Forty-one randomly selected genes were analyzed by quantitative real-time PCR to verify their expression patterns (**Figure 2**). Among these, transcripts encoding the key enzymes involved in ethylene biosynthesis and signaling, such as ACS (L10030076), ACO (L10037576), ETR2 (U20306), EBF (U575), EIN3/EIL (U23712) and ERF (L10026303, L10050388, C2959.2, and U6276), were sharply increased at 1 d after treatment, which were consistent with the results analyzed by DTA (**Figure 2**).

TABLE 1 | Statistics of digital transcript abundance library sequencing and read mapping.

\*Number and percentage of reads mapped onto litchi genome (unpublished data). CK and ETH mean the untreated control and ethephon treatment, the numbers following the CK and ETH are the days after treatment.

Similarly, we verified the increased expression of genes encoding those enzymes related to cell wall degradation, TFs, cytokinin metabolism and ROS production, such as BGLU (U21052), BXL (L10010659), XTH (C5711.4 and U17815), PE/PEI (C5841.2, L10049394, and U1533), PG (L10048763), HD-ZIP (U10783), NAC (U13457), WRKY (U18045 and U9115), CKX (L10048833) and Rboh (L10001757), as well as the down-regulated genes involved in auxin transport and signaling, cytokinin signaling, ROS scavenging and calcium signaling, such as PIN (U11774), AUX1 (U9940), Aux/IAA (C3897.1 and U9140), SAUR (C5333.1), ILR1 (U1508), AHK (U11693), AHP (C4349.2), ARRA (C3809.1), POD (U20379 and U15492), AO (U10993), and CML (U2171 and U4839). The expression level of one auxin early responsive gene (GH3, C4818.1) and one calcium signaling gene (CML, U1498) were increased by ETH treatment, which was identical to the DTA results. One calcium transport gene (CNGC, L10064767) showed down-regulated at 1 d after treatment and increased at 3 d after treatment (**Figure 2**). However, the expression level of one gene encoding auxin response factor (ARF, L10058057) was contrasted to the DTA result (**Figure 2**). Thus, except one ARF gene, all the other 40 genes showed similar expression patterns as detected by the DTA, indicating that the DTA analysis results were effective.

# Transcirptome Responses during Fruit Abscission

After comparing three paired-libraries (CK1 vs. ETH1, CK2 vs. ETH2, and CK3 vs. ETH3), a total of 6167 genes were found to be significantly up- or down-regulated which named as ethephon-responsive genes (**Supplementary Data Excel File 2**). Based on GO enrichment analysis, 3249 genes (53%) were divided into the three principal GO organization categories (**Supplementary Figure 2A**): biological process (2437 genes), cellular component (1459 genes) and molecular function (2825 genes). The enriched GO terms included carbohydrate metabolic process, photosynthesis, extracellular region, cell wall, thylakoid, catalytic activity and oxygen binding. KEGG pathway enrichment analyses showed that a total of 3420 genes were assigned to the 118 related pathways, and the top 20 enriched pathways including plant hormone signal transduction (ko04075) and starch and sucrose metabolism (ko00500) were illustrated in **Supplementary Figure 2B**.

# Analysis of the Candidate Genes Involved in the Fruit Abscission Process

A total of 2471 and 2344 genes were selected in GO (FDR ≤ 0.05) and KEGG enrichment analyses (Q-value ≤ 0.05) with a significant level, respectively. After eliminating duplicated, 2730 genes were identified as the candidate genes involved in the fruit abscission process regulated by ethephon (**Supplementary Data Excel File 3**). The up- and down-regulated genes accounted for 37.44% (1022 genes) and 62.56% (1708 genes) of them, respectively.

Based on their patterns of expression, these candidate genes could be classified into four groups, which consisted of genes with similar temporal patterns of expression kinetics (**Figure 3**). Group I included 1867 early-responsive genes whose expression were up- or down-regulated early at 1 d after treatment; Group II had 148 middle-responsive genes whose expression were not induced or suppressed until 2 d after treatment; Group III contained 258 late-responsive genes that were not regulated until 3 d after treatment; Group IV consisted of 457 constant-responsive genes that up- or down-regulated early and whose expression was maintained constant during the treatment. By hierarchical cluster analysis, each group could be subsequently divided into two to six clusters, for example, Group I included cluster 1A, 1B, 1C, 1D, 1E, and 1F which had 172, 261, 55, 776, 434, and 169 genes, respectively. In total, 723 up-regulated and 1601 down-regulated genes were found at 1 d after treatment, and 299 up-regulated and 107 down-regulated genes were found at 2 d or 3 d after treatment. These results showed that the majority (85.13%) of those candidate genes made a quick response to the ETH treatment in 24 h when no significant difference on fruit abscission rate was found between the control and the ETH treatment.

Except 101 genes encoding proteins of unknown functions, the other 2629 genes had unambiguous annotations. Those genes related to plant hormones, cell wall metabolism TFs, carbohydrate metabolism, ROS and calcium signaling were further analyzed as follows.

## Genes Related to Plant Hormone Biosynthesis and Signaling Pathway

A total of 195 candidate genes were found related to plant hormone biosynthesis and signaling pathway (**Figure 4**, **Supplementary Data Excel File 4**). Of these, 124, 16, 17, and 38 genes belonged to the Group I, Group II, Group III, and Group IV, respectively. Sixty and fifty-five genes were related to auxin and ethylene, among them, 47 auxin-related genes were down-regulated and 39 ethylene-related genes were up-regulated. These genes should be closely associated with fruitlet abscission, including those encoding auxin efflux carrier component (PIN), auxin influx carrier (AUX1), AUX/IAA protein, auxin response factor (ARF), SAUR family protein, GH3 protein, 1-aminocyclopropane-1-carboxylate oxidase (ACO), 1-aminocyclopropane-1-carboxylate synthase (ACS), AP2/ERF transcription factor and ethylene receptor (ETR), et cetera. Twenty-eight gibberellins (GA) related and 18 cytokinin-related genes were found, most of them were down-regulated, such as genes encoding gibberellin 20 oxidase (GA20ox), gibberellin receptor GID1, cytokinin hydroxylase (CYP735A), histidine kinase (AHK) and two-component response regulator (AARA), etc. . . Eight of eighteen abscisic acid (ABA) related genes were upregulated including those encoding zeaxanthin epoxidase (ZEP), abscisic acid receptor (PYR/PYL) and protein phosphatase 2C (PP2C), and the repressed genes mainly encoded 9 cis-epoxycarotenoid dioxygenase (NCED), carotenoid cleavage dioxygenase (CCD), and abscisic acid 8′ -hydroxylase (CYP707). Moreover, six salicylic acid (SA) related genes encoded with salicylic acid-binding protein (SBP) and regulatory protein NPR1 were highly up-regulated, and five jasmonic acid (JA) related genes mainly encoded allene oxide cyclise (AOC) and jasmonate ZIM domain-containing protein (JAZ) were decreased. These results suggested that seven classes of plant hormones were involved in the process of fruitlet abscission induced by the ETH treatment. The most important hormones were ethylene and auxin, followed by GA, cytokinin and ABA. JA and SA ranked the last according to the number of DEGs.

# Genes Encoding for Transcription Factors

Except for 58 genes putatively encoding TFs related to plant hormones, there were 127 candidate TFs belonged to diverse families including ABI3/VP1, bHLH, BLH1, bZIP, GRAS, HD-ZIP, HSF, KNOX, LBD, MADS-box, MYB, NAC, WRKY, Trihelix, and zinc finger (**Figure 5**, **Supplementary Data Excel File 5**). Of these, 51 and 76 genes were up- and down-regulated, respectively. And 78, 9, 14, and 26 belonged to Group I, Group II, Group III and Group IV, respectively. Remarkably, 71 out of 114 TFs in Group I and Group IV were down-regulated, indicating that most TFs were repressed at 1 d after the ETH treatment. There were more than 10 members in those families including bHLH, MYB, WRKY, NAC, LBD, and HD-ZIP. Most members of bHLH and HD-ZIP families were down-regulated, while 13 of 15 members in the family of WRKY were up-regulated, and all members in the families of BLH1, MADS-box, KNOX, and Trihelix were down-regulated. Moreover, 33 of the 51 up-regulated TFs were induced at 1 d after treatment, and the largest TF family was the WRKY (10 genes), followed by MYB (6 genes) and NAC (5 genes), implying that those genes from these TF families could be related with triggering the transcriptional chain reaction during ETH-induced abscission.

# Genes Related to Cell Wall Biosynthesis, Degradation, Loosening, and Modification

A total of 208 genes including 56, 104, 21, and 27 genes related to cell wall biosynthesis, degradation, loosing and modification were found, respectively (**Supplementary Data Excel File 6**). Of these, 72 and 136 genes were up- and down-regulated, respectively. Of the up-regulated genes, there were 51 cell wall degradation, loosing and modification related transcripts

(**Figure 6**). Among them, 4 genes were related to callose degradation, like endo-1,3-β-glucosidases (ENGs) and β-1,3 glucanases (BGN13s); 5 genes were involved in cellulose degradation, such as endo-1,4-β-D-glucanases (CELs) and βglucosidases (BGLUs); 11 genes like endo-1,4-β-mannosidase (MAN), xyloglucan endotransglucosylase/hydrolases (XTHs) and β-D-xylosidases (BXLs) were related to hemicellulose degradation; 13 genes such as polygalacturonases (PGs) and pectate lyase (PLs) were associated with pectin degradation; 12 expansins (EXPs) related to cell wall loosening and 6 pectinesterase/pectinesterase inhibitors (PE/PEIs) genes were involved in cell wall modification. Of the down-regulated genes, there were 35 cell wall biosynthesis-related genes (**Figure 6**), including those encoding cellulose synthase, extensin, glycinerich cell wall structural protein, UDP-glucuronate 4-epimerase and xyloglucan glycosyltransferase. There were not surprised

gene-expression level in clusters is shown by the red solid lines.

that those genes encoding enzymes mentioned above may be involved in the process of fruitlet abscission induced by ethephon. However, 101 down-regulated genes related to cell wall degradation, loosening and modification, and 21 up-regulated genes related to cell wall biosynthesis were also found in our study.

# Genes Related to Photosynthesis, Carbohydrate, and Energy Metabolism

The expression of over 93% (103 out of 110 genes) genes involved in photosynthesis pathway were strongly decreased, especially repressed exclusively at 1 day after the ETH treatment, and most of them belonged to Group I (**Supplementary Data Excel File 7**).

The affected genes function in chlorophyll biosynthesis, thylakoid formation, chlorophyll a/b binding protein, light harvesting (PSI and PSII), electron transport and carbon fixation. These results suggested repressed expression of those encoding for chloroplast and light harvesting associated proteins by ethephon might lead to the process of chloroplast malfunction and photosynthesis inhibition.

Not surprisingly, the down-regulated photosynthesis related genes are linked with changes in the expression of genes in carbohydrate metabolism. A total of 137 candidate genes were found in this section (58 up-regulated and 79 down-regulated, **Supplementary Data Excel File 7**). Overall, 24 out of 32 genes involved in glycolysis and all members of the TCA cycle (7 genes), such as alcohol dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase and ATP-citrate synthase, were strongly downregulated within Group I, indicating that the degradation of sugar was inhibited during the early ETH-induced abscission. However, an increased expression of seven genes associated with fructose and mannose degradation and four genes involved in galactose hydrolysis were also found during the late induction of abscission. These results suggested that sugar degradation was inhibited during the early induction but increased during the late induction. Moreover, our data also showed that 13 out of 17 genes encoding ATPase were repressed after the ETH treatment, especially down-regulated exclusively at 1 day post-treatment (**Supplementary Data Excel File 7**). These results indicated that ethephon might lead to the interdiction of ATP synthesis and the high energy supply.

# Genes Related to ROS Response, Calcium Signal Transduction, and Protein Ubiquitination

A total of 88 transcripts belonging to the reactive oxygen species (ROS) response were preferentially regulated in the ETH-treated FAZ-enriched pedicels (**Supplementary Data Excel File 8**). Of these, 39 genes were up-regulated and the other 49 genes were down-regulated. Among the up-regulated genes (**Figure 6**), we found eight respiratory burst oxidase homolog proteins (Rboh) and one peroxisomal-(S)-2-hydroxy-acid oxidase (GLO) involved in ROS production, indicating a high ROS content might be produced in FAZ. Not surprisingly, 27 genes putatively encoding ROS scavenging enzymes of diverse families showed increased expression, and the most abundant transcripts encoding glutathione S-transferase (GST) and peroxidase (POD). However, the down-regulated ROS scavenging genes were also found, indicating that a competing activity occurred in the regulation of fruit abscission. In addition, 20 genes encoding the key enzymes related to ascorbate synthesis and metabolism, including L-ascorbate oxidase (AO) and inositol oxygenase (MIOX), showed an immediate decreased regulation at the 1 day post-treatment.

As an important signaling molecular, 52 transcripts related to calcium transport and perception displayed altered changes (**Figure 6**, **Supplementary Data Excel File 8**). Among them, 19 and 33 genes were up- and down-regulated respectively, and 46 of them belonged to Group I, indicating that most genes had instantaneous response to the ETH treatment. For example, two calcium influx transporters, CNGC (13 out of 18 genes) and ANX (2 genes), showed strongly decreased expression during the early induction. In contrast, 4 out of 6 transcripts encoding calcium efflux carrier proteins (PMCAs) were increased. These results indicated that a role regulating calcium influx and maintaining calcium level in the FAZ may be associated with the beginning of abscission process. Moreover, two calcium responsive genes, CML (6 out of 10 genes) and CDPK (3 genes), were also repressed during the early induction.

A total of 40 genes involved in protein ubiquitination were found (**Figure 6**, **Supplementary Data Excel File 8**), and 32 of them belonged to Group I and IV, indicating that most genes had a quick response to the ETH treatment. Among them, 26 genes were up-regulated, including those encoding 26S proteasome, cullin, E3 ubiquitin-protein ligase, EIN3-binding f-box protein, U-box domain-containing protein and ubiquitin-conjugating enzyme E2. However, 14 down-regulated genes involved in protein ubiquitination were also found.

# Discussion

Ethephon (2-chloroethylphosphonic acid) is a synthetic plant growth regulator discovered several decades ago, which acts by releasing ethylene when it penetrates plant tissues (Royer et al., 2006). The application of exogenous ethylene inducing fruit abscission has been observed in apple (Yuan, 2007; Kolaricˇ et al., 2011), sweet orange (John-Karuppiah and Burns, 2010), sweet cherry (Smith and Whiting, 2010) and olive (Zahra, 2014). The data herein presented strengthen the direct observation that ethephon aggrandizes both the fruitlet abscission and ethylene

Supplementary Data Excel File 4.

degradation, cell wall loosening and ROS response, and

evolution in litchi. It is speculated that the mechanism of fruitlet abscission after treatment with exogenous ethephon may relate to the action of ethylene. However, a deep knowledge of the molecular events occurring in FAZ during fruit abscission induced by ethephon is still unknown. Although our previous study cloned a LcPG1 gene and found its expression in FAZ was paralleled with the alteration of fruitlet abscission in litchi, induced by the ethephon treatment and inhibited by spraying 2,4-dichlorophenoxyacetic acid (2,4-D) (Peng et al., 2013). So this work mainly focused on genome-wide mining putative fruit abscission related genes regulated by ethylene in litchi.

Litchi fruit growth could be divided into two stages (Li et al., 2003). The first Stage constitutes about two thirds of the whole fruit growth cycle, which is the phase mainly characterized by the growth of pericarp and seedcoat; and the second Stage is the phase mainly characterized by the growth of embryo and the rapid aril growth. In the case of "Feizixiao" litchi used in our study, it needs 70–75 days from female flowering to a mature fruit, which can fluctuate some depending on the female opening date (Li et al., 2004). Fruit weight is about 25–30 g at maturity, but it is approximately 1.0 g when fruit develops at 25 days post anthesis (DPA) which is the time of the ETH treatment in this study. There were three to four waves of physiological fruit drop throughout fruit development in 70–90 days depending on cultivars (Yuan and Huang, 1988). Wave I, wave II, and wave III of abscission occurred around 1 week, 3 weeks and 6 weeks after anthesis, respectively. For a normal inflorescence of "Feizixiao" litchi, there are about 500–800 female flowers. Only 10% of them may set fruit successfully at 1 week after anthesis (wave I), and 30–50% of the surviving fruitlet will drop during wave II, after which no abscission will occur for the next 2–3 weeks. This study focuses on fruitlet abscission occurred at the second wave. The first 3 days after treatment might be coincidence with the peak of fruit drop wave II. It is not surprising that ∼50% of the control fruitlet abscised between 25 and 28 DPA. Moreover, after 28 DPA, the remaining 50% of the fruitlet in the control treatment did not abscise over the next 2–3 weeks. ETH treatment, however, induced abscission of 100% of the fruitlet by 28 DPA. Thus, the ETH treatment largely magnified the second physiological fruit drop, which inducing a significantly higher rate of fruitlet abscission. This is exactly the biological effect expected for ETH. Moreover, ethylene production in fruit between the control and the ETH treatment had the substantial difference. Ethylene production in the control fruitlet remained more or less flat and kept below 8µl·kg−1 ·h −1 in the period of treatment, while which in ETH-treated fruitlet increased rapidly at 1 d after treatment and achieved nearly a ten-fold higher at 3 d when compared with the control. When the pH is above 4.0, ethephon slowly decomposes to release ethylene. The increase in ethylene production was the results of endogenous synthesis and ethephon release, which probably accelerates the fruit drop. In addition, the CFAR had no difference with the control in the first day after the ETH treatment, after then, the CFAR was sharply increased and significantly higher than that of the control. There were two obvious stages during the fruitlet abscission induced by ethephon: the early induction (0–1 d after treatment) that might induce acquisition of ethylene sensitivity and abscission competence, and the late induction (1–3 d after treatment) that might lead to the execution of fruitlet abscission and formation of the defense layer.

A total of 6167 significantly DEGs were screened as ethyleneresponsive genes and 2730 of them were identified as candidate genes involved in the fruitlet abscission process. Over 85% of the candidate genes displayed a significant transient change during the early ethylene-induction. It is generally accepted that ethylene operates as an activator, while auxin act as retardants (Roberts et al., 2002). In agreement with this supposition, 115 out of 195 candidate hormone related genes were involved in biosynthesis and signaling pathway of ethylene and auxin. These evidences were supported by the high expression levels of ethylene signal pathway related genes such as ETR2, EBF, EIN3/EIL and a class of ERF TFs, as previously demonstrated by John-Karuppiah and Burns (2010) in sweet orange fruit and leave abscission zone; and the repressed expression of transcript levels for auxin polar transport carriers (PIN and AUX1) and auxin responsive genes (TIR1, Aux/IAA, ARF, and SAUR). A decline in the abundance of auxin efflux carrier might be responsible for fruitlet abscission induced by shading and NAA in apple (Zhu et al., 2011) and mature-fruit abscission in melon (Corbacho et al., 2013). Moreover, Zhu et al. (2011) found that genes involved with cytokinin and gibberellic acid (GA) signaling pathways were down-regulated by shading and NAA in apple fruitlet FAZ. Similarly, a strongly decreased expression of large number of genes related to cytokinin and GA biosynthesis and signaling, such as CYP735A, AHK, AHP, GA20ox, and GID1, were also found in our study, probably implying that the metabolism of the cytokinin and GA in FAZ were affected in the early ethephon-promoted abscission process. On the other hand, it has been proposed that ABA and JA might be correlated with the abscission activation in citrus fruitlet (Gomez-Cadenas et al., 2000) or leaves (Agustí et al., 2009). They exhibited exactly the opposite results on mature fruit abscission, six of eight DEGs involved in ABA biosynthesis were up-regulated in melon (Corbacho et al., 2013), and six out of the seven DEGs were down-regulated in olive (Gil-Amado and Gomez-Jimenez, 2013). Our result was quite different from them, half of the eight DEGs involved in ABA biosynthesis showed increased transcript abundance during the ethephon-promoted fruitlet abscission in this study. All together, the mentioned above plant hormones were involved in the process of fruitlet abscission induced by the ETH treatment and the most important hormones were ethylene and auxin.

TFs are concerned as major switches of regulatory cascades during development, and the changes in the expression of such genes may affect various biological processes (Riechmann et al., 2000). A total of 185 different TF genes transcript such as AP2/ERF, Aux/IAA, bHLH, MYB, WRKY, NAC, LBD, and HD-ZIP, were affected by the ETH treatment. Those thought to be directly involved in ethylene and auxin signal transduction, such as AP2/ERF, Aux/IAA, and ARF, were already discussed before. The expression of most genes belonging to the family of bHLH was sharply down-regulation, and it was consist with the reports in the flower abscission zone in tomato after flower removal (Meir et al., 2010). Other up-regulated TFs in our work such as NAC and WRKY, were previously reported to be involved in mature fruit abscission in melon and olive (Corbacho et al., 2013; Gil-Amado and Gomez-Jimenez, 2013). Many reports have shown that numerous characterized NAC and WRKY genes are involved in response to environmental stimuli and play various roles in response to biotic and abiotic stress (He et al., 2005; Jensen et al., 2010; Zhao et al., 2012). Thus, these up-regulated NAC and WRKY genes might be similar to ethylene- or stressinduced TFs found in other species (Yang et al., 2009; Jensen et al., 2010) and putatively involved in the downstream of ethylene signaling. Concerning MYB, Corbacho et al. (2013) reported that MYB was the most represent up-regulated TFs during the late mature-fruit abscission in melon, while only 7 members of the 15 affected MYB genes were induced in litchi. All these differential expression of genes encoding TFs belonging to different families might act as early regulators of the abscission induction, but the exact roles of these regulatory factors responding to ethylene induction remain to be further investigated.

ROS are versatile molecules related to a wide range of cellular processes, including programmed cell death, development, and hormonal signaling (Kwak et al., 2006). Previously, some reports supported a link between ROS and abscission. In tobacco, Henry et al. (1974) found that POD activity was increased during the ethylene-induced pedicel abscission. In tomato, delayed abscission of flowers and fruits was related to the increase of ROS-scavenging enzymes (Djanaguiraman et al., 2004). In pepper, Sakamoto et al. (2008) reported that H2O2 was involved in stress-induced petioles abscission, indicating that H2O2 acts downstream abscission signaling from ethylene. In ethylenetreated citrus leaves, a set of transcripts belonging to the ROS scavenging machinery have been reported to be over-represented in petioles rather than the laminar abscission zone (Agustí et al., 2008, 2009). Results in this study showed that a number of Rboh (role in ROS production) and genes encoding ROS scavenging enzymes were induced, suggesting the burst of ROS caused by ethephon treatment.

Calcium has been considered as an important intracellular messenger in plants and is essential for the maintenance of structural integrity of biomembrane and cell wall (Poovaiah and Rasmussen, 1973), and is also required for a variety of ethylene-dependent abscission processes (Raz and Fluhr, 1992). Xu et al. (2009) reported that direct application of calcium on tomato pedicel explants under ethylene would accelerate abscission but there are a number of reports describing that calcium delayed organ abscission (Poovaiah and Leopold, 1973; Beyer and Quebedeaux, 1974; Iwahori and Van Steveninck, 1988). In fact, Poovaiah and Rasmussen (1973) showed that ethephon treatment for bean leaf explants decreased calcium level in the AZ just prior to separation. The role of this element in organ abscission is still controversial. But how does the calcium signaling communicate with the fruit abscission? Our results showed that ethephon treatment up-regulated genes encoding calcium efflux carrier proteins (PMCA), down-regulated different genes encoding calcium influx carrier proteins such as cyclic nucleotide-gated channel (CNGC) genes and calcium responsive genes (CML and CDPK) in the litchi FAZ-enriched pedicel. These molecular results strongly suggested that regulating calcium influx and maintaining calcium level in the FAZ might be associated with the onset of ethephon-promoted litchi fruitlet abscission process. It is speculated that ethylene might lead to a high extracellular calcium level in FAZ, and result in the deposition of calcium on cell wall and the deficiency on the cell inside.

Ubiquitylation-dependent proteolysis is a major event during both the induction and execution of cell death (Estelle, 2001), and could be triggered by H2O2 in tobacco (Vandenabeele et al., 2003). In ethylene-treated citrus leaves, a group of transcripts involved in the ubiquitin/proteasome system have been reported to be induced in both laminar abscission zone and petiolar cortical tissue (Agustí et al., 2008, 2009). The up-regulation of both ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) after the ETH treatment in litchi FAZ-enriched pedicel, suggested that a similar proteolytic mechanism might be involved in ethephon-induced abscission. Also, two 26S proteasome components and a large number of proteins with Fbox and U-box domain like potential E3 ligases were induced by ethephon. In Arabidopsis, abscission of floral organs is arrested with suppressed expression of a F-box protein (González-Carranza et al., 2007). These observations strongly suggested that a general proteasome-related mechanism might play a role in ethephon-induced abscission.

Zhu et al. (2011) found both shading and NAA treatment for apple tree resulted in a large number of photosynthesisrelated genes were down-regulated in the FAZ. For instance, 90 out of the 94 DEGs were repressed in shading-treated FAZ. In our study, 103 out of the 110 DEGs involved in photosynthesis pathway were strongly decreased. These results also indicated that photosynthesis was one of the GO terms enriched in FAZ. The structural features of AZ cells were described by Sexton and Roberts (1982) as densely protoplasmic, with small intercellular spaces, containing large deposits of starch, and with a high density of branched plasmodesmata. Then, AZ cells should not contain photosynthetically active chloroplasts. However, handdissected AZ-enriched young and green fruitlet pedicle (see **Supplementary Figure 1**) used in our study should contain photosynthetically active chloroplasts, which is why so many down-regulated of photosynthesis-related genes were found. In order to uncover the abscission-associated metabolism of AZ cells we should take a laser capture microdissection (LCM) approach to obtain AZ- specific cells sample for performing an accurate study of the abscission events in the future. Thus, the roles of these genes on fruit abscission need to be further evaluated.

Some evidences supported a strong connection between the carbohydrate amounts available for the fruit and their probability of abscission (Yuan and Huang, 1988; Iglesias et al., 2003; Zhou et al., 2008). One of the reasons of abscission may be due to a lack of carbohydrate. Not surprisingly, ETH treatment also affected the carbohydrate and energy metabolism. Affected genes within these groups include those associated with glycolysis, TCA cycle and ATPase. These results indicated that ethephon might lead to the carbohydrate stress and the interdiction of ATP synthesis during the early abscission induction. Moreover, an increased ROS production may be linked to the inhibition of ATPase, as a consequence to the mitochondrial damage (Roy et al., 2008), and the release of cytochrome c from mitochondria, which have been implicated as regulator of programmed cell death (Tiwari et al., 2002).

After the early induction, there was a lot of fruitlet dropped in paralleled with a continuous highly release of ethylene yield during the late induction (1–3 d after ETH treatment). Ethylene may act as the signal generated within the fruit, FAZ, or released from ethephon, through diffusion, triggering abscission event. It was supported by the abundant of several transcripts (ACS, ACO, ERF) involved in ethylene biosynthesis and transductive pathway. Several up-regulated genes were linked to ABA biosynthesis and signaling transduction, such as ZEP and PP2C. It is proposed that ABA might be corrected with the late stage of abscission process. Similarly, ethylene appeared to be a positive regulator of SA action during the abscission induction, since the expression level of SBP and NPR1 genes were highly up-regulated, as found during melon mature-fruit abscission (Corbacho et al., 2013). It assumed that SA might be as an endogenous signaling molecule for stress response or the formation of protective layer after AZ cell separation. Homologs of LBD and WRKY TFs were also highly expressed at 1–3 d after ETH treatment, showing similar change to that reported in tomato flower abscission zone (Wang et al., 2013). The LBD family played a possible role in lateral meristem initiation (Shuai et al., 2002), while the WRKY family hold central positions mediating the regulation of disease resistance (Robatzek and Somssich, 2001). These TFs might have functions in stress defense and the formation of protective layers after fruitlet abscission. For fruit to be shed, cell separation must occur in FAZ, and the abscission is paralleled with intercellular space increase and middle lamella lysis in the FAZ which is the result of the degradation and/or remodeling of cell wall (Lee et al., 2008; Bowling and Vaughn, 2011). There were 43 cell wall degradation and loosing related transcripts up-regulated and 29 cell wall biosynthesis related genes down-regulated, and most

of them had significantly differential expression throughout the whole ethephon treatment, indicating that these genes might be involved in the process of fruitlet abscission induced by ethephon. Several researchers have already reported that the expression of genes encoding for cell wall-hydrolyzing enzymes was associated with abscission and also regulated by ethylene (Lashbrook et al., 1994; Kalaitzis et al., 1997; Roberts and Gonzalez-Carranza, 2009). However, 92 down-regulated genes related to cell wall degradation and loosing, and 15 up-regulated genes related to cell wall biosynthesis were also found in our study, which might be involved in the new cell wall synthesis and reconstruction for the formation of protective layers after fruitlet abscission.

This study only focus on the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon. In conclusion, a total of 2730 candidate genes were involved in the process of litchi fruit abscission induced by ethephon treatment. A preliminary molecular regulatory scheme was herein prompted out for litchi fruitlet abscission induced by ethephon based on our results (**Figure 7**). At the early beginning, the ethylene evolution in fruitlet was greatly increased by ETH treatment, which would suppress the synthesis and polar transport of auxin and trigger abscission signaling. At the same time, FAZ might perceive the abscission signals, and then, 1867 early-responsive genes were up- or down-regulated from 0 to 1 d after ETH treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, TFs, protein ubiquitination, ROS response, calcium signal transduction and etc. . . Then, a lot of genes related to cell wall degradation, TFs, ethylene, ABA and JA biosynthesis and signaling cascade were upregulated. At the last, cell separation happened and the fruitlet abscission was enhanced. To the best of our knowledge, this study provides the first global monitoring of gene expression changes occurring in FAZ-enriched pedicel during litchi fruit abscission.

# Author Contributions

JL was responsible for the overall concept and experimental design, and revising and finalizing the manuscript. CL carried out ethephon treatment, DTA data integration and analysis, performed qRT-PCR experiments, and drafted the manuscript. YW was responsible for bioinformatics analysis. PY and WM performed the ethephon treatment and sample collection. All the authors read and approved the final manuscript.

# References

This work was supported by the National Natural Science Foundation of China (31171920) and China Agricultural Research System (CARS-33-11). We are grateful to the editor and two reviewers for their comments and suggestions.

# Supplementary Material

Acknowledgments

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015. 00502

Supplementary Figure 1 | The pedicel of a litchi fruitlet showing the pre-existing fruit abscission zone (FAZ) with a visible sunken line (thick arrow). The section between the solid lines (∼2 mm) was sample for DTA analysis.

Supplementary Figure 2 | Functional enrichment analysis for ethephon-responsive genes. (A) Enriched GO terms. Hierarchical tree graph of enriched GO terms in agriGO were used to simplify the analysis. The color bar shows the statistical significance, with enrichment significance level FDR ≤ 0.05. (B) KEGG pathway enrichment analysis. The top 20 enriched pathway are showed. The color bar shows the statistical significance, with enrichment significance level Q-value ≤ 0.05. The size of the circle is proportional to the number of genes in the KEGG pathway.

Supplementary Data Excel File 1 | Specific primers details for 41 genes selected for qRT-PCR. Representative genes selected for the analysis were those involved in plant hormone regulation pathway, cell wall modification, transcription factors regulation, ROS, and calcium signaling. All the gene ID, forward, and reverse primer sequences are included.

Supplementary Data Excel File 2 | List of 6167 differential expressed genes in the three pair-comparisons (CK1 vs. ETH1, CK2 vs. ETH2, and CK3 vs. ETH3). We used a false discovery rate (FDR) ≤0.001 and the absolute value of log2 ratio ≥2 as the threshold to judge the significance of transcript abundance differences, and then eliminated the control background influences (CK0 vs. CK1, CK0 vs. CK2, and CK0 vs. CK3).

Supplementary Data Excel File 3 | List of 2730 candidate genes involved in the ethephon-promoted fruitlet abscission process by GO (FDR ≤ 0.05) and KEGG (Q-value ≤ 0.05) enrichment analysis.

Supplementary Data Excel File 4 | Plant hormones-related genes induced or repressed in FAZ-enriched pedicel after ethephon treatment.

Supplementary Data Excel File 5 | Transcription factor genes induced or repressed in FAZ-enriched pedicel after ethephon treatment.

Supplementary Data Excel File 6 | Cell wall-related genes induced or repressed in FAZ-enriched pedicel after ethephon treatment.

Supplementary Data Excel File 7 | Photosynthesis, carbohydrate metabolism, and mitochondria-related genes induced or repressed in FAZ-enriched pedicel after ethephon treatment.

Supplementary Data Excel File 8 | ROS, calcium, and protein ubiquitination-related genes induced or repressed in FAZ-enriched pedicel after ethephon treatment.

abscission in citrus leaves. BMC Plant Biol. 9:127. doi: 10.1186/1471-222 9-9-127

in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments. BMC Plant Biol. 9:68. doi: 10.1186/1471-2229-9-68

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Li, Wang, Ying, Ma and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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.

# Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm

#### Edited by:

Jose I. Hormaza, Instituto de Hortofruticultura Subtropical y Mediterranea la Mayora-Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Spain

#### Reviewed by:

Xuming Huang, South China Agricultural University, China Hiroaki Iwai, University of Tsukuba, Japan

> \*Correspondence: Timothy J. Tranbarger<EMAIL_ADDRESS>
#### †Present Address:

Peerapat Roongsattham, Department of Biology, Prince of Songkla University, Songkhla, Thailand

#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 20 November 2015 Accepted: 05 April 2016 Published: 26 April 2016

#### Citation:

Roongsattham P, Morcillo F, Fooyontphanich K, Jantasuriyarat C, Tragoonrung S, Amblard P, Collin M, Mouille G, Verdeil J-L and Tranbarger TJ (2016) Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm. Front. Plant Sci. 7:540. doi: 10.3389/fpls.2016.00540 Peerapat Roongsattham1 †, Fabienne Morcillo2 , Kim Fooyontphanich1 , Chatchawan Jantasuriyarat 3 , Somvong Tragoonrung4 , Philippe Amblard5 , Myriam Collin1 , Gregory Mouille6 , Jean-Luc Verdeil 7 and Timothy J. Tranbarger 1 \*

1 UMR DIADE, Institut de Recherche pour le Développement, Montpellier, France, 2 UMR DIADE, CIRAD, Montpellier, France, 3 Department of Genetics, Kasetsart University, Bangkok, Thailand, 4 National Center for Genetic Engineering and Biotechnology, Genome Institute, Pathum Thani, Thailand, 5 PalmElit SAS, Montferrier-sur-Lez, France, 6 Institut Jean-Pierre Bourgin, UMR1318 Institut National de la Recherche Agronomique -AgroParisTech, ERL3559 Centre National de la Recherche Scientifique, France, 7 UMR AGAP, PHIV-MRI CIRAD, Montpellier, France

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

Keywords: fruit abscission, abscission zone, pectin, oil palm, cell wall

# INTRODUCTION

Plant organ abscission is a highly regulated developmental process that results in the loss of various organs throughout the life cycle of the plant. The abscission process is complex with many overlapping points of regulation, and involves the integration of multiple external and internal signals that depend on the overall status of the plant (Roberts et al., 2002; Estornell et al., 2013). In particular, seed and fruit abscission are important to coordinate seed dispersal for plant reproductive success. If fruit are shed prematurely before seed development is complete, or too late in relation to seasonal climate changes, reproductive success can be jeopardized. For crop species, seed and fruit abscission are important traits to consider, if seed or fruit abscission occur too early or late, the economic consequences can be significant.

Central to the process is the role of the abscission zone (AZ) where cell separation occurs that leads to organ detachment. Cell separation that occurs during abscission is a tissue and cellular process, which involves the differentiation of the AZ located at the base of the organ to be shed. Generally, the plant AZ consists of one to several cell layers but can also consist of multiple layers as observed in the leaflet rachis of Sambucus nigra (common elder) with up to 30–40 layers (Osborne and Sargent, 1976). AZ cells are typically isodiametrically shaped with dense cytoplasms (Addicott, 1982; Sexton and Roberts, 1982; Roberts et al., 2002). Neighboring cells are joined together by the middle lamella composed primarily of pectin, the most structurally complex family of cell wall polysaccharides and a major component of primary walls of both monocots and dicots (Mohnen, 2008). Pectin, principally homogalacturonan (HG), is also the main component of the middle lamella between adjacent cells and is of paramount importance for cell adhesion and during cell separation (Willats et al., 2001a; Jarvis et al., 2003; Ogawa et al., 2009; Iwai et al., 2013; Daher and Braybrook, 2015). In addition, pectin derived oligogalacturonide degradation products can also act as signaling molecules, possibly through the action of ethylene (Baldwin and Biggs, 1988; Brecht and Huber, 1988; Campbell and Labavitch, 1991; Melotto et al., 1994; Ridley et al., 2001). However, the structural characteristics of pectin and how it functions during cell separation underlying organ abscission are not completely understood.

The methylesterification of HG plays an important role during plant development, can modulate the functionality of pectin, in particular for cell adhesion and for cell separation to occur (Willats et al., 2001b; Jarvis et al., 2003; Mouille et al., 2007). HG is thought to be synthesized in the Golgi complex, targeted through vesicles via the plasma membrane to the apoplast and finally inserted into the cell wall in a highly methylesterified form (Zhang and Staehelin, 1992; Atmodjo et al., 2013). After cell divisions, pectin undergoes demethylesterification at cell junctions where cell separation takes place for intercellular space formation (Willats et al., 2001b; Jarvis et al., 2003). The demethylesterification of HG is catalyzed by pectin methylesterases (PME, EC <IP_ADDRESS>), which modulate HG methylation status and consequently plant development (Wolf et al., 2009). It is believed that demethylesterification allows the formation of calcium (Ca2+) cross-links and the "egg-box" pectin configuration between adjacent HG polymers, which can lead to the formation of rigid pectin gels or HG degradation by pectin degrading polygalacturonases (PGs, EC <IP_ADDRESS>) (Grant et al., 1973; Cosgrove, 2005; Senechal et al., 2014). Indeed, PGs modify the texture and rigidity of the cell wall and also have roles during cell separation processes such as those controlling organ abscission (Hadfield et al., 1998; Ogawa et al., 2009; Swain et al., 2011).

The oil palm (Elaeis guineensis Jacq.) fruit has two types of AZs, one large multilayer primary AZ and up to four adjacent AZs that are less distinguishable. The primary AZ is in the boundary between the pedicel and mesocarp tissues at the base of the oil palm fruit, while the adjacent AZs are at the periphery of the primary AZ at the base of the outer whorl organs including the rudimentary androecium, tepals, and the bracteole (Henderson and Osborne, 1990, 1994; Henderson et al., 2001). The primary AZ of ripe oil palm fruit has high levels of un-methylesterified pectin proposed to contribute to the spatial specificity of cell separation (Henderson et al., 2001). Separation takes place first in the primary AZ while adjacent less distinguishable AZs separate only after the primary zone has separated, which suggested a signal generated in the primary AZ is necessary for adjacent AZ cells to separate (Henderson and Osborne, 1994). The primary AZ is larger and clearly more visible than the adjacent AZs, which are far less distinguishable. A previous study indicated that the primary AZ arises early during floral development, but more detailed information about the morphogenesis of this exceptionally large primary AZ is lacking (Henderson and Osborne, 1990).

Previous data showed the AZ response to ethylene depends on the stage of fruit development; the ripest fruit AZ responds quickest and function to allow rapid cell separation (Roongsattham et al., 2012). The objectives of this work were to examine in detail the intra- and extra-cellular changes during the development and function of the ripe fruit AZ to provide insight in to the underlying mechanisms. We use histological and immunohistochemical methods combined with a multi-imaging approach including confocal, electron, and Fourier-transformed infrared microscopy to examine the cellular characteristics of the oil palm fruit AZ during development and abscission.

# MATERIALS AND METHODS

# Plant Material

Oil palm E. guineensis fruits (type dura of Deli Dabou origin) from CRA-PP Pobé plantation, Benin were used for classical histology experiments. Oil palm E. guineensis fruits (type tenera clone C) from Golden Tenera Plantation in Krabi Province, Thailand were used for immunocharacterization experiments. Fruit sampled at different stages of development were selected from bunches as described previously (Roongsattham et al., 2012).

# Light Microscopy and Image Acquisition

Samples were fixed in 0.2 M phosphate buffer containing 2% (w/v) paraformaldehyde, 1% (w/v) caffeine, and 2% (v/v) glutaraldehyde 25% for a minimum of 2 days at 4◦C as previously described (Buffard-Morel et al., 1992). Serial dehydration with ethanol (EtOH) from 30 to 100%, then 100% butanol/100% EtOH (v/v), and finally 100% butanol was performed for each sample and followed by impregnation and embedding in Technovit 7100 resin (Heraeus Kulzer). Semi-thin sections of 3 µm were cut using a microtome. Each section was stained with toluidine blue, ruthenium red, alcian blue (AB), AB, and periodic acid-Schiff (ABS). Ruthenium red is a commonly used dye to visualize pectin, which selectively binds to the intramolecular spaces of carboxyl groups of pectin (Sterling, 1970; Hou et al., 1999; Leroux et al., 2007). Toluidine blue is a metachromasia compound that stains lignin and phenols to bluish-green with a pH-independent covalent bonding and nonlignin cell wall components to reddish-violet (acid; e.g., pectin) and bluish-violet (neutral; Conrad, 2008). For ABS, sections were double-stained with Periodic Acid-Schiff (PAS) reagent, combined with AB. A description of the stains used in the study is included in Supplementary Table 1. Photomicrographs were taken with a Leica camera on a Leica (LEITZ DMRB) light microscope (x20/0.5; x40/0.7; and x100/1.3). Samples were taken and observations were made with fruit bases containing the AZ from at least three different fruit collected.

# Electron Microscopy

The samples were prepared and analyzed as described previously (Verdeil et al., 2001). Samples taken from the central cell layers in the center of the primary AZ between vascular strand parenchyma cells (1 mm3 cubes of tissue) were fixed in a 0.05 M Sorensen buffer solution (pH 7.3) containing 2.5% glutaraldehyde and gently stirred for 16 h at 4◦C. Cubes were then rapidly rinsed with distilled water (3 × 10 min), then post-fixed in 1% aqueous osmium tetroxide containing 3% sucrose for 2 h at 20 ◦C in the dark. They were then dehydrated in an EtOH dilution series (30, 50, 70, and 90% EtOH; 10 min each) and finally for 15 min in pure EtOH; samples were then embedded in Epon EmBed 812 using an Automated Microwave Tissue Processor (Leica EM AMW). Ultrathin sections (thickness around 80 nm) were obtained with a Leica-Reichert Ultracut E ultramicrotome, and then stained with uranyl acetate in EtOH. Sections were then mounted on Ni-grids and examined with a Hitachi 7100 electron microscope.

# Immunohistochemistry

Samples (base of the fruit including the AZ) were fixed in 1X Phosphate-buffered saline (PBS) buffer containing 4% (w/v) paraformaldehyde for a maximum of 16 h at 4◦C in dark. After fixation, samples were washed twice in 1X PBS buffer with glycine (2%), then twice in 1X PBS buffer. The samples were then dehydrated in EtOH (incubations in 50% EtOH for 1 and 2 h, then 70% EtOH for 1 and 2 h at RT) and samples were preserved in 70% EtOH at 4◦C. Stored tissues were cut at 150 µm thickness with a vibratome (Microm HM 650V, Walldorf, Germany). The sections were placed in glass wells containing PBS solution (pH 7.4), then treated with blocking buffer (BB; 6% bovine serum albumin):PBS = 1:1 (500–1000 µl) and shook gently on a shaker for 3 h at room RT. BB:PBS:primary rat antibody (JIM5, JIM7, LM7, or LM8; Willats et al., 2000, 2004; Clausen et al., 2003) at 9:9:2 was prepared. The PBS-BB solution was removed and replaced with BB:PBS:primary rat antibody. Samples were agitated gently on a shaker overnight (18 h) at 4 ◦C. After incubation, the BB:PBS:primary rat antibody solution was removed and replaced with PBS solution and shook gently on a shaker for 15 min, then repeat this step two times. After this step, all preparations were performed in the dark. Prepare BB:PBS:secondary antibody (Alexa Fluor R 546 Goat Anti-Rat, INVITROGEN, https://www.lifetechnologies.com) at 499:499:2. Alexa 546 was chosen because minimal autofluorescence was found in oil palm fruit tissue corresponding to the windows of spectral emission of this dye. The tissue observation was performed on a Zeiss LSM 510 Meta Confocal Microscope. Microscope imaging was performed at IGH, Montpellier RIO Imaging Platform (www.mri.cnrs.fr) with a multiphoton laser scanning Axiovert 200M 510 META NLO microscope (Carl Zeiss MicroImaging, Jena, Germany) using the multitrack image acquisition. Excitation was at 543 nm with laser power 65% for Alexa 546 and at 405 nm with laser power 7% for DAPI, a fluorescent nuclear stain that binds to A/T rich DNA repeats, with an emission peak of 460 nm (Kapuscinski, 1995). Sections were observed with an immersion objective C-Apochromat 40X/1.2 W and pictures were acquired using the ZEISS LSM image browser software (Carl Zeis MicroImaging GmbH Standort Göttingen—Vertrieb Deutschland, Göttingen, Germany; www.zeiss.com). A description of the antibodies used in the study is included in Supplementary Table 1.

# Cell Parameter Measurements

Cell parameter measurements were performed for cell width, cell wall width, and middle lamella width by using the ImageJ program (http://rsb.info.nih.gov/ij/). The cell width measurements were performed from the histology samples stained with toluidine blue or ruthenium red. The cell wall and the middle lamella width measurements were performed from the confocal microscope images from immunolocalization studies. The cell parameter data were statistically treated with one way ANOVA analyses and then Post Hoc tests were performed for multiple comparisons with DunnettT3 by using the Statistical Package for the Social Sciences (SPSS) Statistics 17.0 (http://www-01.ibm.com/software/analytics/spss/).

# Fourier-Transform Infrared (FT-IR) Microspectroscopy and Degree of Esterification Measurements

The FT-IR microspectroscopy was performed as described previously using an IN10 Thermo Infrared Imaging system (Mouille et al., 2003). Sections of 50 µm were cut using a vibratome. From the average spectra data for each sample, the degree of methylesterification (DME) was estimated using the formula DME = A1740/(A1740 + A1630) described previously, where absorbance at 1630 cm−1 corresponds to the COO− group and absorbance at 1740 corresponds to the carbonyl group from both COOH and COOCH3 (Chatjigakis et al., 1998; Manrique and Lajolo, 2002; Gribaa et al., 2013). A total of 7–11 replicates for each sample were analyzed by FT-IR and used to quantify the DME. The A1630-value was obtained by the average between A1628 and A1632. Statistical significance of the DME between sample groups was calculated by ANOVA at P = 0.01.

# RESULTS

# Distinct Developmental Characters Accompany the Capacity for AZ Cells to Function in Ripe Fruit

In this study, we examined in detail the morphogenesis of the primary AZ compared to adjacent pedicel and mesocarp tissues. The results presented here are based on observations of at least three or more samples and the photos were selected to represent the most consistent features observed. Longitudinal sections through the base of the fruit were made to examine the cellular characteristics of the primary AZ compared with the adjacent mesocarp and pedicel tissues (**Figures 1A–F**). The primary AZ was easily observed at 30 days after pollination (DAP) in fruit sections stained with toluidine blue and consisted of small densely cytoplasmic cells as previously described (**Figure 1A**; Henderson and Osborne, 1990). At 30 DAP, vascular parenchyma tissues were not lignified in any of the tissues (**Figure 1A**). By 120 DAP, vascular bundles were differentiated and lignified in the pedicel and mesocarp, while lignification was interrupted in the AZ vascular bundles at both 120 and 180 DAP (**Figures 1C,E**, dots connected by white lines). As with a previous study, phenolic containing cells were observed near the primary AZ, but unlike the previous study, these specialized cells were also located within the primary AZ layers (**Figures 1A,C,E**, red arrows; Henderson and Osborne, 1990). The polyphenol-containing cells appeared larger by 120 DAP and were often found grouped in two or more cells (**Figures 1C,E**).

Longitudinal sections of 30 DAP fruit stained with DAPI revealed that the nuclei of the AZ layer cells were aligned, particularly in the developing vascular bundles at this stage, then became more visibly aligned throughout the primary AZ during development at 120 and 180 DAP (**Figures 1B,D,F**). At 30 DAP, recently divided cells in the mesocarp, AZ and pedicel tissues were observed (**Figures 2A,D,G**). In the AZ, the plane of recent cell division was periclinal (parallel to the outer fruit surface), while in the mesocarp and pedicel cell divisions were anticlinal (plane perpendicular to the outer fruit surface). At 120 DAP cell divisions were no longer evident, and cells appear elongated in all tissues, and AZ cells appeared narrower than mesocarp and pedicel cells (**Figures 2B,E,H**).

During development, AZ cells had dark staining intercellular spaces often located to the tips of cells suggesting concentrated areas of secretion related to cell wall biosynthesis activity

(**Figures 2E,F, 3**). In contrast, mesocarp and pedicel cells had intercellular spaces that stain less intensively and appeared often to become regions of detachment that led to airspace formation (Willats et al., 2001b; Jarvis et al., 2003). Intercellular airspaces were rarely observed in the AZ cell layers (**Figure 3**). Also during development, dark staining particles in AZ cells appeared to be localized in the cytoplasm that became evident during ripening at 120 and 180 DAP. These intracellular particles appeared to contain pectin, based on coloration with both toluidine blue (**Figures 2E,F**) and ruthenium red staining (**Figure 3**). Similarly stained particles were not observed in either the mesocarp or pedicel cells at 120 or 180 DAP (**Figures 2B,C,H,I**, **3**).

Scale bar for schematic fruit diagrams is 10 mm.

To complement this comparative study of the pedicel, AZ and mesocarp tissues, we measured several quantitative parameters of the cells including: cell width, cell wall width, and middle lamella width. The cell width measurement data from longitudinal sections revealed that at 30 DAP, the average width of a mesocarp cell was 28.29 µm and significantly increased to 36.67 µm at 120 DAP. In contrast, the average pedicel cell width was 30.94 µm at 30 DAP and did not significantly increase at 120 or 180 DAP while in the AZ, the average cell width was 16.05 µm at 30 DAP and significantly increased to 17.84 µm at 120 DAP and to a maximum of 21.87 µm at 180 DAP. Notably, the average AZ cell width was less than the other tissues at all developmental stages examined (**Figure 3**, Supplementary Tables 2–5).

For the cell wall, the average mesocarp cell wall width was 0.59 µm at 30 DAP and significantly increased to 1.25 µm at 120 DAP and 2.59 µm at 180 DAP. In contrast, the average pedicel cell wall width was 1.05 µm at 30 DAP, did not change significantly at 120 DAP, but increased to 2.61 µm at 180 DAP. In contrast to these two adjacent tissues, the average AZ cell wall width was 0.91 µm at 30 DAP and significantly increased to 2.12 µm at 120 DAP, thicker than both the mesocarp and pedicel cells, which suggests more rapid cell wall biosynthesis occurs in the AZ between 30 and 120 DAP than the adjacent tissues. At 180 DAP, the AZ cell wall width was at a maximum of 2.58 µm, while all tissues display the maximum width and there was no statistically significant difference between the tissues at this stage (**Figure 3**, Supplementary Tables 6–9).

At 30 DAP the middle lamella width was too small to measure by our method at this early stage of development. At 120 DAP, the average mesocarp cell middle lamella width was 0.58 µm and significantly increased to 0.93 µm by 180 DAP. In the AZ, the average middle lamella width was 0.59 µm at 120 DAP and significantly increased to 0.94 µm by 180 DAP. In the pedicel, the average middle lamella width was 0.65 µm at 120 DAP and significantly increased to 0.99 µm by 180 DAP. Interestingly, the middle lamella width of each cell type was similar at each stage of development, and increased similarly between 120 and 180 DAP (**Figure 3**, Supplementary Tables 10–13).

To examine the ultrastructure changes that occur during AZ morphogenesis, electron microscopy was performed at 30 and 120 DAP. Thin longitudinal section preparations for electron microscopy analysis of the AZ of 30 and 120 DAP fruit stained reddish-violet with toluidine blue corroborating the presence of pectin material within AZ cells, and an increase cell wall width apparently rich in pectin, particularly at the tips of cells between AZ layers (**Figures 4A,B**). Electron microscopy analysis revealed a dramatic increase in cell wall width that occurs between 30 and 120 DAP, which corroborates the increase in cell wall width measured (**Figures 3**, **4A–D**, **5A–D**).

Accompanying these changes, AZ cell shape at 30 and 120 DAP cells were rectangular but more elongated at 120 DAP (**Figures 4C,D**). The AZ cell nuclei at 30 DAP were rounded or slightly elliptical, while those at 120 DAP were more amoeboid shaped (**Figures 4E,F**). Nucleoli could be observed at 30 DAP but were rarely found in 120 DAP (**Figures 4E,F**). At 30 DAP, nuclei with highly condensed chromatin were observed (**Figure 4E**). Interestingly, at the tip of the elongated AZ parenchyma cells, the cell wall was thickened with the appearance of multiple striated layers or ripples that could be either continuous with the middle lamella or the primary cell wall (**Figure 5E**). Finally, ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles were observed at the periphery of the cytoplasm, in particularly at the tip of the cells (**Figure 5E**), and a fibrous matrix can be found throughout the cell interior (**Figures 4D,F**, **5E**). Taken together, these ultrastructural features suggest extensive cellular activity in the AZ elongated cell tips including high protein synthesis and metabolic activity possibly related to exocytosis.

Plasmodesmata (PD) in the cell walls at 30 DAP between cells in the same AZ layer (intra-layer PD) were frequently observed, but less frequent between cells in adjacent AZ layers (interlayer PD, **Figures 5A,C**). PD appear to be grouped together in areas of cell wall constriction which became more pronounced by 120 DAP (**Figures 5A–D**). At 120 DAP, PD appeared less frequent but the constricted cell wall regions where PD were localized increased in width and were situated in the middle of the cell between cells of the same layer. The constricted regions with PD increased almost twofold between 30 and 120 DAP (**Figures 5A–D**). Nuclei were often closely associated to PD, while vesicle fusion to the plasma membrane could be observed adjacent to thickened cell walls at 120 DAP (**Figure 5C**).

# Cellular Changes Occur in the AZ Cells of Ripe Fruit during Abscission

To assess the intra- and extra-cellular changes that occur in the AZ during abscission, we examined ripe fruit AZ cells before and after cell separation induced by ethylene (Roongsattham et al., 2012). Cell separation was commonly observed within AZ cell layers and not between the AZ layers and adjacent mesocarp or pedicel tissues (**Figures 6A,C**). In the example presented, cell separation occurred closest to the mesocarp, but this was variable and not always the case; separation can also be in the middle of the AZ layers or closer to the pedicel tissue, but was not observed between AZ/pedicel or AZ/mesocarp cells.

During and after cell separation occurs, no evidence for cell rupture or cell division was observed (**Figure 6**). One prominent characteristic after cell separation was the change

of toluidine blue staining from reddish-violet to bluish violet in the AZ cell walls, which suggests a change of cationic environment (i.e., Ca2+ and/or H+) in this region that could be related to pectin modification in the AZ (**Figures 6A–C,E,F**). In contrast, pedicel and mesocarp cell walls stained dark with toluidine blue even after separation in the AZ occurred (**Figures 6A–C**). After separation in the AZ, the nuclei remained aligned and intact and there is no visible evidence of cell death based on the integrity of the nuclei (**Figure 6D**). Another prominent characteristic of AZ cells after separation was the change in the intracellular particles that accumulated in the AZ cells during development (**Figures 2D–F**, **3**, **6A–C,E–L**). Once separation occurred, the AZ cells appeared to have fewer particles as visualized by a decreased coloration with different histochemical stains (**Figures 6A,C,E–L**). Finally, cells in the AZ layers appeared to undergo tip elongation and there was evidence of exocytosis adjacent to separation sites (**Figures 6F,H,J,L**).

We then measured the changes in cell, cell wall and middle lamella widths after ethylene induced cell separation in 180 DAP fruit (**Figure 6**). The average cell width of mesocarp and pedicel cells did not significantly change in 180 DAP fruit after ethylene treatment. In contrast, the average cell width of AZ layer cells before ethylene treatment (21.87 µm) decreased significantly in unseparated AZ cells (17.99 µm), and non-significantly in cells that separated (**Figure 6**, Supplementary Tables 14–17). The average mesocarp and pedicel cell wall width at 180 DAP (2.59 and 2.61 µm, respectively) did not change significantly after ethylene treatment. In contrast, the average cell wall width (2.58 µm) decreased significantly after ethylene treatment in both the separation layer and unseparated AZ layer cells (1.76 and 1.84 µm, respectively, Supplementary Tables 18–21). Finally, the average middle lamella width of mesocarp cells at 180 DAP (0.93 µm) decreased significantly (0.52 µm) after ethylene treatment. A similar decrease was observed in pedicel cells; the average width of the middle lamella was 0.99 µm and significantly decreased to 0.58 µm after ethylene treatment. In contrast, the average middle lamella width of the AZ cells was 0.94 µm, and significantly increased after ethylene treatment to 1.49 µm in separated layer cells, while insignificantly increased to 1.10 µm in cells of the unseparated layers (**Figure 6**, Supplementary Tables 22–25). The increase in the AZ cell middle lamella width suggests this area is undergoing expansion prior to cell separation. The results reveal distinct and dynamic changes occur in the AZ cells in association with the cell separation and abscission process in contrast to adjacent mesocarp and pedicel tissues.

# FT-IR Microspectroscopy Reveal Changes in Methylesterification Status Occur in AZ Cell Walls during Cell Separation

To examine specific changes that occur in the AZ cell walls during cell separation in more detail, FT-IR microscopy was used to compare unseparated and separated AZ cells (**Figure 7**). The spectra revealed that mesocarp cells had higher absorbance at 1731–1749 (a range that corresponds to both the COOH and methylester carbonyl group) than either separated or unseparated AZ cells, which suggested a higher esterification in mesocarp cells (Chatjigakis et al., 1998; Manrique and Lajolo, 2002; Gribaa et al., 2013). Furthermore, unseparated AZ cells had a higher absorbance at 1731–1749 than separated cells in both the distal (close to mesocarp) or proximal (close to pedicel) AZ separation layer cells, which suggested a higher esterification in unseparated cells. From the spectra data, the DME was estimated (see Material and Methods for description) that revealed a significant decrease in DME occurs within separated AZ cells in the distal layers after separation (**Figure 7**, Graph inset).

# The Jim5 Epitope Signal Increases Prior to Cell Separation and is High at Cell Edges and Cells that Have Undergone Separation

The classical histology, electron microscopy, and cell parameter analyses described above provide evidence for changes in the cell wall of AZ layer cells during ethylene-induced abscission, in particular changes related to pectin. We used the antibodies JIM5, JIM7, and LM7 to distinguish the cellular localization of partially methylesterified HG, and LM8 to recognize xylogalacturonan (XGA), another form of pectin that is associated with detached cells (Knox et al., 1990; Clausen et al., 2004; Willats et al., 2004). For each experiment, the fluorescent nuclear stain DAPI was used to ensure the region examined was the AZ by the observation of the nuclear alignment of the AZ layer cells (**Figures 1B,D,F**). The immunohistological controls included incubation without primary antibodies, with secondary antibodies with the different tissues examined (Supplementary Figure 1).

The signal intensity that corresponds to the JIM5 epitope in the AZ from ripe fruit (180 DAP) after 3 h with air treatment was very low (**Figures 8A,B**). In contrast, by 3 and 6 h after ethylene treatment, the signal obtained with JIM5 increased on the inner side of primary cell wall (**Figures 8C–F**). By 9 h after ethylene treatment when separation had occurred, the signal increased dramatically at sites of separation of AZ cells on both the mesocarp (distal) and pedicel (proximal) sides (**Figures 8G–J**). Signals on the inner side of the primary cell wall and in cytoplasm could be detected, in addition to line like signals that arise from deeper within the AZ cells and/or cell layers. A film was made to give a three-dimensional overview of the JIM5 signal in AZ after separation at 9 h (Supplementary Movie 1). The film allows visualization of the high JIM5 signal along the separated cell surfaces, in addition to the lower signals deeper in the AZ layers where cells have not yet separated. By 12 h after ethylene treatment, the signal at the separated primary cell wall of separation layer cells of both the mesocarp and pedicel sides remained high, while the line like signals were lost from the mesocarp side (**Figures 8K–N**). In samples where both separated and non-separated cells could be observed at 9 h with ethylene treatment, the signal was not only observed on the exposed separated cell surfaces, but also appeared to increase in nonseparated cells which may indicate the future separation sites (**Figures 8O,P**). In addition, the signal intensity was seen all along the separated cell layer surface (Supplementary Figure 2). Moreover, the signals at possible future separation sites within the non-separated zone were quantified using the profile option of the Zeiss LSM image browser software and the results suggested that indeed the JIM5 epitope increased in certain AZ cells prior to separation, and may predict the future sites of cell separation (Supplementary Figure 3).

No JIM7 epitope signal was detected in the AZ of 180 DAP fruit at 3 h without ethylene treatment or at 3 and 6 h with ethylene treatment (Supplementary Figures 4A–F). By contrast, at 9 h with ethylene treatment and with separation occurring, the signal increased at the separated primary cell wall of separation layer cells on both mesocarp and pedicel side (Supplementary Figures 4G–J). Similar to the signal at 9 h, the signal at 12 h with ethylene treatment at the separated primary cell wall of

PD, plasmodesmata; R, ribosome; M, mitochondria; ML, middle lamella; FM, fibrous matrix material; PS, polysome; dots connected by white lines, cell wall expansion layers at cell tips. Scale bars in (A–E) are 1 µm and 0.1 µm in C.

separation layer cells in both mesocarp and pedicel side of AZ remained high (Supplementary Figures 4K–N). However, the signal interior to the separation face decreased on the mesocarp side while the signal on the pedicel side remained high. In contrast to the JIM5 epitope, the JIM7 epitope did not reveal an increase in cells prior to separation, only in separated cells (Supplementary Figures 2, 4O,P).

The signal corresponding to the LM7 epitope in the AZ of all samples (3 h without ethylene treatment, 3, 6, 9, 12 h with ethylene treatment) was very low and not associated with cell separation (Supplementary Figures 5A–N).

The signal corresponding to the LM8 epitope after 3 h without ethylene treatment was undetectable but becomes stronger after 9 and 12 h of ethylene treatment and only after AZ cell separation (Supplementary Figures 6A–N). By contrast, the pattern of the signal observed was spotted and appeared to be associated to the vascular tissue cells (Supplementary Figures 2I–L, 6G–N). In comparison, the JIM5 and JIM7 epitopes were observed all along the separation surfaces on both the mesocarp and pedicel sides of the AZ (Supplementary Figures 2A–H). Notably, the JIM5 epitope appeared to increase earlier than either the JIM7 or the LM8.

# DISCUSSION

# AZ Specific Regulation during Development Targets Cell Wall Biogenesis and Results in Anatomy Specialized for Intra-AZ-layer Cell–Cell Communication

The duration of development from the early to ripe stages at which time the fruit abscise and are shed from bunches is variable and depends on the genetic material (Fooyontphanich et al., in press). In the current studies, the final stages of ripening ranged from 160 to 190 DAP when the fruit could be observed to detach naturally in the field. During ripening, ethylene production begins to increase by 120 DAP in the mesocarp, and is thought to be the signal that initiates and regulates oil palm fruit abscission (Henderson and Osborne, 1994; Tranbarger et al., 2011). Indeed, several studies have shown that ethylene induces cell separation in the oil palm fruit AZ, while the response to ethylene is developmentally dependent; ripe fruit AZ have the highest capacity to respond to ethylene (Henderson and Osborne, 1994; Roongsattham et al., 2012). A primary objective of this study was to identify cellular characters of the oil palm ripe fruit AZ that correspond to the capacity to respond and function

FIGURE 8 | Confocal immunohistological images of the JIM5 epitope signal that increases in the AZ cells prior to and after cell separation on the surfaces of separated cells of 180 DAP fruit. (A) JIM5, and (B) JIM5 and DAPI AZ 3 h without ethylene treatment. (C) JIM5, and (D) JIM5 and DAPI AZ 3 h with ethylene treatment. (E) JIM5, and (F) JIM5 and DAPI AZ 6 h with ethylene treatment. (G) JIM5, and (H) JIM5 and DAPI AZ at M side at 9 h with ethylene treatment and separation occurring. (I) JIM5, and (J) JIM5 and DAPI AZ at P side at 9 h with ethylene treatment and separation occurring. (K) JIM5, and (L) JIM5 and DAPI AZ at M side at 12 h with ethylene treatment and separation occurring. (M) JIM5, and (N) JIM5 and DAPI AZ at P side at 12 h with ethylene treatment and separation occurring. (O) JIM5, and (P) JIM5 and DAPI of samples where both separated and non-separated cells can be observed at 9 h with ethylene treatment. Scale bars are 10 µm.

in response to ethylene. During oil palm AZ development, we observed several cellular features that arise during development with timing that corresponds with the capacity for AZ function and response to ethylene.

During early fruit development, the cell division plane in the AZ layer cells is opposite to that observed in both adjacent tissues; periclinal (parallel to the outer fruit surface) in the AZ, and anticlinal in both the pedicel and mesocarp cells. This suggests that AZ expansion is under distinct regulation compared with adjacent tissues from an early stage. Consequently, periclinal cell divisions in the AZ may give rise to the unusual alignment of nuclei between adjacent cells within each AZ layer. Nuclear alignment appears to originate early during vascular tissue differentiation in the base of the fruit then extends across the primary AZ during development. To the best of our knowledge, this is the first observation of nuclear alignment in an AZ. In contrast, no nuclear alignment is observed in the tomato pedicel AZ where cells appear to undergo cell death during the abscission process (Bar-Dror et al., 2011). In the oil palm fruit AZ, our analysis found no evidence for nuclear degradation well after cell separation took place. Interestingly the AZ cell nuclei are closely associated with PD, which could provide an efficient intra-AZ-layer structural capacity to communicate signals and coordinate transcriptional responses, analogous to the observed role for PD in defense signaling (Sager and Lee, 2014; Lee, 2015). Indeed, the timing of PD structural changes between 30 and 120 DAP corresponds to the period when AZ cells acquire the capacity to respond to abscission signals, and may provide the structural anatomical basis to coordinate signals that promote a rapid intra-AZ-layer transcriptional response that leads to cell separation.

The observations that cell wall constrictions are associated with groups of PD between adjacent cells, and vascular cell walls are less lignified within AZ layers, suggests that cell wall biosynthesis and differentiation are under specific control in AZ cells. Indeed, we also show cell wall expansion occurs more rapidly in the AZ than in adjacent mesocarp and pedicel cells, and that this expansion occurs in a polarized manner at the tips of AZ cells (**Figures 3**, **5**). Importantly, the increase in cell wall biosynthesis between 30 and 120 DAP corresponds to the period during which AZ acquire the capacity to respond to ethylene (Roongsattham et al., 2012). Overall, these observations indicate that the control of AZ cell wall biosynthesis is coordinated differentially compared with adjacent tissues, which may be related to the spatial capacity of AZ cells to separate. Differential cell wall construction in the AZ associated with PD development may provide a structural basis for strong intra-layer cell–cell communication required to coordinate abscission signals, while differential cell wall expansion between cell layers may provide the appropriate apoplastic environment for cell separation to take place, analogous to the cell separation that occurs during intercellular space formation (Willats et al., 2001b; Jarvis et al., 2003). Finally, less lignified and differentiated vascular cells in the AZ makes them more susceptible to pectin dissolution by PG. Taken together, AZ cell layer differentiation is controlled by distinct regulation that gives rise to cellular characters that may be important for the capacity to undergo cell separation.

The oil palm AZ consists of a very large primary multicell layer AZ that forms a boundary between two fruit tissues with distinct functions; the pedicel for support of the fruit and vascular connection to the bunch, and the mesocarp which has an unusually high capacity for lipid biosynthesis and storage (Tranbarger et al., 2011). As discussed above, we provide evidence that the AZ is under distinct development controls compared to adjacent tissues. Indeed it is known from dicot model species that AZ development and function are highly regulated processes (Estornell et al., 2013). Arabidopsis, boundary AZs are under the control of specific transcription factors including BLADE-ON-PETIOLE1/2 (BOP1/2), ASYMMETRIC LEAVES1 (AS1), and HAWAIIAN SKIRT (HWS) which determine the differentiation, placement and function of floral organ AZs (Gonzalez-Carranza et al., 2007; McKim et al., 2008; Gubert et al., 2014). In the tomato, flower and fruit pedicel AZ differentiation is controlled by a completely different basis including a complex of MADS-box transcription factors (Butler, 1936; Mao et al., 2000; Nakano et al., 2012; Ito and Nakano, 2015). While the molecular basis for these AZ types may be different, it is clear that in both cases the development and function of the AZ is tightly linked; disruption of development affects the capacity to function in the promotion of cell separation and ultimately organ abscission. The current study highlights that strong spatial regulation leads to differential cell wall biosynthesis and assembly processes (e.g., inhibition of the cell wall lignification of vascular bundles across the AZ and extensive cell wall biogenesis at the tips of AZ cells) in the oil palm ripe fruit AZ. Given that the oil palm fruit AZ is multi-cell layered and very large compared to other plant AZs, it provides an interesting experimental system to examine in detail the cellular and biochemical processes that take place during cell separation and abscission, in particular for a monocot fruit species.

# Pectin Biosynthesis, Metabolism, Esterification Status, and Other Cell Wall Parameters in the Oil Palm AZ are Regulated Differently than Adjacent Tissues

Previous studies revealed that oil palm fruit AZ cell walls are rich in unmethylated pectin and that PG activity and the EgPG4 transcript is highly expressed in the AZ in response to ethylene (Henderson and Osborne, 1994; Henderson et al., 2001; Roongsattham et al., 2012). These features were proposed as a mechanism to specify cell separation to the AZ in the oil palm fruit base. In the current study, we provide several lines of evidence for the importance of pectin and pectin methylesterification status for the abscission process, and provide a link between ethylene and effect on the AZ cell walls. Firstly, during AZ development, we observed an increase in intracellular particles that stained strongly for pectin with ruthenium red. Furthermore, these particles decreased in separated AZ cells after abscission, suggesting a functional importance for the abscission process. Secondly, we observed an increase in the JIM5 epitope by 3 h after ethylene treatment localized to cells in the AZ region before separation. Once separation occurs, the JIM5 epitope increases dramatically on both proximal and distal AZ layer cell surfaces that have undergone cell separation. A previous study described nodular structures thought to be pectin material that appear on the separated cell surfaces (Henderson and Osborne, 1990). The current study provides further evidence that the material that accumulates on the separated cell surfaces is pectin. JIM5 binds strongly to HG, the most abundant pectin polymer of galacturonic acid, with a relatively low methylesterfication status and binds weakly to unesterfied oligogalacturonides (Willats et al., 2000; Clausen et al., 2003). In parallel experiments, we did not see the same pattern of epitope signal with JIM7, which only increased after cell separation. JIM7 recognizes higher order methylesterified HG than JIM5 (Willats et al., 2000; Clausen et al., 2003). In addition, the absence of an increase in the LM7 signal rules out the low methylesterified epitope (four contiguous unesterified galacturonic acid residues adjacent to or flanked by residues with methylester groups) recognized by both JIM5 and LM7, which leaves the moderately methylesterified epitope (three contiguous unesterified galacturonic acid residues adjacent to or flanked by residues with methylester groups) or unesterfied HG as the main epitopes detected by JIM5 in AZ cells prior to and after cell separation. Finally, the LM8 epitope consists of a region of highly substituted xylose in xyloglacturonan (XGA) that is observed in regions of cell separation and complete cell detachment (Willats et al., 2004). In the current study, the LM8 epitope does not increase in cells prior to separation, but is mainly localized to vascular strand cells that have separated, suggesting changes to xyloglacturonan are important for these cell types to separate in the oil palm AZ.

Several studies have examined the cell wall structure before, during and after cell separation in different species undergoing various organ abscission processes (Uheda and Nakamura, 2000; Lee et al., 2008; Bowling and Vaughn, 2011; Iwai et al., 2013). During Azolla branch abscission, the JIM5 epitope signal disappeared in the fracture surface of the separated cell (Uheda and Nakamura, 2000). Similarly, during impatiens leaf abscission, a loss of the JIM5 (unmethylesterfied HG) epitope signal was observed in the cell wall/middle lamella at the face of the separation surface of separated cells (Bowling and Vaughn, 2011). In contrast, during the induction of AZ differentiation in the poinsettia leaf base, a demethylesterification of HG was inferred by the differential signals observed with JIM5 (high signal for unmethylesterified HG) and JIM7 (low signal for partially methylesterified HG) in the AZ cell walls and on the cells distal to the AZ 7 days after induction (Lee et al., 2008). The results suggested the possible involvement of PMEs for the activation of the AZ. However, the study of poinsettia leaf abscission did not examine events after cell separation, and in addition, changes in other cell wall components such as RGI, hemicellulose, and lignin could be more important than pectin (Lee et al., 2008). In tomato, no change in JIM5 or JIM7 was observed during flower or fruit abscission, while increases in xyloglucan, pectic galactan, and arabinan were observed during flower abscission (Iwai et al., 2013). Notably, no changes in signals from any antibodies tested in the previous study were observed during tomato fruit abscission.

In the current study, we provide evidence for a preferential accumulation of JIM5 labeled HG at the edges of separated cells. During development, we observed rough endoplasmic reticulum, mitochondria, and vesicles, in addition to striated cell wall formation at the tips of AZ cells. Together, these results suggest that during development AZ cells undergo polarized oriented cell wall building activity, which culminates with the secretion of HG (JIM5 signal) during abscission in a polarized manner. These functions could either be a part of the mechanism for the abscission process, or part of the defense mechanism to protect the scar after separation. However, these two possible functions are not mutually exclusive and could be linked. Indeed, several lines of evidence suggest an active role for HG in the abscission process. First, the JIM5 epitope increases in the AZ prior to separation and the signal continues to increase after AZ cells separate. Second, pectin accumulation detected in AZ cells prior to separation decreases after separation takes place, suggesting pectin is secreted during abscission. We also observed a high JIM5 signal on both the distal and proximal separated AZ cells, which is not consistent solely with a role for defense, given that a defense response would not be expected in the distal AZ cells of the shed fruit. Finally, a significant decrease in the DME in separated AZ cells closest to the mesocarp suggests that methyl ester groups are being actively removed from HG, possibly by PME activity, in these AZ cell layers, and could explain the high JIM5 signal observed prior to and after AZ cell separation.

During abscission, cell elongation and expansion have been observed but it is unclear whether they are an active part of the abscission process (Wright and Osborne, 1974; Butenko et al., 2003; Patterson and Bleecker, 2004). The current results provide evidence for cell elongation accompanied by a decrease in cell and cell wall width, and an expansion of the middle lamella width in AZ cells that separate. While these changes are significantly quantified in the AZ cells that separate, similar changes do not occur in mesocarp or pedicel cells, again highlighting the differential regulation that controls AZ cell function and fate. Interestingly, during pollen tube and root hair tip growth the polar secretion of HG plays key roles in the elongation process (Rounds and Bezanilla, 2013). The tip growth model proposes that methylated HG is secreted into the wall where it undergoes de-methylesterification by PME, which allows the de-methylesterified HG to form Ca2+ crosslinks that results in more rigid cell walls. In the current study, we provide evidence that functional components for tip growth are present in the AZ cells; cell elongation, tip oriented pectin biosynthesis and a decrease in methylesterification, all associated with cells that have undergone separation. While in the tip growth systems cells are elongated, in AZ cells elongation may be a source of HG secretion that plays an active role in cell separation.

# AUTHOR CONTRIBUTIONS

TT and FM devised and participated in all aspects of the study. TT, CJ, and ST coordinated the logistics for study. TT, FM, PR, and CJ performed the ethylene experiments and collected samples for studies. PR, KF, MC, and JV performed the immunolocalization studies. PR, MC, and JV performed the light microscopy and electron microscopy analyses. GM and PR performed the FT-IR microscopy analysis. ST, CJ, PR, and PA participated in the data analysis and critically read the manuscript. TT, JV, FM, and PR participated in writing the article. All authors read and approved the final submitted manuscript.

# ACKNOWLEDGMENTS

This work was supported by a Fondation Agropolis RTRA doctoral grant to PR (project number 07030). Financial support for the project also came from PHC Thaïlande projects 2007– 2010 (codes 16589YK and 16589YK) to TT and ST, from PalmElit SAS/IRD/CIRAD to FM, TT, and KF, and doctoral scholarship support from the French Embassy in Thailand for KF. We thank Chloé Guerin for technical assistance for the immunolocalization studies. We thank Chantal Cazevieille (MRI-Inserm) for technical assistance with the electron microscopy analysis. We especially thank Anek Limsrivilai and the staff at GoldenTenera Oil Palm Plantation in Thailand, and the personnel at the Institut National de Recherche Agricole du Bénin, Centre de Recherche Agricoles Plantes Pérennes (Pobe, Benin), for their technical and logistical support during the collection of the genetic material used in this study.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00540

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Roongsattham, Morcillo, Fooyontphanich, Jantasuriyarat, Tragoonrung, Amblard, Collin, Mouille, Verdeil and Tranbarger. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). 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.

# Two abscission zones proximal to Lansium domesticum fruit: one more sensitive to exogenous ethylene than the other

#### Prapinporn Taesakul 1, 2, Jingtair Siriphanich1, 2 \* and Wouter G. van Doorn1, 3

1 Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom, Thailand, 2 Postharvest Technology Innovation Center, Commission of Higher Education, Bangkok, Thailand, 3 Mann Laboratory, Department of Plant Sciences, University of California, Davis, Davis, CA, USA

Longkong (Lansium domesticum) fruit grows in bunches and is also sold as bunches. Individual fruit can separate from the bunch both before and after commercial harvest. The fruit has two separation sites. The first is located between bracts on the stem and the fused sepals (separation zone 1: SZ1) and the second between the fused sepals and the fruit (separation zone 2: SZ2). True abscission occurred at both zones. We investigated whether the two zones were active at different stages of development and if they were differentially sensitive to ethylene. Abscission occurred in the SZ1 in very young fruit (fruit still at the ovary stage), during early fruit development (5 weeks after full bloom; WAFB), and in ripe and overripe fruit (15–17 WAFB). Abscission did not spontaneously occur in the SZ2, but by the time the fruit was fully ripe, 15 WAFB, and later, a slight mechanical force was sufficient to break this zone. In fruit bunches severed from the tree at 5, 8, and 13 WAFB, break strength (BS) in SZ1 decreased much more after exogenous ethylene treatment than that in SZ2. Ethylene induced abscission in the SZ1, but not in SZ2. At 5, 8, and 13 WAFB, treatment with 1-methylcyclopropane (1-MCP; an inhibitor of ethylene perception) had a small effect on BS in the SZ1 and no effect in the SZ2. It is concluded that abscission in the SZ1 was much more sensitive to ethylene than that in the SZ2. In intact plants SZ1 reacts to endogenous ethylene, e.g., as a result of stress, while SZ2 apparently allows animals to remove the ripe fruit from the tree with minimal force.

#### Edited by:

Timothy John Tranbarger, Institut de Recherche pour le Développement, France

#### Reviewed by:

Siddharth Kaushal Tripathi, University of Mississippi, USA Vadim Volkov, London Metropolitan University, UK

#### \*Correspondence:

Jingtair Siriphanich, Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, 1 Malaiman Rd., Nakhon Pathom 73140, Thailand<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 08 December 2014 Accepted: 02 April 2015 Published: 21 April 2015

#### Citation:

Taesakul P, Siriphanich J and van Doorn WG (2015) Two abscission zones proximal to Lansium domesticum fruit: one more sensitive to exogenous ethylene than the other. Front. Plant Sci. 6:264. doi: 10.3389/fpls.2015.00264 Keywords: abscission, ethylene, fruit drop, Lansium domesticum, longkong, 1-methylcyclopropene, ultrastructure

# Introduction

Longkong (Lansium domesticum Correa) is a tropical fruit of the Meliaceae family. The pulp of longkong is consumed fresh. The peel contains oleoresin, which is used to counter diarrhea, while the crushed seeds are used to cure fever. The fruit is widespread throughout South East Asia (Yaacob and Bamroongrugsa, 1992; Paull, 2004). Currently longkong is only traded within the South East Asian region. Shipping the fruit to distant markets must be done by air, to reduce the problem of fruit abscission.

There has been some discussion about nomenclature, e.g., whether Lansium domesticum is synonym with Aglaia dookoo. Muellner et al. (2005) separated Aglaia from Lansium whereby the former has 1–3 ovary locules, while Lansium has five. Fruit called langsat is also L. domesticum, but has a thinner fruit peel than longkong. Two fruit called duku and duku-langsat, are also L. domesticum; with a morphology similar to that of longkong and langsat (Song et al., 2000). Lim (2012) gave a detailed description of the various fruit in the langsat-longkong group.

Longkong fruit are berries which develop on inflorescences, bunches contain about 25–40 fruit. The ripe fruit is round, about 3–5 cm in diameter. When immature, the peel is green and the fruit pulp is white. When ripe, the peel turns yellow and the pulp becomes translucent with a juicy texture and a typical smell. The fruit pulp is then sweet with a slightly sour tone (Paull, 2004).

Longkong fruit is non-climacteric, i.e., it does not produce a peak in respiration rate or in ethylene production, and fruit ripening is not sensitive to exogenous ethylene. Normally, the fruit is harvested before full maturity. If sold on local markets, recommended harvesting is about 15 weeks after full bloom (WAFB) (Lichanporn et al., 2009). When intended for markets at greater distances, the fruit should be harvested at 13 WAFB. Fruit harvested at 15 WAFB shows a high percentage of fruit abscission (Taesakul et al., 2012).

A few species reportedly contain more than one fruit abscission zone. In citrus, abscission zones were found at the base of the pedicel, and between the sepals and the fruit. The pedicel is defined as a stem that attaches single flowers/fruit to a bigger stem of the inflorescence/fruit bunch (The stem or branch that holds a group of pedicels is called a peduncle). After exposure to ethylene, increased cell wall degrading enzyme activity was found in both abscission zones (Goren, 1993a,b; Burns, 1997). Tomato fruit also has two abscission zones (Biain de Elizalde, 1980), while peach has three (Rascio et al., 1985).

Longkong has sessile fruit, hence the pedicel is absent. The stem branches of the inflorescence/fruit bunch are called peduncles. A separation zone was found between the peduncle and the sepals (SZ1) and a second between the sepals and the fruit (SZ2; Taesakul et al., 2012). Preliminary experiments showed that in the absence of exogenous ethylene, drop of mature fruit occurred when using a small mechanical force, at the SZ2. When exogenous ethylene was present, fruit drop occurred solely at the SZ1.

We studied the separation process at the two sites in more detail. We describe anatomical changes during flower and fruit development, using light and electron microscopy and measured the break strength (BS) at the separation zones. Both ethylene and 1-MCP (which inhibits ethylene perception) were applied to test the role of ethylene in this process.

# Materials and Methods

### Plant Material

Bunches of longkong fruit (Lansium domesticum Corr.) were severed from 5 to 10 randomly selected trees, which were 12 years old and had similar canopy size and apparent health. The trees grew in an orchard in Wang Saem District of Chanthaburi Province (Thailand). One bunch was randomly severed per tree every 2 weeks, starting 3 WAFB and ending 17 WAFB. The means of average daily temperature and relative humidity during the 4 months fruit growth period were 28 ± 2.5◦C and 85 ± 10%, respectively (Figure S1). Bunches were placed in perforated plastic crates (36×60 × 33 cm) lined with paper, and were held at ambient temperature for 1–3 h. Crates were then transported to the laboratory, using an air-conditioned van at 25–27◦C. Transport took 5 h. Upon arrival the fruit was held dry in the crates, at 24–26◦C for about 3 h until use.

# Physical Properties During Fruit Development

In 10 fruit from each of the ten bunches we determined fresh weight, length, diameter of the fruit, and diameter of the fused sepals, using a digital scale (TANITA 1579, Tokyo, Japan) for weight, and Vernier calipers (Mitutoyo 532-121, Kanagawa, Japan) for length.

# Flower Structure and the Presence of Abscission Zones in the Flowers

An Olympus 302990 stereo-microscope (Tokyo, Japan) with two Olympus TGHM lateral light sources were used to assess flower structure and the presence of abscission zones in the flowers. Pictures were recorded using a Dinoeye AM423X digital eyepiece (Taipei, Taiwan ROC) connected to a computer.

### Abscission Zone Morphology and Ultrastructure

Longkong bunches were severed from the trees at 3, 8, and 13 WAFB and separated into two groups. One group was evaluated using light microscopy, the other using scanning electron microscopy.

### Cell Size

The two separation zones were removed by cutting, using a sharp razor blade. A small part of the peduncle remained attached at the proximal side of the SZ1. A small part of the fruit remained attached at the distal side of the SZ2. The isolated parts were then cut in the middle between SZ1 and SZ2. The segments were cut longitudinally, fixed in 3.7% (v/v) formaldehyde, 50% (v/v) ethanol, and 5% (v/v) acetic acid (FAA) solution, embedded in paraffin, cut into 16µm thick slices, stained with Fast-green and Safranin-O and mounted on glass slides for observation using light microscopy (Axiostar plus, Carl Zeiss, Aalen, Germany). Cell sizes in the peduncle at the proximal side of the SZ1, in the SZ1, in the SZ2, and at the distal side of the SZ2 were determined using an ocular micrometer. Three samples (replicates) were used per treatment; nine cells were measured in each sample.

## Ultrastructure, Ethylene Treatment

Eighteen longkong bunches were divided into two groups. One group was used as control. The second group was exposed to 1µL L−1 ethylene for 12 h. After the ethylene treatment, the fruit were forcefully removed from the fused sepals. The sepals were also forcefully removed from the peduncle. The tissues near the separation site were collected and were dipped immediately in 2% glutaraldehyde in 0.05 M sodium phosphate buffer overnight, then treated for 2 h with 2% osmium tetroxide. The samples were dehydrated in an ethanol series, and were critical-point dried with CO2, and coated with 60 nm of gold/palladium. Observations were made using scanning electron microscopy (JEOL JSM-S410LV, Tokyo, Japan). Three samples were used for each stage.

# Break Strength and Abscission During Fruit Development on the Tree

The number of abscised fruit was determined both during development on the tree and after the harvest of almost ripe fruit. Abscission and BS were observed in bunches severed from the trees 3–17 WAFB. Abscission from the trees was low, as indicated by the low number of abscission zone scars. Abscission was determined by the drop of fruit during transportation from the trees to the laboratory. BS was determined using a push-pull force gauge (John Chatillon and Sons, Greensboro, NC), following the method of Gersch et al. (1998). To determine the BS at SZ1, the peduncle was attached to a drill clip which was mounted to the end of the push-pull force gauge. For SZ2 BS, the peduncle was removed using a razor blade. The fused sepals were attached to the drill clip. BS was determined by slowly pulling the sepal or the fruit, straight from the abscission planes, to separate them from the peduncle or the sepal. Care was taken not to pull the fruit at an angle under the abscission plane. The number of replicates was 25, consisting of five bunches (each cut from a different tree) and five fruit per bunch, at each sampling time.

### During Fruit Storage

Longkong bunches were severed from five trees at 5, 8, and 13 WAFB, and brought to the laboratory. Bunches were divided into three groups. The first group was used as the control, the second group was exposed to 1µL L−1 ethylene for 12 h and the third was treated with 1µL L−1 1-MCP (Ethylbloc, Floralife Inc., Walterboro, SC) for 6 h. Treatments occurred at 25◦C. Fruit was then stored at 25◦C for 4 days. The BS and fruit drop at SZ1 and SZ2 were determined at intervals, as described. The number of replicates was 25, consisting of five bunches (each cut from a different tree) and five fruit per bunch, at each sampling time.

# Statistical Analysis

Analysis of variance was performed on all data. Mean separation was carried out using paired t-test and Duncan's multiple range test (SPSS 15 program, Landau and Everitt, 2004). Differences at P < 0.05 were considered to be significant.

# Results

# Abscission of Young Ovaries

In the field many young ovaries were found to abscise. **Figures 1**, **2A** show the position of the ovary, and the structure of the flower. The flower had five fleshy petals, fused at their base (**Figures 1A–C,L**, **2A**). The petals of flowers that had just opened were off-white with creamy shades (**Figure 1A**). Fused with the inner base of the petals was a ring of very thick, fused filaments (**Figures 1L**, **2A**), bearing ten anthers (**Figure 1L**, white arrows). Subtending the petals were five green sepals, which are also fused (**Figures 1A–H,L**, **2A**). The sepals were distal to three bracts (**Figures 1C,I–K**).

The following stages of flower development were distinguished: (a) flower opening, (b) petals plus stamens separate from the flower, (c) stigma separates from the flower, (d) ovary or young fruit may separate from the flower. In **Figures 1L**, **2A** the petals plus stamens had separated from the rest of the flower, but had not yet fallen off. In **Figure 1D** the petals and fused stamens had fallen. An apparent abscission zone at the petal base is indicated by arrowheads in **Figure 1L**. Initially, separated petals plus stamens can be removed by little mechanical force, as they were still surrounded by the sepals (**Figures 1D,L**, **2A**). The petals were still whitish by the time of their separation. If the separated petals and stamens did not fall early (thus remain kept in check by the sepals), they showed discoloration to brown (**Figure 1B**), followed by desiccation (**Figure 1C**). After the fall of the petals plus stamens the female reproductive organs were visible (**Figures 1D–H**). The top of the stigma was flat, somewhat irregularly shaped at the edges (**Figures 1D,F**). The style was short and thick. The stigma + style separated at their junction with the hypogynous ovary. It usually fell off after slight discoloration to brown (**Figures 1G,H**). The ovary was situated on top of, and was fused with, the sepals (**Figures 1D–H,L**, **2A**). This entire structure (ovary and sepals) could abscise, whereby the abscission zone was localized at the proximal side of the sepals, which was the distal side of the three subtending bracts (**Figures 1I–K**). The localization of this abscission zone (SZ1), after abscission, is indicated in **Figures 1I,K**. The three bracts are indicated and the abscission area is shown by arrows. It should be noted that the flower as well as the fruit were situated under an angle less than 90◦ with respect to the peduncle (**Figures 1I,J**, **2B**).

# Position of the Two Separation Zones of the Fruit and Fruit Development

Abscission of ovaries (very young fruit) occurs at SZ1; it usually takes place in less than 5% of the ovaries on a bunch. The ovaries abscise together with the subtending sepals. A longitudinal section of SZ1 and the future separation zone SZ2 are indicated in **Figure 2A**. SZ1 is located just below the ovary, but had apparently not become fully developed by the time of ovary abscission. SZ2 extends, at its circumference, into the separation zone of the petals plus stamens. Thus, the SZ1 was located between the bracts on the peduncle and the fused sepals, while the SZ2 was between the sepals and the fruit. **Figure 2B** gives a scheme of the position of the two separation zones in full-grown fruit. As the fruit is sessile (has no pedicel), only the sepals separate the ovary from the distal part of the bracts.

The length and diameter of longkong fruit increased almost linearly from 3 to 15 WAFB, while fruit weight increased exponentially. The diameter of the fused sepals (0.4 cm by week 3 after full bloom) only slightly increased during this period (Table S1). Fruit development included a change of the peel from green to yellow by about 10 WAFB (**Figure 3**).

# Cell Size in and Ultrastructure of the Separation Zones

### Cell Size

At 3 WAFB, the cells in the peduncle above the SZ1 were about 32µm long. Cell size increased to 44µm by 13 WAFB. At 3 WAFB the cells in SZ1 were considerably smaller (20µm) than in those in the peduncle. Their size had increased to about 30µm by 13 WAFB. In SZ2 the cells were about the same size as in SZ1 (Table S2).

#### Ultrastructure

All cells in SZ1 at 3 and 8 WAFB had ruptured when the fruit together with the fused sepals (calyx) were pulled off the bunch. At these periods rupture occurred irrespective of ethylene treatment (**Figures 4A,B,D,E**). By contrast, at 13 WAFB and no ethylene treatment, half of the cells had separated (**Figure 4C**).

zone of the petal base, indicated by dashed arrows, apparently is an extension (outer ring) of the future SZ2. Key: as in Figure 1, bar equals 1 mm. (B) Scheme of the abscission zones in mature fruit. Key: 1, peduncle; 2, fused sepals (calyx); 3, fruit; 4, SZ1; 5, SZ2; 6, area proximal to SZ1; 7, area distal to SZ2; 8, bract.

After ethylene treatment at 13 WAFB all cells showed smooth separation (**Figure 4F**).

At 3 WAFB all cells in the SZ2 had ruptured when the fruit were pulled from the fused sepals, irrespective of ethylene treatment (**Figures 4G,J**). At 8 WAFB about half of the cells in the SZ2 showed smooth separation, while the remainder of the cells ruptured (**Figures 4H,K**). This was observed irrespective of ethylene exposure. At 13 WAFB, most of the cells had smoothly separated in the control (**Figure 4I**). All cells smoothly separated after ethylene treatment (**Figure 4L**).

#### Fruit Drop and BS in Intact Plants

By 3 WAFB and later, fruit abscission from the trees was low, as only a small number of abscission zone scars were observed. Abscission was determined by the drop of fruit during transportation from the trees to the laboratory. **Figure 5A** shows that, in the season investigated, fruit drop at the SZ1 took place during 5–7 WAFB (immature fruit with green peel) and during 15–17 WAFB (mature with full yellow peel). No fruit drop was found at the SZ2 during fruit development on the trees (**Figure 5A**). The BS at SZ1 and SZ2 increased from 3 to 9 WAFB (**Figure 5B**). The BS at SZ1 slightly decreased from 9 to 15 WAFB (**Figure 5B**). By contrast, the BS at SZ2 decreased sharply from 9 to 15 WAFB (**Figure 5B**).

### Effects of Ethylene and 1-MCP

Effects of ethylene and 1-MCP were determined using bunches that were severed from the trees at 5, 8, and 13 WAFB. Bunches picked at 5 WAFB, and kept at 25◦C, fruit drop was low in untreated controls, higher after ethylene treatment, and lower than the controls in the 1-MCP treatment (**Figure 6A**). In bunches severed at 8 WAFB, and kept at 25◦C, fruit drop in the controls was 20% by day 4. After ethylene treatment it was about 30% by day 4 (**Figure 6C**). When bunches were taken at 13 WAFB, and kept at 25◦C, fruit drop in the controls was absent by day 4 (**Figure 6E**), and was still very low by day 7 (data not shown). In fruit exposed to 1µL L−1 ethylene, fruit drop was more than 20% by day 4 (**Figure 6E**), and more than 40% by day 7 (data not shown), while 1-MCP treatment had no effect (**Figure 6E**). At any of the picking dates no spontaneous fruit drop during storage was found at the SZ2, in controls, ethylene treatment, and 1-MCP treatment (data not shown).

BS was determined at 5, 8, and 13 WAFB. Each ethylene treatment resulted in a rapid drop in BS at the SZ1 (**Figures 6B,D,E**). By contrast, ethylene had only a slight effect at SZ2 (**Figures 6B,D,E**). Treatment with 1-MCP (carried out on bunches severed at 5 and 13 WAFB) had little effect, both at SZ1 and SZ2 (**Figures 6B,D,E**).

# Discussion

Several separation zones were observed in the flowers and fruits of longkong: one at the base of the style, one at the base of the petals and attached stamens, one between the fused sepals and three bracts on the peduncle (SZ1), and one between the sepals and the fruit (SZ2). Separation of the petals and stamens occurred together. This was apparently due to true abscission as small cells were observed, but the separation process was not studied in detail hence it cannot be excluded that the tissue was torn. Tearing off instead of abscission has been described in petals, stamens, and styles in some species. This tearing is due to the mechanical forces generated by the growing fruit (Reiche, 1885; Wacker, 1911; van Doorn and Stead, 1997).

SZ1 and SZ2 of longkong fruit contained cells that were much smaller than the adjacent cells. Abscission zones usually contain small cells, but the presence of small cells is by itself inadequate proof of true abscission. After fruit drop at SZ1 (either without exogenous ethylene or after ethylene treatment) the zone showed smooth separation rather than cell breakage. This strongly suggests that SZ1 is a true abscission zone. After slight mechanical force is exerted on ripe longkong fruit, this leads to fruit drop at SZ2. In addition, most cells were easily separated, strongly indicating that an abscission process was in progress. Nonetheless, as the fruit did not abscise spontaneously, a number of cells apparently still needed to be broken. It was previously suggested that there was no true abscission zone at SZ2 (Taesakul et al., 2012). This is correct insofar the zone did not show spontaneous abscission, but at the same time not true because most of the zone cells showed smooth cell walls (**Figure 4L**) after separating the fruit from the bunch at 13 WAFB. The data show that middle lamella separation (abscission) occurred in many cells at SZ2.

FIGURE 3 | Morphology of longkong fruit (Lansium domesticum) bunches. (A) 5 WAFB, (B) 7 WAFB, (C) 10 WAFB, (D) 13 WAFB. Color break occurred at 10 WAFB. Bar in (A) is 5 cm. The bar also applies to (B–D).

In many abscission zones the vascular bundle is the only part not undergoing wall separation (Roberts et al., 2002), but this does not hamper abscission. This may also be true for SZ1. However, because of the lack of spontaneous abscission even by 15 WAFB, SZ2 seems to contain more cells that have not undergone cell separation than SZ1 by the time of abscission at that zone.

During fruit development on the trees, the BS at SZ1, at 15 WAFB, was clearly higher than SZ2, but fruit drop and abscission was higher at SZ1 (**Figure 5**). The results seem conflicting, but they could be explained either by tearing off of the fruit due to growth, or by normal abscission. We found smooth abscission scars, which seems to exclude tearing off of the fruit. The explanation of the apparent discrepancy may possibly be that a subset of the fruit was attacked by pests such as fungi, causing a local increase in ethylene production and thus abscission. This fruit then dropped, hence their BS must have been close to zero.

However, the majority of the fruit that remained was healthy, thus had high BS. This means that the average BS, determined in the remaining fruit, was high. However, one may ask why fruit dropped at SZ1 and not at SZ2, while both zones were equally exposed to fungal-induced ethylene. The explanation might be that SZ2 is less ethylene-sensitive. The difference in ethylene sensitivity between SZ1 and SZ2 is shown in **Figure 6**, where harvested fruit was exposed to ethylene.

After ethylene treatment of fruit harvested at the harvest time for local consumption (i.e., 15 WAFB) the BS at SZ1 had decreased significantly. The cells that had separated in the SZ1 showed smooth cell walls, indicative of a natural abscission processes. These data indicate that SZ1 contains an abscission zone that is normally sensitive to ethylene, similar to the zones in many other fruit, for example tomato (Tabuchi et al., 2000).

The anatomical data on longkong separation zones are similar to those in citrus (Goren, 1993a,b). One citrus fruit abscission zone (called A) is located at the proximal part of the pedicel, which is similar to the location of SZ1 in longkong. A more distal abscission zone in citrus is located between the sepals (calyx) and the fruit (called zone C), which is similar to the position of the SZ2 in longkong. The main difference between longkong and citrus is the closer proximity of the two abscission zones in longkong, as longkong has no pedicel. In longkong the two abscission zones are located at both sides (distal and proximal) of the fused sepals. Similarly, tomato has two abscission zones in the fruit stem. A proximal zone is located between the pedicel and peduncle (comparable to SZ1), and a distal zone between fruit and pedicel (slightly comparable to SZ2; Biain de Elizalde, 1980). In peach fruit stems three abscission zones have been found, the most distal one is at the junction of the fruit with the pedicel (slightly similar to SZ2 in longkong), and two others are in the pedicel (Rascio et al., 1985).

During growth on the trees, ovary and fruit drop occurs during three periods. Very young fruit (ovaries) abscised just after the petal life span had terminated. Young fruitlets abscised about 5 WAFB. Mature fruit abscised at about 15 WAFB. Similar abscission periods have been described for several other fruit. In Citrus lemon, for example, a period of massive abscission results in loss of flowers and ovaries. About a week later, fruit having a diameter similar to that of full-grown peas abscised. The third period of abscission occurred in the spring, reducing the number of growing fruitlets, which were by then about the size of a golf ball. This is similar to what is called June fruit drop in many species in the Northern Hemisphere (Goren, 1993a,b; Iglesias et al., 2006). Citrus fruit remained on the tree until over-mature, but seems to eventually abscise.

Flower abscission and early fruit abscission is usually not determined by the absence of pollination/fertilization but by competition between pollinated flowers and between fertilized ovaries. Abscission of fertilized flowers and very young fruit was found to be regulated by the availability, to the organ, of mineral nutrients or carbohydrates (Stephenson, 1981; Racskó et al., 2006; McFadyen et al., 2012). Abscission of the ovaries in longkong also does not seem related to the absence of fertilization, as longkong fruit is thought to be predominantly parthenocarpic (Bernardo et al., 1961). Ovary abscission could possibly therefore relate to

competition. Similarly, it could be suggested that abscission of longkong fruit at the immature stage is mainly due to competition (Stephenson, 1981; Wright, 1989; Iglesias et al., 2006).

The longkong ovaries are fused with the subtending sepals. Ovaries therefore abscise together with the sepals, at an abscission zone that is located between the sepals and three subtending bracts (thus at SZ1). We found that (a) abscission of ovaries in longkong flowers occurred only at SZ1, (b) abscission of immature fruit by 5 WAFB took place only at SZ1, and (c) spontaneous abscission of ripe fruit (not the fall after slight mechanical force) was also restricted to SZ1. Only after a slight mechanical force was exerted on ripe (mature) fruit, was abscission found at SZ2. This is quite different from citrus, where abscission of the ovaries and that of very small fruit, i.e., during 6–8 weeks after fruit set, took place exclusively at abscission zone A (similar to SZ1), while by the time of the June abscission period abscission zone A had become progressively inactivated while abscission zone C (similar to SZ2) began to operate and later on all abscission took place at abscission zone C. Only during 2–3 intermediate weeks is abscission found at both sites (Goren, 1993a,b; Iglesias et al., 2006). The data on longkong also differ from those in tomato and peach. Biain de Elizalde (1980) reported that small immature tomato

fruit abscised at the proximal zone (similar to SZ1) while ripe fruit showed abscission at the distal zone (slightly similar to SZ2). In peach Rascio et al. (1985) found that the abscission zone at the junction of the fruit and the pedicel (similar to SZ1) is responsible for abscission of floral buds, open flowers, and very young fruit. Two abscission zones in the pedicel become activated later on, one during early June drop, the other during late June drop.

At 5, 8, and 13 WAFB we observed increased abscission at SZ1 after ethylene treatment while SZ2 did not respond to ethylene by increasing abscission. Ethylene only induced a small decrease in the BS at SZ2 (where abscission of mature fruit took place, after slight mechanical force only), but did not induce abscission at SZ2. These data show that SZ1 was highly sensitive to ethylene, whereas SZ2 was only slightly sensitive, not enough to induce abscission. This is quite different from tomato fruit where ethephon (ethylene) treatment increased the abscission of ripe fruit but not of small immature fruit (Biain de Elizalde, 1980). In citrus ethylene increased abscission in both abscission zone A (although only until week 6–8 after fruit set, and no longer thereafter) and abscission zone C (Goren, 1993b; Iglesias et al., 2006). This is also different from our findings in longkong fruit.

In citrus it depends on the developmental stage of zone A whether ethylene induces abscission in this zone or not (Goren, 1993b; Iglesias et al., 2006). The present data on longkong show that exogenous ethylene was also not able to induce abscission in the SZ2, even though the BS became slightly lower after the ethylene treatment. SZ2 was considerably less sensitive to exogenous ethylene than the SZ1. It is therefore possible that the SZ2 is relatively insensitive to endogenous ethylene. This needs to be determined in further experiments.

After a short transportation period (from the field to the laboratory) of bunches harvested at 13–15 WAFB, fruit drop during transportation was low. In the present-tests (**Figure 6**), which lasted only 4 days, no fruit drop was found in controls harvested at 13 WAFB. By contrast, in other (unpublished) postharvest storage simulation experiments in the laboratory, using fruit harvested at 13 WAFB, abscission mainly increased after day 4 of storage, and varied between and 10 and 70% by day 10. Drop occurred mostly at SZ1 with about 5% at SZ2. High rates of abscission might be due mainly to ethylene production of fruit infected by fungus. An additional practical test (also unpublished data) found that fruit harvested at 13 WAFB and undergoing long sea transportation in a container (7 days at 18◦C) showed about 70–80% fruit drop by the end of transportation, increasing to 100% after transfer to ambient temperature for 2 days. Most of the drop (about 95%) occurred at SZ1. These data also suggested that fruit infected with fungus produced relatively high ethylene concentrations in the closed container, inducing abscission at SZ1. This abscission can largely be prevented, as it was previously reported that a 6 h treatment with 1µL L−1 1- MCP drastically reduces fruit drop at the SZ1 (Taesakul et al., 2012).

A hypothesis might be proposed as to the possible function of the two separation zones in longkong fruit. As the separation at SZ1 by 13 WAFB was significantly promoted by exogenous ethylene, this zone could possibly function in response to changes in endogenous ethylene levels or to changes in ethylene sensitivity. It could therefore be responsive to factors that cause physiological stress. Infection of longkong fruit with fungi, of which Phomopsis sp. and Lasiodiplodia theobromae are the most important

# References

(Kaewsorn and Sangchote, 2003), might induce such a stress. SZ2 was much less affected by the ethylene treatment than SZ1. Separation at SZ2 had already started before the change in peel color (**Figure 6B**), as indicated by the presence of smooth cell walls after pulling the fruit from the fused sepals (**Figure 4H**). By 15 WAFB the BS at SZ2 was quite low, although not low enough for spontaneous abscission. This low BS possibly allows animals to remove healthy and ripe fruit from the tree, using only slight mechanical force.

It is concluded that longkong ovary and fruit drop on the tree only occurred at SZ1, which contains an abscission zone that is highly sensitive to exogenous ethylene. By contrast, fruit drop at SZ2 was not induced by treatments with exogenous ethylene, even though cell separation in this zone had already started by the time of fruit coloration, and was slightly increased by the ethylene treatment. Incomplete cell separation at SZ2 seems to explain why no spontaneous fruit drop occurred at this zone. By 15 WAFB only a small mechanical force induced full separation at SZ2. The two zones are thus functionally different: one shows abscission in response to an increase in exogenous ethylene concentrations while the other does not. We suggested a hypothesis to account for the presence of two abscission zones with different characteristics.

# Author Contributions

PT did the experimental work, carried out data analysis, and drafted the manuscript; WD interpreted the data and revised the manuscript; JS designed the work, acquired funding, supervised experiments, interpreted the data, and was involved in revising the manuscript. The three authors approved the final version and agree to be accountable for all aspects of the work.

# Acknowledgments

The work was financially supported by (a) the Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok Thailand and (b) the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0041/2554).

# Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015. 00264/abstract

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Taesakul, Siriphanich and van Doorn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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.

# **Burst of reactive oxygen species in pedicel-mediated fruit abscission after carbohydrate supply was cut off in longan (***Dimocarpus longan***)**

*Ziqin Yang 1,2, Xiumei Zhong 1,3, Yan Fan 3 , Huicong Wang 1 , Jianguo Li 1 and Xuming Huang 1 \**

*1 Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou, China, 2 Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, China, 3 Section of Fruit Crops, Dongguan Agricultural Research Center, Dongguan, China*

#### *Edited by:*

*Timothy J. Tranbarger, Institut de Recherche pour le Développement, France*

#### *Reviewed by:*

*Grama N. Dhanapal, University of Agricultural Sciences, India Joshua D. Klein, Agricultural Research Organization-Volcani Center, Israel Michael F. Cohen, Sonoma State University, USA*

#### *\*Correspondence:*