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

Abstract

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.

Figure 1

Effect of ethylene and 1-aminocyclopropane- 1-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.

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).

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.

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 multiple alignments of proteins using the profile alignment function of ClustalW (http://www.ch.embnet.org/software/ClustalW-XXL.html) and were generated with MEGA7 (Kumar et al., 2016) using the neighbor-joining algorithm with 1000 bootstrap replicates. Only bootstrap supports higher than 50% were considered and are shown in the nodes. Sequences are color-coded as follows: $\text{CitXXXXX}$, up-regulated exclusively in AZ-C cells; $\text{CitXXXXX}$, down-regulated exclusively in AZ-C cells; $\text{CitXXXXX}$, represented in the 20 K citrus microarray (Martinez-Godoy et al., 2008) but without hybridization results. $\text{ATXGXXXXX}$ and $\text{ATXGXXXXX}$, up- and down-regulated, respectively, in Arabidopsis stamen-AZ cells (Lashbrook and Cai, 2008); $\text{XXXXX}$, and $\text{XXXXX}$, up- and down-regulated, respectively, during AZ activation in other plant species. CEL-a1 and CEL-b1, cellulases previously identified and characterized in citrus AZs (Burns et al., 1998). $\text{LAZ}$, up-regulated in the citrus laminar AZ (LAZ) cells during ethylene- and water stress-promoted leaf abscission (Agustí et al., 2008, 2012); $\text{AZ-C tissues}$, up-regulated in citrus AZ-C and surrounding tissues (Cheng et al., 2015). Transcripts of NtCEL5 were down-regulated in the corolla base of tobacco plants over-expressing an antisense-oriented sequence of NtBOP2 ($\mathit{\text{NtBOP2-AS}}$; Wu et al., 2012). Transcripts of AtGH9B3 and AtGH9B4 were down-regulated in receptacles of $\mathit{\text{ida-2}}$ mutant plants (Liu et al., 2013) and $\mathit{\text{hae-2/hsl2-3}}$ double mutant plants (Niederhuth et al., 2013) while transcripts of AtGH9B17 were down-regulated only in receptacles of hae-2/hsl2-3 double mutant plants (Niederhuth et al., 2013). (B,C) CitCEL6 expression by in situ hybridization in the AZ-C of Ricalate Navel fruits after 24 h of ACC treatment (B, sense probe; C, anti-sense probe). Hybridization is indicated by the presence of a dark purple precipitate (). AZ-C, abscission zone C; FR, fruit rind. Scale bars: 500 μm.

Figure 5

Phylogenetic relationships between polygalacturonases (PGs) of the Group A (GH28) and gene expression changes in response to ethylene in AZ-C and FR cells. PGs of the Group A located at the Citrus clementina haploid genome, 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 Arabidopsis (Lashbrook and Cai, 2008), tomato (Kalaitzis et al., ; Li et al., ; Hong and Tucker, 1998), apple (Atkinson et al., ; Li and Yuan, 2008), oilseed rape (Petersen et al., ; Sander et al., ; Gonzalez-Carranza et al., 2002), melon (Hadfield et al., 1998), oil palm (Roongsattham et al., 2012), and banana (Mbéguié-a-Mbéguié et al., 2009) are shown. Phylogenetic trees are based on multiple alignments of proteins using the profile alignment function of ClustalW (http://www.ch.embnet.org/software/ClustalW-XXL.html) and were generated with MEGA7 (Kumar et al., 2016) using the neighbor-joining algorithm with 1000 bootstrap replicates. Only bootstrap supports higher than 50% were considered and are shown in the nodes. Sequences are color-coded as follows: $\text{CitXXXXX}$, up-regulated exclusively in AZ-C cells; $\text{CitXXXXX}$, localization of transcripts in the citrus fruit AZ-C by in situ hybridization; $\text{CitXXXXX}$, represented in the 20 K citrus microarray (Martinez-Godoy et al., 2008) but without hybridization results. $\text{AZ-C tissues}$, up-regulated in citrus AZ-C and surrounding tissues (Cheng et al., 2015). $\text{ATXGXXXXX}$, up-regulated in Arabidopsis stamen-AZ cells (Lashbrook and Cai, 2008); $\text{XXXXX}$, up-regulated during AZ activation in other plant species. Transcripts of ADPG2 (AT2G41850) and QRT2 (AT3G07970) were down-regulated in receptacles of $\mathit{\text{ida-2}}$ mutant plants (Liu et al., 2013) and hae-2/hsl2-3 double mutant plants (Niederhuth et al., 2013) while transcripts of AT2G43880, AT2G43890 and AT3G59850 were down-regulated only in receptacles of $\mathit{\text{hae-2/hsl2-3}}$ double mutant plants (Niederhuth et al., 2013). (B,C) CitPG20 expression by in situ hybridization in the AZ-C of Ricalate Navel fruits after 24 h of ACC treatment (B, sense probe; C, anti-sense probe). Hybridization is indicated by the presence of a dark purple precipitate (). AZ-C, abscission zone C; FR, fruit rind. Scale bars: 500 μm.

Figure 6

Phylogenetic relationships between PGs from Groups B and C (GH28) and gene expression changes in response to ethylene in AZ-C and FR cells. PGs of the Groups B and C located at the Citrus clementina haploid genome, 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 Arabidopsis (Lashbrook and Cai, 2008), soybean (Tucker et al., 2007), and lychee (Peng et al., 2013). Phylogenetic trees are based on multiple alignments of proteins using the profile alignment function of ClustalW (http://www.ch.embnet.org/software/ClustalW-XXL.html) and were generated with MEGA7 (Kumar et al., 2016) using the neighbor-joining algorithm with 1000 bootstrap replicates. Only bootstrap supports higher than 50% were considered and are shown in the nodes. Sequences are color-coded as follows: $\text{CitXXXXX}$, up-regulated exclusively in AZ-C cells; $\text{CitXXXXX}$, down-regulated by exclusively in AZ-C cells; $\text{CitXXXXX}$, up-regulated in both AZ-C and fruit rind (FR) cells; $\text{CitXXXXX}$, down-regulated exclusively in FR cells. $\text{ATXGXXXXX}$, down-regulated in Arabidopsis stamen-AZ cells (Lashbrook and Cai, 2008); $\text{XXXXX}$, up-regulated during AZ activation in other plant species. $\text{LAZ}$, preferentially expressed in the citrus laminar AZ (LAZ) cells during ethylene-promoted leaf abscission (Agustí et al., 2009); Petiole, preferentially expressed in the citrus leaf petiolar cells during ethylene-promoted leaf abscission (Agustí et al., 2009); $\text{AZ-C tissues}$, up-regulated in citrus AZ-C and surrounding tissues (Cheng et al., 2015). Transcripts of AT4G23500 were down-regulated in receptacles of $\mathit{\text{ida-2}}$ mutant plants (Liu et al., 2013) and $\mathit{\text{hae-2/hsl2-3}}$ double mutant plants (Niederhuth et al., 2013).

Figure 7

Immunolocalization of pectic polysaccharides in the AZ-C. Longitudinal sections of tissue containing the AZ-C of Ricalate Navel maturing fruits were incubated with the monoclonal antibodies (mAb) LM5 (A,E,H,K), LM6 (B,F,I,L), and JIM5 (C,G,J,M) to detect (1,4)-β-D-galactans, (1,5)-α-L-arabinans and partially methylesterified/de-esterified HGs, respectively, after 0 (A, B and C), 24 (E,F,G) and 48 h (H–M) of ACC treatment. Control did not show immunofluorescence (D). Scale bars: 5 μm. Key labeling: walls at the two AZ-C cell areas (divided cells area [DCA] and starch-rich area [SA]) and at the fruit rind (FR) cell layers just below the SA showing fluorescence due to each of the mAbs after 0 (), 24 () or 48 () h of ACC treatment. Micrographs represent the merger of images from pectic epitopes detection by mAbs (green) and from cellulose detection by calcofluor white (blue).

Figure 8

Lignin biosynthesis and deposition in the abscission zone area during citrus fruit abscission. (A) Tissue localization of lignin through phloroglucinol-HCl staining in longitudinal sections of the AZ-C from Washington Navel fruits treated for 48 h with ethylene. Lignin is deposited at the central core of the AZ-C, at the separation line, and spreads to the adjacent cells of the fruit rind through the distal side of the AZ-C. Scale bar: 500 μm. Key labeling: AZ-C, abscission zone C; FR, fruit rind; VB, vascular bundles. (B) Lignin biosynthesis intermediates were quantified through UPLC-MS/MS in AZ-C cells at 0, 12, 24, and 36 h after ACC treatment. Data are expressed as ng of coumaric acid, caffeic acid and ferulic acid per mm³ of microdissected tissue. The results are means of three independent samples containing ~40,000 pooled AZ-C cells ± SE. (C) Genes belonging to the general phenylpropanoid and monolignol biosynthesis pathways and lignin polymerization up- $\text{CitXXXXX}$ or down-regulated $\text{CitXXXXX}$ exclusively in the fruit AZ-C cells during ethylene-promoted citrus fruit abscission. Enzymes and proteins associated with monolignol biosynthesis and polymerization are: phenylalanine ammonia lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT), coniferaldehyde dehydrogenase/sinapaldehyde dehydrogenase (CALDH/SALDH), caffeoyl shikimate esterase (CSE), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), cinnamyl alcohol dehydrogenase (CAD), Casparian strip membrane domain protein-like (CASPL), laccase (LAC) and peroxidase (PRX).

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.