figure 1A), phloroglucinol staining (Figures 1B,C) and SEM (Figure 2) data suggested that the activation of the fruit AZ-C by ethylene/ACC occurred early after treatment, probably prior to 24 h. The events related to cell wall loosening might start at 24 h, while cell separation seemed to begin at 48 h and to be completed at 96 h after treatment. Gene Expression Regulated by Ethylene in AZ-C and FR Cells For gene expression analysis, we used the 20 K citrus cDNA microarray (Martinez-Godoy et al., 2008). We isolated cells from the central core of the AZ-C as well as from the FR located beneath the AZ-C through LM (Figure S2) to perform a timecourse experiment (0, 12, and 24 h-ethylene) and compared data from each analysis. Results showed that ethylene differentially regulated 2280 genes exclusively in the AZ-C cells, 1742 genes exclusively in the FR cells, and 2001 genes were regulated by ethylene in both the AZ-C and the FR cells (Table S1). All differentially regulated genes were grouped into functional categories according to the Munich Information Frontiers in Plant Science | www.frontiersin.org 6February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission TABLE 1 | Relative gene expression values (AZ-Ct vs. AZ-C0) of genes involved in cell wall modification exclusively regulated by ethylene in AZ-C cells. Name Contig/singleton ID Microarray probe Putative Ath orthologue Relative expression [log2 (AZ-Ct/AZ-C0)] 12 h 24 h ENDO-1,4-β-GLUCANASES|CELLULASES (GH9s) CitCEL3 aCL1687Contig1 IC0AAA38AD03 AT2G32990 – 0.69 CitCEL6* aCL1347Contig1 C21007H10 AT4G02290 – 4.15 CitCEL10 aCL7597Contig1 IC0AAA68DE06 AT4G02290 – 0.60 CitCEL17 aCL1288Contig1 C32011E04 AT1G75680 −0.88 – CitCEL22 aC20010F01SK_c C20010F01 AT1G23210 – 0.72 POLYGALACTURONASES (GH28s) CitPG6 aCL2029Contig1 C03009E03 AT4G23820 –1.58 – CitPG16 aCL5261Contig1 C01018A12 AT3G61490 1.02 – CitPG20* AT3G07970 in-situ hybridization CitPG41 aC18008C05Rv_c C18008C05 AT3G57790 1.10 1.49 aCL1063Contig1 IC0AAA19CA02 AT3G57790 0.88 0.95 CitPG42 aIC0AAA85AB02RM1_c IC0AAA85AB02 AT3G48950 – 0.63 CitPG43 aCL675Contig4 IC0AAA67DG09 AT2G43870 – 2.34 PECTATE-LYASES (PL1s) CitPL1 aC03011D06SK_c C03011D06 AT5G63180 −0.86 −0.80 CitPL5 aIC0AAA15AF11RM1_c IC0AAA15AF11 AT1G67750 2.66 PECTIN-METHYLESTERASES (CE8s) CitPME8 aC05807A09SK_c C05807A09 AT4G33220 −3.13 −2.57 CitPME11 aCL1691Contig1 C08033H07 AT1G11580 −2.30 – CitPME13 aCL4116Contig2 C01011H09 AT5G53370 −2.15 −2.90 CitPME24 aCL1451Contig1 IC0AAA40DF03 AT1G69940 0.71 0.71 CitPME41 aCL2379Contig1 C32102B03 AT5G09760 – 1.41 PECTIN-ACETYLESTERASES (CE18s) CitPAE1 aKN0AAP13YN19FM1_c KN0AAP13YN19 AT3G62060 −0.72 – CitPAE4 aCL67Contig4 C08028G04 AT4G19420 0.58 0.51 CitPAE6 aCL7344Contig1 C02003B05 AT5G26670 -1.57 – aKN0AAI1DH10FM2_c KN0AAI1DH10 AT5G26670 −2.07 – β-GALACTOSIDASES (GH35s) CitGBAL16 aC31805H10EF_c C31805H10 AT4G36360 −1.59 aCL7104Contig1 C02004B02 AT4G36360 −1.01 β-GALACTOSIDASES (GH2s) CitGH22 aCL4443Contig1 C31401H10 AT3G54440 0.71 0.95 β-GLUCOSIDASES (GH1s) CitBGLU17 aCL5744Contig1 C31007D10 AT2G44480 −0.59 −0.55 CitBGLU24 aCL1136Contig3 IC0AAA1CB06 AT3G06510 – 0.62 β-MANNOSIDASES (GH5s) CitMAN4 aC04002G09SK_c C04002G09 AT1G02310 1.41 – XYLOGLUCAN ENDOTRANSGLYCOSYLASES/HYDROLASES (GH16s) CitXTH16 aC02023G10SK_c C02023G10 AT4G03210 −1.55 −2.33 CitXTH24 aIC0AAA99CH05RM1_c IC0AAA99CH05 AT1G32170 1.07 1.03 CitXTH28 aCL6772Contig1 C01009B04 AT4G37800 – −0.88 α-XYLOSIDASES (GH31s) CitXYL4 aCL6235Contig1 C05075C11 AT1G68560 −0.98 – β-XYLOSIDASES (GH3s) CitBXL13 aCL3345Contig1 C02024D10 AT1G78060 −0.63 – CitBXL16 aCL8110Contig1 IC0AAA75AA10 AT5G20950 −0.99 – EXPANSINS CitEXP14 aCL2131Contig1 IC0AAA14BD04 AT2G40610 – 3.01 aIC0AAA87BH09RM1_c IC0AAA87BH09 AT2G40610 1.63 3.19 (Continued) Frontiers in Plant Science | www.frontiersin.org 7February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission TABLE 1 | Continued Name Contig/singleton ID Microarray probe Putative Ath orthologue Relative expression [log2 (AZ-Ct/AZ-C0)] 12 h 24 h CitEXP15 aKN0AAQ1YG09RM1_c KN0AAQ1YG09 AT4G17030 1.69 1.40 CitEXP19 aC02006G07SK_c C02006G07 AT1G20190 – 0.75 CELLULOSE SYNTHASES/CELLULOSE SYNTHASE-LIKE PROTEINS CitCes1 aC16012C03SK_c C16012C03 AT4G24010 1.82 – CitCes2 aIC0AAA5DG11RM1_c IC0AAA5DG11 AT5G05170 -1.74 – CitCsl3 aCL5293Contig1 C05070F01 AT3G03050 – 0.68 UDP-GLUCOSE 4-EPIMERASE CitUGE1 aC31108G08EF_c C31108G08 AT4G10960 – 0.95 CitUGE2 aCL6604Contig1 C03009D04 AT1G12780 – 0.82 MANNAN SYNTHASES CitManS1 aCL3377Contig1 IC0AAA16BH03 AT5G22740 – 1.47 CitManS2 aCL3377Contig2 IC0AAA99AD02 AT5G22740 – 1.36 GALACTOMANNAN GALACTOSYLTRANSFERASE (GMGT) CitGMGT1 aC34004D03EF_c IC0AAA58DH01 AT2G22900 – 1.35 RHAMNOSE BIOSYNTHETIC ENZYME CitRHM1 aCL4478Contig1 KN0AAI3AD11 AT1G78570 −0.52 – GLUCOSYLTRANSFERASES CitGTF1 aCL3010Contig2 IC0AAA58BE08 AT1G77130 0.82 – CitGTF2 aCL1592Contig1 IC0AAA30DE02 AT1G16570 0.58 – CitGTF3 aCL3054Contig2 KN0AAK3DE03 AT3G50060 – 1.21 CitGTF4 aCL6931Contig1 IC0AAA42BE09 AT3G25140 – 0.65 CitGTF5 aCL5570Contig1 C05056H08 AT1G77990 −1.42 −1.51 CitGTF6 aCL6758Contig1 C31701H10 AT3G02100 – −1.23 CitGTF7 aKN0AAB3DB09ZM1_c KN0AAB3DB09 AT3G45400 – 0.60 GDP-L-FUCOSE SYNTHASE CitGLFS1 aCL790Contig1 IC0AAA85AB07 AT1G17890 – 0.73 GALACTOSIDE 2-ALPHA-L-FUCOSYLTRANSFERASE CitGLFT1 aIC0AAA69BA06RM1_c IC0AAA69BA06 AT1G74420 – 0.58 CitGLFT2 aCL5210Contig1 C08029G10 AT1G05575 −0.87 −1.24 UDP-GLUCOSE DEHYDROGENASE CitUGD1 aKN0AAP5YD20FM1_c KN0AAP5YD20 AT5G15490 – 0.51 GLUCURONOSYL TRANSFERASE-LIKE PROTEIN CitGluT1 aCL8573Contig1 C02015B05 AT3G55700 – −0.54 UDP-GLUCURONATE DECARBOXYLASE CitUGluD1 aCL1799Contig2 C02011A11 AT2G28760 1.67 1.57 MANNOSYLTRANSFERASE-LIKE PROTEIN CitManT1 aIC0AAA25BC01RM1_c IC0AAA25BC01 AT2G27100 0.77 0.60 –, No significant regulation. (*) Localization of gene expression in AZ-C cells by in-situ hybridization. Putative gene identifications are based on sequence homology with Arabidopsis thaliana. Additional data are shown in Figures S3–S6. Center for Protein Sequences (MIPS). The categories sugar, glucoside, polyol, and carboxylate metabolism and polysaccharide metabolism showed a higher percentage of regulation in the AZ-C compared with the FR (Table S2). The set of genes discussed below was selected because of its prominent representation in the AZ-C or its particular biological interest. These gene families included those associated with cell wall metabolism and monolignol biosynthesis and polymerization (Table S3). Many Genes Related to Cell Wall Modification Are Regulated during Fruit Abscission A high number of genes encoding cell wall modification enzymes were differentially regulated by ethylene exclusively in the fruit AZ-C. Our data suggested that this strong activation of cell wall metabolism occurred through both degradation and biosynthesis (Table 1) as we previously showed in the laminar abscission zone (LAZ) during leaf abscission (Agustí et al., 2008, 2009, 2012). Genes encoding enzymes that hydrolyze the cell wall and Frontiers in Plant Science | www.frontiersin.org 8February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission FIGURE 4 | Phylogenetic relationships between cellulases/endo-1,4-β-glucanases (CELs) and gene expression changes in response to ethylene inAZ-C and FR cells. (A) CELs annotated in the Citrus clementina haploid genome (Wu et al., 2014), regulated by ethylene in AZ-C cells and/or FR cells of Washington Navel maturing fruits and previously described as related to the abscission process in other plant species are shown. Phylogenetic trees are based on (Continued) Frontiers in Plant Science | www.frontiersin.org 9February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission FIGURE 9 | Specific cellular and molecular events involved in the dissolution of the middle lamella, the disassembly of cell walls and the synthesis and deposition of lignin in the AZ-C during ethylene-promoted abscission. The AZ-C consists of two different cell areas, the Divided Cells Area (DCA) and the Starch-rich Area (SA). The early cellular and molecular events associated with citrus fruit abscission occur in the central core of the AZ-C between the axial vascular bundles and spread up to the calyx button periphery reaching then the floral disc. The final outcome of this cell separation process is the shedding of the fruit remaining the calyx button attached to the tree as shown in the inset of the upper-left corner of the figure. Two parallel cellular events involving cell wall dissolution and synthesis and deposition of lignin occurred specifically in the SA of the AZ-C cells during abscission. These cellular events are potentially promoted by the tissue-specific expression of particular members of several gene families that have been clearly involved in those metabolic pathways. Changes in the pectic polysaccharides distribution in the AZ-C cell walls enabled us to correlate evidences of enzymatic activity with gene expression results. In particular, based on immunodetection of partially methylesterified/de-esterified HG and expression results, we propose that PMEs CitPME24 and CitPME41 and PAE CitPAE4 may act on de-esterification of HGs in the AZ-C cell walls (Table 1,Figure S3). The activity of PMEs and PAEs is thought to facilitate the subsequent action of pectin hydrolases (Chen and Mart, 1996). Thus, the PGs CitPG43,CitPG16,CitPG20,CitPG41, and CitPG42, and the PLs CitPL5, and CitPL19 may potentially hydrolyze the HGs highly accessible after CitPME24,CitPME41, and CitPAE4 activity (Table 1,Figures 5,6,Figures S3–S5). Finally, the only α-Larabinofuranosidase identified in citrus (CitASD1) did not show significant changes in gene expression based on the statistical cutoff mentioned in materials and methods (Figure S4). In Arabidopsis, it has been reported that AtBXL1 and AtBXL3 acted as bifunctional α-L-arabinofuranosidase/β-D-xylosidases (Minic et al., 2004; Arsovski et al., 2009). Therefore, changes observed in (1, 5)-α-L-arabinans at the AZ-C might be due to the dual activity of β-XYLs such as CitBXL11 and CitBXL19, which were in the same clade as AtBXL1 and AtBXL3 (Figure S4). Frontiers in Plant Science | www.frontiersin.org 16 February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission A Set of Genes Involved in Lignin Biosynthesis and Polymerization are Regulated in the AZ-C Cells Significant expressed genes belonging to different gene families involved the monolignol biosynthesis pathway were upregulated by ethylene exclusively in the AZ-C (Table 2, Figure 8,Figure S7). These included a phenylalanine ammonialyase (CitPAL5), a p-coumarate 3-hydroxylase (CitC3H1), a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (CitHCT2), a 4-coumarate-CoA ligase-like protein (Cit4CL7), a cinnamoyl-CoA reductase-like protein (CitCCR1), and a cinnamyl alcohol dehydrogenase (CitCAD3). In addition, three genes encoding proteins involved in the oxidative coupling of monolignols and belonging to the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN family (CitCASPL1D1a, CitCASPL2B2, and CitCASPL4A4) were also up-regulated by ethylene exclusively in the AZ-C (Table 2,Figure 8, Figure S7). These results correlated with the observation of lignin deposition in the AZ-C after 24 and 48 h of ethylene/ACC treatment (Figures 1B,C) and the increase in the level of lignin intermediates detected by UPLC-MS/MS in AZ-C cells (Figure 8B). A significant increase of coumaric acid was observed at 12 h after ACC treatment. In addition, levels of caffeic acid and ferulic acid, compounds that are synthesized from coumaric acid, increased at 12 h and were maintained up to 36 h. Taken together, these data mainly reflected the activation of the H lignin pathway which results from the incorporation of p-hydroxyphenyl (H) units into the lignin polymer (Figure 8C). However, G lignin (lignin with guaiacyl units) biosynthesis would be also possible in activated AZ-C cells since an increase in CitC3H1 and CitHCT2 expression and caffeic and ferulic acids levels also occurred in AZ-C cells despite any member of the CCoAOMTs gene family were up-regulated (Figure 8). Regarding S lignin biosynthesis, four caffeic acid O-methyltransferases (CitCOMT2,CitCOMT3, CitCOMT6, and CitCOMT12) were down-regulated exclusively in AZ-C cells (Table 2,Figure 8C). CitCOMT3 was closely related in sequence to AtCOMT1 (Figure S7), a member of the Arabidopsis COMT gene family with 5-hydroxyconiferaldehyde O-methyltransferase activity that has been implicated directly in S lignin synthesis (Nakatsubo et al., 2008). These results suggest that the S lignin pathway might be inactive in AZ-C cells during fruit abscission. In woody dicotyledons, such as citrus, lignin is polymerized from mostly G and S lignin subunits (Sarkanen and Ludwig, 1971). However, lignin composition can differ among cell types (Nakashima et al., 2008; Ruel et al., 2009) and expression data suggested that cell walls of AZ-C cells might be mainly enriched in H lignin and probably also in G lignin subunits. The role of lignin deposition has been associated with the generation of protective layers at the tissues remaining in the plant during the last step of the abscission process (Addicot, 1982; Agustí et al., 2008; Van Nocker, 2009). However, it has been suggested that lignification could also facilitate the mechanical cell wall breakage during cell separation processes (Sexton, 1979; Liljegren et al., 2000). In the AZC, lignin deposition only occurred at the distal side of the AZ-C (Figure 8A). This differential deposition of lignin strongly suggests that this polymer mainly acts by generating a tension in the fracture plane to facilitate cell wall breakage during citrus fruit abscission rather than forming protective layers. CONCLUSION In this work, the isolation of specialized cell types through LM, combined with the global transcriptional analysis of ethylenepromoted AZ-C cells and the comparison with adjacent FR cells, enabled us to identify an AZ-C-exclusive gene set potentially involved in citrus fruit abscission. This set of genes includes those related to cell wall remodeling as well as lignin biosynthesis and polymerization. The combined function of these genes would enable cell wall modifications necessary for organ detachment (Figure 9). These results, together with the anatomical and morphological analysis of the AZ-C, the determination of changes in pectic polysaccharides distribution and the deposition of extracellular polymers observed in the activated AZ-C, lead to the most comprehensive characterization of citrus fruit abscission performed to date. In addition, our work shows the first classification in citrus of gene families involved in cell wall modification, which are crucial in abscission, and reveals a robust nexus between phylogenetic proximity and expression pattern during abscission in citrus and other plant species, not previously described. Therefore, this study strongly suggests that different plant species use common genes to control the abscission process. The dataset provided in this study is a highly valuable resource for guiding future functional analyses in order to answer specific abscission-related questions. In particular, those cell wall-related genes, which are evolutionarily conserved in citrus and other plant species with similar expression pattern during abscission, would represent major candidate genes for further biotechnological approaches. AUTHOR CONTRIBUTIONS PM, JA, MT, and FT conceived the survey and designed the experiments; PM performed laser microdissection of citrus tissues; VA and AG performed metabolite profiling analyses; PM, JA, and CD performed microarray experiments and analyses; MC, SC, and FT performed immunolocalization experiments; LE, MG, MP, and FT performed in situ hybridization experiments; PM, JA, MT, and FT wrote the article with contributions of all the authors; all authors discussed the results and edited the article. ACKNOWLEDGMENTS We thank E. Blázquez, I. Sanchís, A. Boix, and M. A. Argomániz for the help on all of the assays and experiments performed both in the laboratory and in the field. This study was financially supported by the Spanish Instituto Nacional de Investigaciones Agrarias (grant RTA2008-00065-00-00 to FT [including a PhD fellowship for PM] and RTA2014-00071-C06-01 to MT), the Frontiers in Plant Science | www.frontiersin.org 17 February 2017 | Volume 8 | Article 126 Merelo et al. Cell Wall Remodeling during Abscission Spanish Ministerio de Economia e Innovación (grants PSE060000-2009-8 and IPT-010000-2010-43 to MT and BIO201126302 to MP) and the Spanish Ministerio de Industria (grant AGL2011-30240 to MT). VA and CD were recipients of a “Juan de la Cierva” and an INIA/CCAA postdoctoral contract, respectively. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00126/full#supplementary-material Table S1 | Specific primers used for sqRT-PCR. Table S2 | Genes regulated by ethylene exclusively in AZ-C or FR cells, and in both AZ-C and FR cells. Table S3 | Functional categories related to polysaccharide metabolism and subcellular localization regulated in the AZ-C and the FR. Table S4 | Identification of citrus genes belonging to different families of cell wall remodeling enzymes, and monolignol biosynthesis and polymerization. Figure S1 | sqRT-PCR-based relative expression in the AZ-C and the FR. Figure S2 | LM isolation of AZ-C and FR cells. Figure S3 | Phylogenetic relationships between Carbohydrate esterases. Figure S4 | Phylogenetic relationships between Glycoside hydrolases. Figure S5 | Phylogenetic relationships between Pectate lyases. Figure S6 | Phylogenetic relationships between Expansins. Figure S7 | Phylogenetic relationships between members of gene families associated with monolignol biosynthesis and polymerization. REFERENCES Addicot, F. T. (1982). Abscission. University of California Press, Berkeley. Agustí, J., Gimeno, J., Merelo, P., Serrano, R., Cercos, M., Conesa, A., et al. (2012). 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 20 February 2017 | Volume 8 | Article 126