Article pubs.acs.org/JAFC

Hormones, Polyamines, and Cell Wall Metabolism during Oil Palm Fruit Mesocarp Development and Ripening Huey Fang Teh, Bee Keat Neoh, Yick Ching Wong, Qi Bin Kwong, Tony Eng Keong Ooi, Theresa Lee Mei Ng, Soon Huat Tiong, Jaime Yoke Sum Low, Asma Dazni Danial, Mohd. Amiron Ersad, Harikrishna Kulaveerasingam, and David R. Appleton* Sime Darby Technology Centre Sdn Bhd, First Floor, Block B, UPM-MTDC Technology Centre III, Universiti Putra Malaysia, Serdang, 43400 Selangor, Malaysia S Supporting Information *

ABSTRACT: Oil palm is one of the most productive oil-producing crops and can store up to 90% oil in its fruit mesocarp. Oil palm fruit is a sessile drupe consisting of a fleshy mesocarp from which palm oil is extracted. Biochemical changes in the mesocarp cell walls, polyamines, and hormones at different ripening stages of oil palm fruits were studied, and the relationship between the structural and the biochemical metabolism of oil palm fruits during ripening is discussed. Time-course analysis of the changes in expression of polyamines, hormones, and cell-wall-related genes and metabolites provided insights into the complex processes and interactions involved in fruit development. Overall, a strong reduction in auxin-responsive gene expression was observed from 18 to 22 weeks after pollination. High polyamine concentrations coincided with fruit enlargement during lipid accumulation and latter stages of maturation. The trend of abscisic acid (ABA) concentration was concordant with GA4 but opposite to the GA3 profile such that as ABA levels increase the resulting elevated ABA/GA3 ratio clearly coincides with maturation. Polygalacturonase, expansin, and actin gene expressions were also observed to increase during fruit maturation. The identification of the master regulators of these coordinated processes may allow screening for oil palm variants with altered ripening profiles. KEYWORDS: oil palm, Elaeis guineensis, polyamines, hormones, cell wall metabolism

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INTRODUCTION Fruit development is a complex process, being a combination of events in physiology, gene and protein expression, and biochemical changes toward maturation. To date, metabolite profiling of soft fleshy fruits such as strawberry,1 peach,2 and tomatoes3 during ripening is more widely studied compared to oil fruits such as olive, avocado, and oil palm. This is partly because soft fruits are metabolite rich but also because of their importance to the human diet.1−3 Studies reported on soft fruit start from the fertilized ovary through to the mature fruit stage. These studies have suggested cross-talk between hormones, polyamines, and cell wall metabolism in fruits during development and ripening.4 The fruit of the oil palm (Elaeis guineensis Jacq.) is a source of two edible oils and is currently the most productive oil crop with average annual yields above 4 ton/ha. Unlike most temperate oil-bearing crops where oil is stored in the seed, the majority of palm oil is produced in the fleshy mesocarp of the fruit, with relatively less as kernel oil. The oil palm fruit consists of the exocarp-outer skin, mesocarpouter oily fibrous layer, the endocarp layer covering the kernel seed, and the kernel itself. During the later phase of fruit development, most fruit will accumulate high levels of carbohydrates in the form of starch or sugars, whereas oil palm fruits accumulate oil, almost exclusively. Oil palm fruit is considered to be nonclimacteric since no significant ethylene synthesis occurs during the fruit ripening period, i.e., from about 120 to about 150 days post pollination.5 The study of metabolites involved in ripening, such as phytohormones, © 2014 American Chemical Society

polyamines, as well as cell wall metabolism in oil palm mesocarp, may provide useful insights into why it is so productive and lead to the development of varieties that ripen faster through the identification of key controlling genes. Plant hormones are key regulators of many seed processes, including TAG accumulation. It is known that plant phytohormones are also involved in the control of fruit growth, which is demonstrated by the fact that these chemical compounds help to enhance cell division and promote cell expansion or enlargement during different stages of development. Fruit size is due more to cell division than cell enlargement, hence any factors that contribute to cell division will have an influence on fruit growth and final fruit size.6 Many recent studies have shown that the phytohormone abscisic acid (ABA) plays an important role in the regulation of fruit development and ripening,7 and gibberellic acid (GA3) has been proposed as a possible trigger to increase fruit set and fruit size of numerous vine and fruit crops.8 To date in oil palm, hormone analysis has been reported in sap exudate from stumps of preanthesis inflorescences,9 wild-type and truncated leaves,10 and fruit mesocarp.11 In addition to plant hormones, other natural substances, such as the polyamines (PAs), may be involved in controlling fruit growth and ripening. Spermidine Received: Revised: Accepted: Published: 8143

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Metabolite Extraction. The extraction of polar and lipid metabolites from oil palm tissues was carried out in a single integrated procedure according to a previously published method.20a For hormone extraction, ground tissue (100 mg) was extracted with a mixture of 1-propanol:H2O:conc HCl (2:1:0.002, v/v; 500 μL) with internal standards, [2H5]ABA and [2H5] trans-zeatin riboside (50 ng), added, followed by agitation for 30 min at 4 °C. CH2Cl2 (1 mL) was then added, followed by agitation for another 30 min and then centrifugation for 5 min at 4 °C, 13 000g. After centrifugation, two phases formed with plant debris suspended between the layers. An aliquot of the lower layer (1 mL) was concentrated and resolubilized in MeOH for analysis. All extracts were kept dry at −80 °C until further use. The analysis was comprised of targeted and untargeted LC-MS, GC-MS, and CE-MS analysis. Analysis Methods. Targeted Profiling of Hormone Metabolites Using LC-MS. LC-MS data were acquired using UPLC-triplequad MS (Waters, USA). The Waters Acquity UPLC system (Waters Corporation, Milford, MA) was run in gradient mode, as per a previously published method.21 The column used was reversed-phase C18 (1.8 μm, 2.1 × 100 mm; Waters) set at 30 °C. Solvent A was H2O (0.1% formic acid v/v), and solvent B was MeOH (0.1% formic acid v/v); the flow rate was 0.3 mL/min. The gradient was as follows: 30% B (0−2.0 min), linearly increased to 100% B at 20 min. The autosampler temperature was set at 4 °C with a 3.0 μL injection volume. MS analysis was performed on a Xevo TQs triple quadrupole mass spectrometer (Waters Corporation, Milford, MA). MS analysis was carried out in positive and negative ion electrospray ionization (ESI) modes of detection. The electrospray ionization capillary voltage on both modes was set at 2.5 kV, with the cone gas flow set at 150 L/ h, and desolvation temperature of 350 °C. The collision gas flow was set at 0.15 mL/min. The MRM settings in the MS/MS function with corresponding cone voltage and collision energy were optimized for each standard compound. The total acquisition duration for both UPLC and MS was set at 15 and 5 min, respectively. Data were acquired and processed using MassLynx V4.1 and TargetLynx, respectively. [2H5] trans-zeatin riboside was used as an internal standard for positive mode and [2H6] ABA for negative mode. Targeted Profiling of Polyamine Metabolites Using CE-MS. Targeted profiling of 108 primary metabolites using CE-MS was carried out by Human Metabolome Technologies, Inc. (HMT), Japan, as per a previously published method.22 RNA Extraction. Total RNA was extracted according to the San et al. method.23 The extraction buffer contained 0.1 M Tris-HCl, pH 7.6, 0.1 M NaCl, 6% p-aminosalicylic acid, 1% SDS, 0.35% βmercaptoethanol, and 5% phenol. The buffer was added at a rate of 4 mL/g of plant tissue, vortexed, and extracted with phenol:chloroform. The supernatant was precipitated twice using 3 M LiCl followed by ethanol precipitation. The pellet was then redissolved in DEPCtreated distilled water and again precipitated with 3 M LiCl followed by ethanol. The precipitate was finally dissolved in DEPC-treated distilled water and stored at −80 °C. Synthesis of cRNA, Microarray Hybridization, and Scanning. Expression microarray analysis was performed using Agilent’s one color method (Agilent Technologies, Palo Alto, CA, USA).24 Total RNA samples from mesocarp were individually treated and labeled with a one-color Cy3 dye according to the Low Input Quick Amp Labeling protocol version 6.0, December 2009, provided by Agilent. Briefly, a total of 100 ng of total RNA was used to synthesize cRNA labeled with Cy3 dye. Labeled cRNAs were then purified using Qiagen’s RNeasy mini kit as recommended by the manufacturer, and quality and quantity were assessed using the Spectrophotometer ND1000(NanoDrop). The purified labeled cRNA was hybridized onto the Arabidopsis, rice, and custom oil palm mesocarp array at 65 °C for 16 h. After hybridization, 2 steps of washing were performed with wash buffer 1 and 2 for 1 min each. The array was then air-dried for a few seconds before proceeding with image scanning using the Agilent microarray slide scanner (SG11350602). A blank control without RNA, put through the entire hybridization and scanning processes, was also used to provide an estimate of the array background noise when no labeled probe is present.

(Spd), spermine (Spm), putrescine (Put), and ornithine are the most commonly found polyamines in plants with several studies reported on tomatoes,12 mango,13 strawberry,14 and peach.15 Exogenous applications of PAs have also been reported to increase the endogenous levels of PAs and fruit size.16 However, biosynthesis and distribution of PAs in relation to oil palm fruit mesocarp growth and development have not been reported to date. In the context of studying fruit maturation and ripening, hormones, polyamines, and cell wall metabolism have interrelated roles. Mesocarp tissue is composed of thin-walled storage parenchymatous cells that are characterized by a prominent cell wall consisting of a complex network of polysaccharides and proteins giving mechanical strength to the tissues. A wide range of hydrolases that catalyze the process of softening are activated at transcriptional and translational levels. Softening is further enhanced by expansins that break down the multiple polysaccharide networks, thus increasing the accessibility of hydrolases, such as polygalacturonase (PG) or cellulase, to the cell wall polymers.17 Tomato PG has been the most widely studied cell wall hydrolase in relation to ripening, while pectin degradation during ripening has been reported in avocado mesocarp.18 We were also interested in studying hormone levels since they are well-known to play important roles in lipid biosynthesis regulation.19 On the basis of our previous work,20 we had identified several key biosynthetic pathways that are important to the synthesis of lipids and that the study of hormones may provide insights into the regulation of these networks. For this study, hormones and polyamines in oil palm mesocarp were analyzed from 12 to 22 weeks after pollination, representing the main stages of lipid accumulation and ripening. Tandem HPLC and mass spectrometry (MS) with multiple reaction monitoring (MRM) were used, in which several plant hormones (IAA, CKs, ABA, and their metabolites) were quantified using deuterated internal standards. We also extracted RNA from mesocarp tissue to determine gene expression changes during ripening. Transcript levels of the cell wall metabolism genes, pectin, polygalacturonase, cellulose, and expansin, were analyzed using an oil palm mesocarp transcriptome microarray. Further understanding of the biological and physiological mechanisms underlying oil palm fruit ripening may be useful for optimizing harvesting as well as lead to the breeding of palms with improved ripening profiles.

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MATERIALS AND METHODS

Plant Material. Oil palm fruits were derived from commercial crosses of Serdang Avenue dura and AVROS pisifera to yield hybrid tenera progeny. The fruit bunches were sampled from a commercial estate in Carey Island, Malaysia, at different developmental stages preceding, during, and after the major oil biosynthesis period, namely, 12, 14, 16, 18, 20, and 22 WAP. All fruitlets were separated from the bunches, and then 20 were randomly selected from each without bias to location in the bunch. Mesocarp tissues were harvested and snap frozen in liquid N2 to quench the metabolism of the plant tissues and enzyme activity and then stored in the dark at −80 °C for metabolite analysis and mRNA extraction. Prior to extraction, plant material (100 mg) was ground to a fine powder using a pestle and mortar under liquid nitrogen. Chemicals. Zeatin, gibberellins (GA3 and GA4), ICA, IAA, BA, ABA, MeIAA, SA, CA, MeBA, IBA, JA, MeSA, MeCA, MeJA, and OPDA were purchased from either Aldrich or Sigma Chemical Co. (St Louis, USA). Isotopically labeled internal standards including [2H5]IAA and [2H6]ABA and [2H5] trans-zeatin riboside were purchased from OlChemim Ltd. (Olomouc, Czech Republic). 8144

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Data Extraction, Normalization, and Comparisons. Raw microarray data were extracted from scanned images by using the Feature Extraction software (version 10.7.31; Agilent). The extracted microarray data were normalized using the Quantile normalization algorithm and analyzed using R (version 2.13.1). After normalization, comparisons of selected candidates between oil palm mesocarp microarray were carried out based on the normalized signal intensity produced.

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RESULTS AND DISCUSSION Hormone Metabolism during Oil Palm Development. Fruit ripening in oil palm is accompanied by lipid and carotenoid accumulation along with chlorophyll degradation.25 To determine the association of specific hormones with the timing of key ripening events, the major classes of plant hormones (ABA, auxins, cytokinins, and GA) in the mesocarp were profiled during the final stages of fruit development. Endogenous phytohormones occur only in a trace amount in plants. Consequently, for their analysis, sensitive mass spectrometric methods were combined with chromatographic separation techniques such as gas chromatography and high performance liquid chromatography. Unigenes also known to be involved in metabolism and response of multiple hormones including auxin, ethylene, gibberellins (GA), brassinosteroid (BR), jasmonic acid (JA), abscisic acid (ABA), and cytokinins (CK) were identified during mesocarp maturation from microarray expression data. Oil palm mesocarp tissue also had detectable levels of IAA, trans-zeatin, trans-zeatin glucoside, GA3, GA4, ABA, MeJA, and cinnamic acid. Changes in endogenous concentrations of GA, ABA, and cinnamic acid in mesocarp tissues during fruit growth and development are shown in Figure 1. Gibberellins. Gibberellins (GAs) are plant growth regulators that are known to stimulate physiological responses during plant development, such as shoot growth, flower development, dormancy release, and seed germination, as well as alter the source−sink metabolism through their effect on photosynthesis and sink formation.26 GA is also important in controlling seed maturation, germination, and seedling growth. While only trace amounts of GA3 were detected, interestingly GA3 and GA4 exhibited clear opposite trends during oil palm mesocarp development. GA3 appeared to be higher at Week 12 and decreased to almost undetectable levels at ripening.11 However, GA4 exhibited an opposite trend to GA3 as its concentration increased during fruit development and peaked at Week 18, followed by a gradual decline to Week 22. GA4 is the main bioactive hormone in cucumber27 and Arabidopsis28 and is also clearly important in oil palm fruit ripening. Several unigenes with annotated functions in gibberellin biosynthesis and responses were identified in the oil palm mesocarp transcriptome. Three genes that encode “DELLA proteins”, the repressor of GA responses, were identified and showed downregulated expression patterns toward maturation (Figure 2A). Unigenes encoding proteins homologous to GA20ox and GA3ox that convert the inactive precursors to bioactive GAs also showed mostly down-regulated expression patterns in mesocarp during fruit development after peaking at 18 WAP, concordant with the peak concentration of GA4 observed followed by a decline (Figure 1A in the Supporting Information). In particular, GA20ox exhibited much lower expression at 22 WAP. Interestingly GA2ox, which is involved in the catabolic pathway of GA4, exhibits a similar expression trend, peaking at 14−18 WAP and declining thereafter,

Figure 1. Hormone profiles during mesocarp development.

concordant with the lower levels of GA3 observed. The combination of lower expression of GA20ox and GA3ox resulting in less provision of GA4, combined with lower expression of GA2ox after week 18, may contribute to the dramatic lowering of GA3 concentrations at the last stage of ripening. Abscisic Acid. Liu et al. reported that high levels of endogenous ABA occur concurrently with rapid increases in the rate of fresh and dry weight accumulation in soybean seeds and coincide with the maximum rate of cell division.29 Several studies report an increase in free ABA levels during grape ripening concomitant with sugar accumulation and color changes.30 In oil palm, mesocarp also contained higher levels of ABA as lipid accumulated during ripening. ABA has been proposed as a possible trigger of ripening in some fruits as well.31 The specific role of ABA during fruit ripening remains obscure although ABA accumulation appears to be critical and participates in the regulation of ripening of both climacteric and nonclimacteric fruit. Transcription levels of 9-cis-epoxycarotenoid dioxygenase 1 gene, NCED1, in oil palm mesocarp were also found to be congruent with ABA content clearly indicating its importance to the ripening process of oil palm (Figure 1 and Figure 2B). It was previously observed by Chernys et al. that 8145

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Figure 2. (A) Gibberellin, (B) abscisic acid, (C) cytokinin, (D) auxin, (E) ethylene.

NCED1, a key enzyme involved in ABA biosynthesis, is upregulated during the ripening of several fruits, including avocado.32 In addition, two unigenes for ‘abscisic acid 8’hydroxylase were clearly down-regulated in mesocarp at 20−22 weeks, possibly leading to a decrease in ABA degradation (Supporting Information) allowing accumulation. The interaction between GA and ABA can have antagonistic effects according to Gao et al.33 Seed maturation and germination are not determined by ABA alone but instead by the ABA/GA ratio. ABA content increases in Arabidopsis seed as the embryo enters the maturation stage, and the resulting high ABA/GA ratio promotes maturation, induces dormancy, and inhibits cell-cycle progression, embryo growth, and germination.34 While in maize, it also appears to be the ABA/GA ratio and not the absolute hormone concentration that controls germination.35 For oil palm mesocarp, at the beginning of the maturation phase, ABA levels increase in fruits, while GA3 decreases, the resulting elevated ABA/GA3 ratio clearly coinciding with maturation (Figure 3).

Figure 3. ABA/GA3 ratio of palm mesocarp samples during 12−22 WAP. Data obtained from LC-MS-triplequad analysis.

Cytokinins. Cytokinins also have an important regulatory role in plant development and growth. They improve seed germination, release bud from apical dominance, and stimulate 8146

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Figure 4. Changes in polyamine levels and transcript expression in mesocarp across 6 time points.

leaf expansion and development of reproductive organs and retardation of senescence.36 Cytokinins have been reported to increase the number of pods/plant and seed weight in soybean. trans-Zeatin was the predominant cytokinin detected in oil palm mesocarp, followed by trans-zeatin glucoside. Contrary to ABA, the levels of trans-zeatin are high early in fruit development but decrease to very low or undetectable levels at maturation (Figure 1). trans-Zeatin glucoside exhibited different behavior, being detected at low concentration at weeks 12−16, peaking at week 18, followed by a decline until week 22 (Figure 1). Cytokinins are thought to be involved in berry set and in growth promotion and tend to inhibit fruit ripening.37 The levels of zeatin are high early in grape development but decrease rapidly to be low at around the time of ripening.37 Three unigenes encoding proteins homologous to “cytokinin dehydrogenase” and three unigenes for cytokinin-N-(or O)glucosyltransferase, which regulate homeostasis by deactivating cytokinin were identified in mesocarp. Of these, only cytokinin-N-glucosyltransferase 2 exhibited increased expression towards ripening (Figure 2). Hormonal analysis by Habib et al. revealed that various cytokinin derivatives such as transzeatin, trans-zeatin riboside, trans-zeatin O-glucoside, and transzeatin riboside 5′-monophosphate were significantly higher in

truncated oil palm leaf syndrome leaves compared to wild-type oil palm leaves.38 Jasmonic Acid. Jasmonic acid (JA) has been demonstrated to be involved in cross talk with ethylene signaling components in Arabidopsis and has been associated with fruit maturation. In climacteric and nonclimateric fruits, jasmonate levels increase during early fruit developmental stages, suggesting that jasmonate could be involved in cell division and other early fruit expansion processes. Generally, jasmonates and salicylates contribute to local and systemic defenses against pathogens, wounding, and stress responses and are involved in pollen and embryo development, seed germination, senescence, fruit ripening, and allelopathy.39 To our knowledge jasmonate has not been previously reported in the context of oil fruit ripening. Methyl jasmonic acid (MeJA) was found to increase after oil biosynthesis commences until ripeness in oil palm (Figure 1). JA and MeJA have been reported to retard cell division in soybean callus [Glycine max (L.) Merr.].40 We observed that 12-oxophytodienoate reductase 3, lipoxygenase 7, jasmonateinduced protein, and allene oxide synthase 1 and 2 are expressed at a higher level as compared to other jasmonic acid biosynthesis genes (Figure 1D in the Supporting Information). 8147

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Figure 5. Expression microarray data for genes which are involved in cell wall metabolism.

Auxin. In oil palm mesocarp it was reported that indole-3acetic acid (IAA) levels peak after pollination and then decline to very low levels in the ripe fruit.11 IAA was also found to be undetectable in this study of mesocarp after week 12. However, auxin biosynthesis genes were detected using expression microarray. A gene coding for an indole-3-acetic acid-amido synthetase GH3.8 exhibited the highest expression at week 18 but declined in expression toward ripeness (Figure 2D). Many genes coding for auxin and IAA proteins were down-regulated at week 22 suggesting that auxin levels are indeed lowered toward ripening. A transcript encoding IAA-amino acid hydrolase 1 (ILR1, 5, 6, and 7), which is putatively involved in IAA homeostasis, was down-regulated during fruit development (S1E). Auxin-response factors bind auxin-response elements of auxin-responsive genes and thus seem to act as regulators of gene transcription.41 In this study, several auxin response factors (ARFs 1, 2, 4, 6, 7, 9, 11, 12, 15, 16, 18, 19, 23) were down-regulated during the maturation stages except ARF 7, 17, and 23 (Figure 1E in the Supporting Information). The majority of transcripts related to auxin transport and perception

displayed decreased abundance in mesocarp (Supporting Information). Ethylene. Consistent with the critical roles of ethylene during fruit maturation and ripening, the majority of unigenes related to ethylene functions showed up-regulated expression patterns as maturation progressed. We found that one unigene encoding the EIN3-binding F-box protein in oil palm mesocarp samples, a negative regulator of ethylene action, exhibited down-regulated expression patterns (Figure 2E) along with upregulated expression patterns for most ethylene function related unigenes. Ethylene-responsive transcription factor 1 is found to be expressed highest among other genes related to ethylene pathways. Recent research reported by Tranbarger et al. shows that oil palm mesocarp exhibits characteristics of a climacteric fruit with an autocatalytic burst of ethylene coordinated transcriptional activity.11 Brassinosteroids. Lastly, brassinosteroids also regulate various aspects of plant growth and development, including cell elongation, photomorphogenesis, xylem differentiation, and seed germination as well as adaptation to abiotic and biotic environmental stresses.42 Brassinosteroid insentive 1-associated 8148

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processes such as cytoplasmic streaming, cell shape determination, organelle movement, and extension growth.51 We have also noted that amino acid levels are increased in high yielding fruits at weeks 12−14.20a Identified unigenes with annotated functions in cell wall metabolism represent another major transcriptomics change during mesocarp maturation. Fruit softening during ripening is associated with depolymerization and solubilization of cellulose microfibrils, hemicellulose, and pectins in the cell wall.52 Fruit softening during ripening results from the hydrolytic breakdown of cell wall polymers (e.g., celluloses, hemicelluloses, pectins) by hydrolases (e.g., polygalacturonase [PG], pectin methyl esterase, pectate lyase, rhamnogalacturonase, βgalactosidase).53 Pectolytic enzymes constitute a unique group of enzymes that are responsible for the degradation of pectin and pectic substances in plant cell walls. In peach, high levels of polygalacturonase and low levels of cellulose have been reported for the fruit ripening stage.54 Much research has focused on pectin degradation, resulting from the action of the ripening-related enzyme polygalacturonase, as the key element underlying the softening process. Expansin gene A-8, cellulose synthase-like protein, and polygalacturonase genes have the similar pattern of gene expression level in mesocarp fruits across the six time points in oil palm mesocarp (Figure 5). Our results also showed that the expression level of polygalacturonase inhibitor transcript exhibited an opposite trend to polygalacturonase. Polygalacturonase gene family members have been studied in fleshy oil palm fruit ripening and abscission recently by Roongsattham et al.55 They identified the expression level of five transcripts that encode PGs increased during ethylene treatments that induce cell separation. During avocado ripening the amount of high and intermediate sized polymers in the pectin fraction have been reported to decrease.18 β-Galactosidase expression decreased during ripening in oil palm mesocarp (Figure 5). β-Galactosidase may be involved in releasing stored energy for rapid growth, degrading cell wall components during senescence, and releasing free galactose during metabolic recycling of galactolipids, glycoproteins, and cell wall components.56 Caffeic acid 3-O-methyltransferase (COMT) decreased gradually from week 12 to 22 (Figure 5). Caffeoyl CoA 3-O-methyltransferase (CCOMT) also peaked at week 14 and decreased gradually thereafter (Figure 5). Strong down-regulation of COMT and CCOMT has been shown to result in decreased lignin content.57 Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix so that cell wall hydrolase enzymes can access, thus enhancing softening of, tissues.58 Expansins are encoded by a super family of genes that are divided into four families, namely, expansin A, expansin B, expansin like A, and expansin like B.58,59 Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato-ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. We found nine forms of expansin genes expressed during oil palm mesocarp development (Expansin B5, A2, A7, A8, A10, A15, like-A1, like-A2, and like-B1). In particular, expression levels of expansin gene-A8 increased significantly (Figure 5). Xie et al. found that expression and enzyme activity of cell wall hydrolases including

receptor kinase 1, brassinosteroid-regulated protein, and brassinosteroid LRR receptor kinase genes were detected in oil palm mesocarp (Figure 1F in the Supporting Information) and decreased from week 12 to 22. Polyamine Metabolism during Oil Palm Development. Polyamines, generally putrescine (Put), spermidine (Spd), and spermine (Spm), are polycationic compounds of low molecular weight that are present in living organisms. In plants, polyamines are present from micromolar (∼10 μM) to millimolar concentrations.43 They distribute differently among tissues and developmental stages, with Put and Spd being more abundant than Spm and cadeverine (Cad). Plant polyamines are considered as growth regulators, as they play fundamental roles in a wide range of growth, differentiation, and morphogenetic processes during the course of plant ontogeny. They have been shown to be involved in fruit enlargement, maturation, and ripening.15 Thus, the changes in endogenous free polyamines (Put, Spd, Spm, and ornithine) were monitored to examine their role during mesocarp development. Figure 4 shows the changes in polyamine levels and related gene expression in the oil palm mesocarp across the six time points. The concentrations of both ornithine and Spm remained relatively low compared to Spd and Put. Spd was higher than Put across time points, both increasing toward ripeness. Spermidine synthase (SpdSyn) is a key enzyme in polyamine biosynthesis and is responsible for Spd accumulation as also seen in Figure 4. The concentration profile of Spm also followed the expression levels of spermine synthase (SpmSyn). Along with accumulation of polyamines, arginine showed steady consumption after week 12. The pathway to Spd from methionine also revealed similar coordination with ripening. The increase in polyamines during oil palm fruit ripening suggests that endogenous polyamines may be involved in response to ethylene production.13 Quinet et al. found that a high level of Put concentration played an active role in triggering biosynthesis of ethylene.44 For oil palm mesocarp, Put, Spd, and Spm concentrations increased toward maturation along with ethylene.11 Contrary to oil palm, higher levels of polyamines are found in the initial fruit growth period, progressively declining toward maturation in several fruits including apple,16 grapes,45 tomato,12 mango,13 and peach.46 Polyamines are reported to be abundant during the cell division stage and decrease during the subsequent cell enlargement phase, ripening, and postharvest.47 However, the opposite trend seen in oil palm fruit in this study has been reported in mandarins and rambutans.48 In addition, Maria et al. recently reviewed that mature olive fruit abscission is associated with upregulation of polyamine metabolism.49 Cell Wall Metabolism. Plant cell wall metabolism, particularly cell wall degradation and modification, has long been associated with fruit ripening and softening. The complexity of the cell wall is encoded by multigene families that likely contribute to fruit cell wall metabolism including multiple steps such as substrate generation, synthesis and glycosyl transferase activities, secretory pathways, wall assembly, wall dynamics, and wall disassembly. Genes encoding for cell wall metabolism have been studied for their potential roles in fruit ripening and postharvest fruit softening. During oil palm mesocarp development, the cells continue to grow as they accumulate lipid.50 Actin gene expression seemed to increase between week 12 and week 14, remaining high thereafter (Figure 5). This may be related to cell expansion during lipid accumulation as actin is involved in a number of cellular 8149

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Figure 6. Schematic representation of the integration of polyamines, hormones, and their respective genes in mesocarp fruit ripening and abscission events. The numbers between brackets correspond to a gene/metabolite expression ratio of week 20 to week 12. Abbreviations: SAMDC, Sadenosylmethionine decarboxylase; SPDS, spermindine synthase; SPMS, spermine synthase; ODC, ornithine decarboxylase; AIH, agmatine iminohydrolase; ADC, arginine decarboxylase; CPA, N-carbamoylputrescine amidohydrolase; ACC, 1-aminocyclopropane-1-carboxylic acid; DcSAM, decarboxylated S-adenosyl methionine; ROS, reactive oxygen species; TFs, transcription factors; EIN, ethylene-insensitive; ERF, ethyleneresponsive transcription factor; EIL, EIN3-Like.

expansin and polygalacturonase were increased during the final stages of fruit development and abscission.4 Figure 6 shows the temporal expression patterns of hormones and polyamines between weeks 12 and 20. Almost all the polyamines and hormones exhibited increased expression, except GA3, sucrose, arginine, and L-methionine. The ratio of genes coordinated in regulation of these hormones and polyamines was found to be concordant with the ratio value of their metabolites in most cases. Among the hormones and polyamines metabolites, a few were evident as the most critical for ripening in oil palm: Spd (7.9) (driven by SAM (4.4)) and GA4 (7.0) showing the most differential, followed by methyl jasmonate (5.5) and ABA (3.6). As discussed earlier, the effect of ABA may be more pronounced by its ratio with GA3 increasing even more markedly at 20 WAP. Xie et al. reported cross-talk among the hormones, saccharides, and polyamines in apple, mango, and citrus.4 It can be seen that their levels in oil palm can be related to lipid biosynthesis commencement first and later to fruit maturation (softening and abscsission). In summary, this work describes the combined analysis of transcripts and metabolites contributing to the involvement of hormones, polyamines, and cell wall metabolism during oil palm fruit ripening from 12 weeks after pollination (WAP). Figure 7 summarizes the changes in total oil content, amino acids, organic acids, polyamines, and hormones in the three main stages of development: lag phase (12−14 WAP), maturation (16−18 WAP), and finally ripening (20−22 WAP). Lag phase (12−14 WAP) occurs after cell division and cell expansion, where peak lipid biosynthesis starts during maturation (16−18 WAP), reaching maximum lipid concentrations at ripening (20−22 WAP). Figure 7 clearly shows the key developmental changes related to oil biosynthesis at weeks 14−16 and then ripening at weeks 20−22 and indicates how hormones and polyamines are coordinated. Some are

Figure 7. Summary of total oil, amino acids, organic acids, polyamines, and hormones in the mesocarp tissue during oil palm fruit development.

concordant with the onset of lipid biosynthesis at 16 WAP, such as GA4 concentration, while others are concordant with the final stages of ripening (fruit softening and abscission), most notably ABA and the ABA/GA3 ratio as well as 8150

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(7) Hauser, F.; Waadt, R.; Schroeder, J. I. Evolution of Abscisic Acid Synthesis and Signaling Mechanisms. Curr. Biol. 2011, 21 (9), R346− 55. (8) Chao, C.-C. T.; Khuong, T.; Zheng, Y.; Lovatt, C. J. Response of evergreen perennial tree crops to gibberellic acid is crop loaddependent. I: GA3 increases the yield of commercially valuable ‘Nules’ Clementine Mandarin fruit only in the off-crop year of an alternate bearing orchard. Sci. Hortic. (Amsterdam, Neth.) 2011, 130 (4), 743− 752. (9) Huntley, R. P.; Jones, L. H.; Hanke, D. E. Cytokinins and gibberellins in sap exudate of the oil palm. Phytochemistry 2002, 60 (2), 117−27. (10) Habib, S.; Ooi, S. E.; Novák, O.; Tarkowská, D.; Rolčík, J.; Doležal, K.; Syed-Alwee, S.; Ho, C. L.; Namasivayam, P. Comparative mineral and hormonal analyses of wild type and TLS somaclonal variant derived from oil palm (Elaeis guineensis Jacq. var. tenera) tissue culture. Plant Growth Regul. 2012, 68, 313−317. (11) Tranbarger, T. J.; Dussert, S.; Joet, T.; Argout, X.; Summo, M.; Champion, A.; Cros, D.; Omore, A.; Nouy, B.; Morcillo, F. Regulatory mechanisms underlying oil palm fruit mesocarp maturation, ripening, and functional specialization in lipid and carotenoid metabolism. Plant Physiol. 2011, 156 (2), 564−84. (12) Morilla, A.; García, J. M.; Albi, M. A. Free Polyamine Contents and Decarboxylase Activities during Tomato Development and Ripening. J. Agric. Food Chem. 1996, 44 (9), 2608−2611. (13) Malik, A. U.; Singh, Z. Endogenous Free Polyamines of Mangos in Relation to Development and Ripening. J. Am. Soc. Hortic. Sci. 2004, 129 (3), 280−286. (14) Ponappa, T.; Miller, A. R. Polyamines in normal and auxininduced strawberry fruit development. Physiol. Plant. 1996, 98 (3), 447−454. (15) Liu, J.; Nada, K.; Pang, X.; Honda, C.; Kitashiba, H.; Moriguchi, T. Role of polyamines in peach fruit development and storage. Tree Physiol. 2006, 26 (6), 791−8. (16) Biasi, R.; Bagni, N.; Costa, G. Endogenous polyamines in apple and their relationship to fruit set and fruit growth. Physiol. Plant. 1988, 73 (2), 201−205. (17) Payasi, A.; Mishra, N.; Chaves, A.; Singh, R. Biochemistry of fruit softening: an overview. Physiol. Mol. Biol. Plants 2009, 15 (2), 103−113. (18) Sakurai, N.; Nevins, D. J. Relationship between Fruit Softening and Wall Polysaccharides in Avocado (Persea americana Mill) Mesocarp Tissues. Plant Cell Physiol. 1997, 38 (5), 603−610. (19) (a) Sharma, N.; Anderson, M.; Kumar, A.; Zhang, Y.; Giblin, E. M.; Abrams, S. R.; Zaharia, L. I.; Taylor, D. C.; Fobert, P. R. Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts. BMC Genomics 2008, 9, 619. (b) Jadhav, A. S.; Taylor, D. C.; Giblin, M.; Ferrie, A. M.; Ambrose, S. J.; Ross, A. R.; Nelson, K. M.; Irina Zaharia, L.; Sharma, N.; Anderson, M.; Fobert, P. R.; Abrams, S. R. Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7′-, 8′- and 9′-hydroxy ABA in microspore derived embryos of B. napus. Phytochemistry 2008, 69 (15), 2678−88. (c) Gutierrez, L.; Van Wuytswinkel, O.; Castelain, M.; Bellini, C. Combined networks regulating seed maturation. Trends Plant Sci. 2007, 12 (7), 294−300. (20) (a) Teh, H. F.; Neoh, B. K.; Hong, M. P.; Low, J. Y.; Ng, T. L.; Ithnin, N.; Thang, Y. M.; Mohamed, M.; Chew, F. T.; Yusof, H. M.; Kulaveerasingam, H.; Appleton, D. R. Differential metabolite profiles during fruit development in high-yielding oil palm mesocarp. PLoS One 2013, 8 (4), e61344. (b) Loei, H.; Lim, J.; Tan, M.; Lim, T. K.; Lin, Q. S.; Chew, F. T.; Kulaveerasingam, H.; Chung, M. C. Proteomic analysis of the oil palm fruit mesocarp reveals elevated oxidative phosphorylation activity is critical for increased storage oil production. J. Proteome Res. 2013, 12 (11), 5096−109. (21) Pan, X.; Welti, R.; Wang, X. Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 2008, 69 (8), 1773−81.

polyamines. These results provide a valuable contribution for the understanding of key genes and expression timing changes related to the regulation of the complex process of mesocarp ripening, in particular the initiation of peak oil biosynthesis and final ripening before harvesting. In summary, we conclude that (1) high polyamine concentrations coincide with lipid accumulation and maturation; (2) ABA is concordant with GA4 but not GA3 during fruit development, and the resulting elevated ABA/GA3 ratio appears to promote maturation; (3) ethylene-responsive transcription factor 1,9-cis-epoxycarotenoid dioxygenase 1 (NCED1), cytokinin-N-glucosyltransferase 2, and ethylene-responsive transcription factor 1 were all expressed higher in up-related pattern until week 20; (4) polygalacturonase, expansin A-8, cellulose synthase-like protein, and actin genes clearly increase toward ripening. The elucidation of the specific promoter binding sites and transcription factors that are in common between genes that exhibit related expression profiles during ripening or transcription factors modulated by hormone concentrations may lead to the identification of important timing regulators of oil biosynthesis and ripening. These will in turn assist in the development of fruit that ripens faster and/or produces more oil.

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ASSOCIATED CONTENT

* Supporting Information S

Expression microarray data for hormone genes in developing oil palm fruit. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +603-89422641. Fax: +603- 89431867. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank their colleagues in SDRC for their valued input and discussions, especially the Breeding group and the harvesting team (SDRC). We also thank Human Metabolome Technologies (Japan) for CE-MS analysis.

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REFERENCES

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