Identification and Expression of Skinning Injury-responsive Genes in Sweetpotato

in Journal of the American Society for Horticultural Science
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  • 1 Faculty of Agriculture, Pattimura University, Jl. Ir. M. Putuhena, Ambon 97233, Indonesia; and the Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, Dramaga-Bogor, Indonesia
  • 2 School of Plant, Environmental, and Soil Sciences, Louisiana State University Agricultural Center, 131 J.C. Miller Hall, Baton Rouge, LA 70803

Skinning injury in sweetpotatoes (Ipomoea batatas) is responsible for significant postharvest loss resulting from storage diseases and weight loss. Unfortunately, there is no report on the genes involved in wound healing of sweetpotato and a better understanding will facilitate improved breeding strategies. An annealing control primer (ACP) system was used to identify genes expressed after skinning injury of sweetpotato cultivar LA 07-146 storage roots. Using 20 ACPs, 63 differentially expressed genes (DEGs) were identified. Functional annotation of the DEGs revealed that genes previously shown to respond to dehydration, those involved in wounding response, and the lignin and suberin biosynthesis pathways were induced in response to skinning. Expression analysis of 18 DEGs through quantitative reverse transcription–polymerase chain reaction (PCR) showed that DEGs involved in lignin and suberin pathways were up-regulated after 8 and 12 hours of skinning. Other genes showed up- or down-regulation in their transcript abundance depending on the time the storage root was sampled after intentional skinning. The genes up-regulated in response to skinning may be useful to identify expression markers for screening sweetpotato lines tolerant to skinning injury in breeding programs.

Abstract

Skinning injury in sweetpotatoes (Ipomoea batatas) is responsible for significant postharvest loss resulting from storage diseases and weight loss. Unfortunately, there is no report on the genes involved in wound healing of sweetpotato and a better understanding will facilitate improved breeding strategies. An annealing control primer (ACP) system was used to identify genes expressed after skinning injury of sweetpotato cultivar LA 07-146 storage roots. Using 20 ACPs, 63 differentially expressed genes (DEGs) were identified. Functional annotation of the DEGs revealed that genes previously shown to respond to dehydration, those involved in wounding response, and the lignin and suberin biosynthesis pathways were induced in response to skinning. Expression analysis of 18 DEGs through quantitative reverse transcription–polymerase chain reaction (PCR) showed that DEGs involved in lignin and suberin pathways were up-regulated after 8 and 12 hours of skinning. Other genes showed up- or down-regulation in their transcript abundance depending on the time the storage root was sampled after intentional skinning. The genes up-regulated in response to skinning may be useful to identify expression markers for screening sweetpotato lines tolerant to skinning injury in breeding programs.

Sweetpotato is a genetically complex clonal crop with incompatibility that presents a bottleneck in backcrossing desirable traits into otherwise superior cultivars. Breeding programs thus rely on open-pollinated, mass selection techniques to improve the mean performance of successive generations for important traits (Jones et al., 1986). This successful approach, begun in the late 1930s, has surmounted many obstacles limiting industry growth. Nevertheless, sweetpotato breeding programs in Asia rely on biparental crossing to generate a breeding population. Breeding programs have consistently pursued the following goals: 1) higher storage root yield; 2) storage roots with a consistent spindle-like shape and attractive smooth skin; 3) field and storage disease resistance; 4) long-term storage quality; 5) good culinary attributes; and 6) insect resistance (Collins and Hall, 1992). Reducing labor inputs is a more abstract goal today and implies further mechanization in production. Developing a cultivar that can withstand the rigors of bulk harvesting builds on these goals; skinning resistance is a prerequisite.

Our strategy is to screen for and breed for cultivars with a more durable skin, which can resist skinning, cosmetic desiccation at wound sites, and fresh weight loss. This combination would lessen loss and allow for a more roughly handled storage root. There are cultural practices that lessen skinning such as removing vines 5 to 7 d before harvest (La Bonte and Wright, 1993), but entire fields can decay if heavy rains occur between devining and harvest. Tough-skinned lines have been avoided in breeding programs because of presumed marketplace aversion, although no attempts have been made to test acceptability. A tougher skin may enable us to avoid wounding and enhance harvest mechanization, particularly for processors in the United States. From a global perspective, sweetpotato is mostly hand-harvested with minimal use of machinery; however, post-harvest loss is particularly high in developing countries. For example, shelf life of East African sweetpotato lasts no more than 1 to 2 weeks (Rees et al., 2008). Studies showed that reduced root weight by water loss is associated with a higher rate of rot in East Africa cultivars and weight loss ranges from 8% to 30% after only 2 weeks (Rees et al., 2008). This represents a tangible loss of marketable product. Such data are not available for Asia; however, trends are likely similar. Skinning tolerance is thus equally important in developed and developing countries.

Skinning is inevitable and the rapid deposition of wound periderm at the injury site is critical. Wound periderm, which is similar to native periderm, forms best at 28 to 30 °C and relative humidity above 85% (Kushman and Wright, 1969). Wound healing has been negatively correlated with decreased dry matter content (van Oirschot et al., 2006) and positively correlated with increased sugar levels at the wound site (Rees et al., 2008). van Oirschot et al. (2006) found that the thickness of the desiccated cell layer and the depth of the lignified layer are affected by cultivar and humidity. Lignified layers are not always produced (or discontinuous) in some cultivars and continuity is more important than thickness (van Oirschot et al., 2006). A sweetpotato cultivar with a more durable skin and superior wound healing characteristics would reduce postharvest loss and enable the U.S. industry to move toward mechanization, whereas the goal in developing countries is to extend storage, transport, and marketability of sweetpotato. The popular U.S. cultivar Beauregard is superior to local cultivars in East African trials when assayed for wound healing (Rees et al., 2008); however, U.S. producers consider it susceptible to skinning injury and not ideal for highly mechanized harvest.

A critical adjunct to breeding is to understand the genetics behind wound healing. Lignification and increased sugar at the wound site have been shown to be correlated with wound healing (Rees et al., 2008). Little is otherwise known. Among several transcript profiling technologies, an ACP system is a recently developed simple, sensitive, and reproducible PCR-based method that allows identification of differentially expressed genes in certain biological processes (Kim et al., 2004). The ACP system provides a suitable primer with annealing specificity that specifically targets the template sequence for hybridization through a polydeoxyinosine [poly(dI)] linker. The principle of ACP technology is based on a unique tripartite structure of a specific oligonucleotide with its 3′ and 5′ ends separated by a regulator and the interaction of each end of this primer during a two-stage PCR. Because of the high annealing specificity during PCR, the application of the ACP to DEG identification generates reproducible, accurate, and long (100 bp to 2 kb) PCR products that are detectable on agarose gels (Kim et al., 2004). This technique has been used to identify abundant, transient, and rarely expressed candidate genes induced in response to Aspergillus flavus infection in cotton [Gossypium hirsutum (Lee et al., 2012)] and petroleum hydrocarbon exposure in Spartina alterniflora (Ramanarao et al., 2011). The objective in this study was to directly identify critical physiological processes involved in the healing process by examining gene expression changes after intentional skinning.

Materials and Methods

Plant materials and skinning treatment.

Freshly harvested storage roots of sweetpotato cultivar LA 07-146 were washed, blot-dried, and carefully scraped with a razor scraper (Titan 11030; Star Asia-USA, Renton, WA) to remove the thin outer pigmented skin and stored at room temperature (24 ± 1 °C), 50% relative humidity, and 400 lx light. ‘LA 07-146’ was chosen for this study because this cultivar is uniquely suited to sweetpotato french fry processing and skins when bulk-harvested. The entire roots were skinned and the skinned roots were peeled to the same thickness (1.2 mm) at 0 (control), 2, 4, 8, and 12 h after skinning and the peels were immediately frozen in liquid nitrogen and stored at –80 °C for RNA extraction. Three independent roots were used for each time point as replicates.

RNA Isolation, cDNA preparation, and ACP-based gene-fishing PCR.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the instructions of the manufacturer, except for replacing the RLT lysis buffer with RLC lysis buffer. The quantity and quality of the total RNA were determined using a spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE). An aliquot of 2 μg RNA of three biological replicates extracted from storage root tissues for each time point was pooled to capture rare transcripts that may have escaped in one of the replicates as a result of technical errors during gene fishing experiment. Also, 3 μg RNA extracted from storage roots at 8 and 12 h were pooled together to make the sample size four for convenience with PCR handling during gene fishing but were used independently during subsequent gene expression analysis.

First-strand cDNA synthesis was performed using a GeneFishingTM DEG premix kit (Seegene, Rockville, MD) as previously described (Ramanarao et al., 2011). Briefly, 3 μg of RNA representing different time points (0, 2, 4, 8, and 12 h) was reverse-transcribed to first-strand cDNA at 42 °C for 90 min in a final reaction volume of 20 μL containing 1× reaction buffer, 2 mm dNTP mix, 20 U RNase inhibitor (Promega, Madison, WI), 2 μL of 10 μM (dT)15-ACP1 (Seegene), and 1 μL Moloney murine leukemia virus transcriptase [200 U/μL (Promega)]. The first-strand cDNAs were diluted 5× with nuclease-free water for further use.

Second-strand cDNA synthesis and subsequent PCR amplification were performed in a single tube as previously described (Ramanarao et al., 2011). Second-strand cDNA was synthesized by first-stage PCR followed by a second-stage PCR in a final reaction volume of 20 μL containing 50 ng of the diluted first-strand cDNA, 1× SeeAmp ACPTM master mix (Seegene), 1 μL of 10 μM dT-ACP2, and 2 μL of 5 μM arbitrary ACPs [Seegene (Ramanarao et al., 2011)]. The tube containing the reaction mixture was placed in a preheated (94 °C) thermal cycler. The thermal profile for the first-stage PCR was: one cycle at 94 °C for 1 min followed by 50 °C for 3 min and 72 °C for 1 min. The second-stage PCR amplification profile was: 40 cycles of 94 °C for 40 s followed by 65 °C for 40 s, 72 °C for 40 s, and a 5-min final extension at 72 °C. The amplified PCR products were resolved in a 2% agarose gel. Twenty ACP primers were used during the second-stage PCR in the present study to capture the DEGs.

Cloning and sequencing of DEGs.

Seventy fragments corresponding to DEGs, based on their intensity or presence/absence between control (0 h, immediately after skinning) and skinning samples, were excised from the gel and extracted by using a Qiaquick gel extraction kit (Qiagen). The DEGs were cloned into pGEM®-T Easy vector (Promega) following the method described by Baisakh et al. (2006). A total of 250 positive colonies from 70 DEGs were confirmed by colony PCR using M13F/R primers. Plasmids isolated from 119 independent clones were single-pass sequenced with T7 primer in an ABI 3730x1 genetic analyzer (Applied Biosystems, Foster City, CA).

DNA sequences were processed manually to remove the vector backbone and the poly (A) tail. Functional annotation of the DEGs was performed by interrogating their sequences against the non-redundant nucleotide and protein database of National Center for Biotechnology Information using BLASTN and BLASTX (Altschul et al., 1990) with a cutoff e-value of 1e-10.

Semiquantitative reverse transcription–polymerase chain reaction (sqRT-PCR) analysis of DEGs.

Transcript abundance was determined for 18 DEGs with known function annotation by sqRT-PCR (Table 1). For reverse transcription, 1 μg of total RNA isolated from storage roots at different time points was reverse-transcribed independently to first-strand cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Two microliters of the first-strand cDNA was used for PCR analysis using DEG-specific primers (Table 1) designed using Primer 3.0 (Rozen and Skaletsky, 2000). The PCR conditions were the same (annealing at 55 °C for 35 cycles) as described earlier (Ramanarao et al., 2011).

Table 1.

Differentially expressed genes (DEGs) induced in response to skinning in sweetpotato storage roots and the corresponding primer sequences.

Table 1.

Quantitative reverse transcription–PCR (qRT-PCR) of DEGs.

The qRT-PCR, being a more sensitive method for detection of transcript accumulation, was also performed to validate the sqRT-PCR data using the same cDNA samples that were used for sqRT-PCR as per the method described earlier (Ramanarao et al., 2011). PCR analysis was carried out with three independent roots (collected from three different plants) as replicates using SYBR green master mix (Bio-Rad), 2 μL of cDNA, and 3.25 pmol DEG-specific primers (Table 1) in a MyiQ real-time PCR analyses system (Bio-Rad). The relative expression ratio was calculated using the 2-ΔΔCt method (Ramanarao et al., 2011) with sweetpotato elongation factor gene (Solis, 2012) as an internal reference gene and control (0 h) as the calibrator. A serial dilution of cDNA was used to prepare a standard curve for determination of the PCR efficiency as described by Baisakh et al. (2008).

Results and Discussion

Isolation of DEGs under skinning injury.

All 20 ACP primers used in the experiment resulted in amplification of DEGs in storage roots after skinning injury (Fig. 1). ACP2, 6, 14, 15, and 17 resulted in most DEGs, and 8- and 12-h time points produced the most DEGs. In total, 70 unambiguous and reproducible DEGs (58 up-regulated and 12 down-regulated) were identified and 119 independent clones (from 42 DEGs) with an average size of ≈400 bp were sequenced. All sequences produced quality reads. A sequence similarity search revealed that 101 sequences (from 39 DEGs) matched plant-specific cDNA sequences. These DEGs represented 63 unigenes: 19 contigs (assembled sequences that were overlapping by 50 nt) and 44 singlets (that did not have any assemble into a contig) with an overall redundancy (frequency of exactly identical sequences) of 29.7%. Functional annotation showed that 48 DEGs were annotated to genes with known functions in the public domain and the remaining 15 DEGs matched with expressed/hypothetical proteins with unknown functions. The DEGs represented genes involved in dehydration response of plants (transcription factors and protein kinases), storage protein genes, and wound-responsive genes.

Fig. 1.
Fig. 1.

Representative gels from polymerase chain reaction (PCR) with annealing control primers (ACP) showing differentially expressed genes (DEGs) in sweetpotato storage root at 0, 2, 4, and 8 (+12) h after skinning injury. Upward and downward arrows indicate up-regulated and down-regulated DEGs, respectively; M = 1-kb DNA size marker.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.210

The genes encoding wound-induced protein [translationally controlled tumor protein (TCTP)—IbSIn59; transaldolase (TAL)—IbSIn46] and storage protein [sporamin B (Spor B)—IbSIn60] were most abundant followed by the signaling genes. The genes that were identified in response to skinning injury in sweetpotato root were divided into three categories: genes with early response, genes with late response, and genes with transient expression. Genes included in the early response group included those involved in abiotic stresses [early light-inducible protein (ELIP3)—IbSIn61a, glutamate decarboxylase (GDC)—IbSIn61b, serine-rich protein (SRP IbSIn21)] and wound-induced and storage protein genes such as Spor B, cytochrome P450 76C4 (Cyt P450)—IbSIn30b, ribonucleoprotein complex subunit 3 (RNP)—IbSIn56, nitrogen fixation unit (NifU)—IbSIn40, and TCTP. The transitory genes included the ones involved in transcriptional regulation and signaling such as thioredoxin H2 (TH2)—IbSIn04, basic Helix-loop-helix (bHLH)—IbSIn30a, and IbRPK. The late response genes included those involved in the lignin and suberin biosynthesis pathway [caffeic acid 3-O-methyl transferase (CCOMT), phenylalanine ammonia lyase (PAL), extensin (Ext)], which are most likely induced as a healing mechanism by deposition of suberin and lignin at the injury site. In brief, skinning induces a complex temporal genetic response in sweetpotato roots.

Transcript abundance analysis of DEGs.

Semiquantitative and qRT-PCR analysis showed differential transcript accumulation of the 18 DEGs in storage roots at different time points after skinning injury (Figs. 2 and 3). This suggested that all of these genes were involved in stress-responsive pathways. The sq- and qRT-PCR results were comparable (Figs. 2 and 3). Most of the 18 genes showed significant up-regulation in the roots 8 and 12 h after skinning (Figs. 2 and 3). IbSIn40, similar to the nifU gene, was induced only at the 12-h time point. A few transcripts showed increased accumulation immediately on imposition of skinning injury, whereas some others showed low abundance after 2 and 4 h of skinning, but then were highly up-regulated at 8 and/or 12 h.

Fig. 2.
Fig. 2.

Semiquantitative reverse transcription polymerase chain reaction (PCR) analysis of differentially expressed genes in storage root of sweetpotato at 0, 2, 4, 8, and 12 h after skinning injury. Elongation factor gene (EF) was used as the endogenous reference for cDNA loading control.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.210

Fig. 3.
Fig. 3.

Expression of differentially expressed genes in storage root of sweetpotato at 2, 4, 8, and 12 h relative to 0 h after skinning. HRS = hepatocyte growth factor-regulated tyrosine kinase substrate (IbSIn69); Cyc = cyclophilin (IbSIn48); PPI CYP40 = peptidyl-prolyl cis-trans isomerase CYP40 (IbSIn 29); GDC = glutamate decarboxylase (IbSIn61b); TAL = transaldolase (IbSIn46); ELIP3 = early light-inducible protein (IbSIn61a); CCOMT = caffeic acid 3-O-methyl transferase; PAL = phenylalanine ammonia lyase; Ext = extensin, RPK = ribonucleoprotein complex subunit (IbSIn15); SRP = serine-rich protein (IbSIn21); bHLH = basic Helix-loop-helix (IbSIn30a); Cyt P450 = cytochrome P450 76C4 (IbSIn30b); NifU = nitrogen fixation unit (IbSIn40); Spor B = sporamin B (IbSIn60); TCTP = translationally controlled tumor protein (IbSIn59); TH2 = thioredoxin H2 mRNA (IbSIn04); RNP = ribonucleoprotein complex subunit 3 (IbSIn56). The error bars represent se.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 3; 10.21273/JASHS.138.3.210

Transcript abundance analysis of stress-responsive genes involved in abiotic stresses.

A number of DEGs similar to genes known to be involved in biotic and/or abiotic stress responses of plants were differentially expressed under skinning injury in sweetpotato storage root. IbSIn04, the DEG similar to thioredoxin (TH2) mRNA, showed up-regulation in its transcript accumulation in root tissues at 8 and 12 h, which was evident from both sq- and q-RT-PCR. At 2 h after injury, the transcript accumulation was almost undetected in the root. In plants, TH2 has a number of functions: mobilization of protein reserves such as storage proteins and as enzyme inhibitors during germination (Huang et al., 2004). Possible diverse roles and functions of three TH2 genes in storage roots of sweetpotato were substantiated by their differential expression patterns in response to developmental and environmental cues (Huang et al., 2004).

The cDNA (IbSIn21) encoding SRP showed up-regulation in response to skinning injury; however, the fold-change up-regulation was low compared with other transcripts (Fig. 3). SRP has a role in alternative mRNA splicing by binding to specific RNA sequences and assembling the spliceosome at weak splice sites (Golovkin and Reddy, 1998). The precise recognition and removal of introns from precursor mRNA during alternate splicing is an important process essential for the expression of eukaryotic genes under different environmental stresses (Gupta et al., 2005).

The transcript of IbSIn48, similar to cyclophilin gene (Cyc), was at its peak at 8 h before declining at 12 h. Cyc is a housekeeping gene, which was found to be identical to peptidyl-prolyl cis-trans isomerase (PPI) that catalyzes the cis-trans isomerization of proline peptide bonds in oligopeptides by accelerating the rate proteins fold into their native conformation (Helekar and Patrick, 1997). Another DEG, IbSIn29, similar to PPI (PPI CYP40), had 2.4- and 2.5-fold higher mRNA abundance in 8 and 12 h compared with the control (0 h). Cyc has been shown to play important roles in stress response in plants through mRNA processing, protein degradation, and signal transduction (Romano et al., 2004). PPI CYP40 mRNA synthesis was found to be significantly stimulated by chemical stresses in maize (Zea mays) and bean (Phaseolus vulgaris) plants (Marivet et al., 1992).

IbSIn61a was similar to a cDNA for early light-inducible proteins (ELIP3), which showed a sequential increase in its mRNA accumulation with the highest increase at 12 h. Although the function of ELIP3 has not been firmly established, accumulating data suggest a role in endogenous circadian rhythm (Tao et al., 2011) and photoprotection, especially under various stress conditions such as heat, cold, salinity, and desiccation (Pinto et al., 2011; Tao et al., 2011). Alamillo and Bartels (2001) reported that Elip-like protein dsp 22 accumulated in the PSII in response to photoinhibition damage caused by desiccation. The highest expression of ELIP3 at 12 h (7.8-fold) is likely the result of the desiccation after skinning of the root and was probably associated with the carotenoid pigment zeaxanthin, thereby protecting the tissues against further damage.

A cDNA (IbSIn61b) with 75% similarity (3e-12; 53.6 bit score) to Nicotiana tabacum glutamate decarboxylase (GDC) accumulated 3.0-fold higher transcript levels in root at 8 h compared with that at 0 h before declining after 12 h of skinning. Gad has an important role in the gamma aminobutyric acid (GABA) shunt, a Ca2+/CaM-regulated pathway of plants engaged in abiotic stress response (Bouche and Fromm, 2004). Additionally, the root-specific Arabidopsis thaliana isoform Gad1 is involved in GABA synthesis under normal growth conditions and in response to heat stress (Bouche et al., 2004). The evidence that GABA accumulates under several environmental conditions such as hypoxia, cold shock, heat shock, mechanical stimulation, water stress, and darkness (Hoeflich and Ikura, 2002) indicates a potential role of GDC in the desiccation tolerance followed by skinning in sweetpotato.

The cDNA (IbSIn60), similar to sporamin B (Spor B), showed down-regulation of its transcript accumulation up to 8 h after skinning and a slightly increased mRNA accumulation in storage roots at 12 h. Spor B constituted 4.8% of the DEGs isolated in response to skinning injury. Sporamin is the main storage protein found in sweetpotato storage roots (Shewry, 2003), and Chen et al. (2008) showed that sporamin transcripts are systemically induced in leaf tissue of sweetpotato by wounding. In addition to its role as a storage protein, sporamin plays a defense role as a protease inhibitor (Hattori et al., 1989; Maeshima et al., 1985).

The transcript abundance of DEG IbSIn46, which showed sequence similarity to transaldolase (TAL), showed a slight up-regulation at 2 and 4 h after skinning but then increased at 8 and 12 h (2.9- and 3.4-fold, respectively). Studies using various potato tissues showed that TAL accumulation was enhanced in the stem relative to the leaves or tubers (Moehs et al., 1996). TAL’s possible role in increasing flux through the shikimate pathway in response to pathogen-induced wounding suggested that TAL participates in plant defense mechanisms in production of secondary metabolites (Caillau and Quick, 2005). A high occurrence of TAL (6.4% of DEGs) in sweetpotato root and the late overexpression of TAL suggested that its expression was a result of temporal response to wounding caused by skinning injury.

TCTP is often designated as a stress-related protein because of its highly regulated expression. The cDNA IbSIn59, similar to TCTP, showed an up-regulation only at 8 h after skinning. Of 63 DEGs regulated in response to skinning injury in sweetpotato, 7.9% represented TCTPs. Overexpression of TCTP in bacterial cells helped protect the bacteria from heat shock-induced damage by acting as a molecular chaperone (Gnanasekar et al., 2009). High occurrence of TCTPs may have a significant role in binding and protecting the native protein of sweetpotato storage roots from degradation (Gnanasekar et al., 2009).

Transcript abundance analysis of genes involved in lignin and suberin biosynthesis.

The formation of a lignified layer after skinning prompted us to test the expression of other genes involved in the lignin and suberin pathways that were not captured through this ACP-based gene fishing. CCOMT, Ext, and Cyt P450, but not PAL, involved in the lignin and suberin biosynthesis were induced only after 8 h of skinning. Up-regulation of these genes at a later stage of skinning injury indicates an onset of the biosynthetic pathway leading to the development of a suberin and lignin layer after skinning.

The expression of IbSIn30b, similar to cytochrome P450 76C4-like (Cyt P450), was undetectable at 2 and 4 h of skinning but was induced at 8 and 12 h (1.6-fold) of skinning injury. Of the 63 plant-related DEGs, 6.4% are Cyt P450. In plants, the Cyt P450 family protein plays important roles in biosynthetic pathways, including those of oxygenated phenylpropanoids (Schuler, 1996). This Cyt P450 76C4-like gene is a member of superfamily of mono-oxygenases that plays an important role in suberin biosynthesis. Up-regulation of this gene at 12 h of skinning injury indicates an onset of the biosynthetic pathway that would eventually lead to development of a suberin layer after skinning in the root.

Caffeic acid 3-O-methyltransferase transcript was slightly induced at 2 and 4 h of skinning but was highly overexpressed after 8 and 12 h of skinning. CCOMT plays a pivotal role in the lignin biosynthetic pathway by catalyzing the multistep methylation reactions of hydroxylated monomeric lignin precursors. Similarly, IbPAL coding for phenylalanine ammonia lyase in sweetpotato was highly induced in response to skinning injury. PAL catalyzes phenylalanine to produce cinnamic acid, the first rate-limiting step in lignin biosynthesis. Additionally, PAL is induced by various biotic and abiotic stresses and it modulates the resistance to stresses by regulating the biosynthesis of phenolic compounds. Unlike in A. thaliana in which PAL is expressed constitutively, PAL expression in storage roots of sweetpotato was up-regulated within 4 h and reached its maximum level at 12 h in response to skinning injury (Cochrane et al., 2004).

A sweetpotato gene coding for Ext also was induced after 8 h of skinning in sweetpotato. In plants, extensins are structural cell wall proteins that accumulate in cells under mechanical injuries (Guzzardi et al., 2004), in response to wounding, and in cells proliferating under hormone control. It can be hypothesized that IbExt in sweetpotato is induced as the cell wall starts to thicken in response to mechanical wounding caused by skinning.

Transcript abundance analysis of genes involved in transcriptional regulation/signaling.

Differentially expressed genes showing sequence similarity with transcription factors and signaling genes showed low expression levels on skinning (Figs. 2 and 3).

The DEG IbSIn30a, similar to basic helix-loop-helix (bHLH), was induced at low levels after 2 and 4 h of skinning and almost doubled its expression at 8 h and maintained this level of expression through 12 h (Figs. 2 and 3). Plant bHLHs are a multigene family of transcription factors, which have been implicated in wounding stress response in different plant species such as rice [Oryza sativa (Kiribuchi et al., 2005)]. The OsbHLH148 transcription factor positively regulated OsJAZ proteins involved in drought and wound tolerance in rice (Seo et al., 2011). Seo et al. (2011) showed that the induction of OsbHLH148 in response to wounding was “slight,” which is consistent with the present sweetpotato bHLH expression results. This suggested that the bHLH gene was induced in response to the wound injury caused by skinning in sweetpotato.

Transcript abundance of IbSIn56, which showed sequence similarity to ribonucleoprotein (RNP), was down-regulated immediately after skinning but showed a slight up-regulation only at 12 h (Figs. 2 and 3). RNP complexes regulate post-transcriptional control mechanisms in the cell nucleus and cytoplasm, including protein localization, sequestration, and turnover. Chinnusamy et al. (2007) and Lorkovic (2009) showed that several nuclear RNA-binding proteins, which are members of the RNP complex, are crucial for plant development and stress responses.

IbSIn15, which was similar to a member of the receptor protein kinase family (RPK), showed low expression at 2 h and a further down-regulation at 4 and 8 h before up-regulation at 12 h of skinning. Only a few among hundreds of RPK genes have been studied for their physiological roles; several RPK transcripts were reported to accumulate after wounding (He et al., 2005; Nishiguchi et al., 2002). Takabatake et al. (2006) showed that receptor-like protein kinase is involved in wound signaling in plants through salicylic acid-induced protein kinase and wound-induced protein kinase.

Conclusions

The present study provides novel information about early expression of dehydration-responsive genes followed by genes involved in the lignification and suberization as a post-skinning healing mechanism in sweetpotato storage root. It is likely similarities exist in potato (Solanum tuberosum). Research involving gene expression in known skinning-resistant and -sensitive cultivars will provide clues to identify the genetic and physiological network involved in the skinning resistance mechanism in sweetpotato. The up-regulated genes involved in these resistance modules of a skinning-resistant cultivar may prove useful to identify expression markers for screening sweetpotato lines tolerant to skinning injury in breeding programs.

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  • Gupta, S., Wang, B.B., Strykera, G.A., Zanetti, M.E. & Lal, S.K. 2005 Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites Biochim. Biophys. Acta 1728 105 114

    • Search Google Scholar
    • Export Citation
  • Guzzardi, P., Genot, G. & Jamet, E. 2004 The Nicotiana sylvestris extensin gene, Ext 1.2A, is expressed in the root transition zone and upon wounding Biochim. Biophys. Acta 1680 83 92

    • Search Google Scholar
    • Export Citation
  • Hattori, T., Yoshida, N. & Nakamura, K. 1989 Structural relationship among the members of multigene family coding for the sweetpotato tuberous roots storage proteins Plant Mol. Biol. 13 563 572

    • Search Google Scholar
    • Export Citation
  • He, G., Tarui, Y. & Iino, M. 2005 A novel receptor kinase involved in jasmonate-mediated wound and phytochrome signaling in maize coleoptiles Plant Cell Physiol. 46 870 883

    • Search Google Scholar
    • Export Citation
  • Helekar, S.A. & Patrick, J. 1997 Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression Proc. Natl. Acad. Sci. USA 94 5432 5437

    • Search Google Scholar
    • Export Citation
  • Hoeflich, K.P. & Ikura, M. 2002 Calmodulin in action: Diversity in target recognition and activation mechanisms Cell 108 739 742

  • Huang, D.-J., Chen, H.-J., Hou, W.-C. & Lin, Y.-H. 2004 Isolation and characterization of thioredoxin h cDNA from sweet potato Plant Sci. 166 515 523

  • Jones, A., Dukes, P.D. & Schalk, J.M. 1986 Sweet potato breeding, p. 1–35. In: Bassett, J.M. (ed.). Breeding vegetable crops. AVI Publ., Westport, CT

  • Kim, Y.J., Kwak, C.I., Gu, Y.Y., Hwang, I.T. & Chun, J.Y. 2004 Annealing control primer system for identification of differentially expressed genes on agarose gels Biotechniques 36 424 434

    • Search Google Scholar
    • Export Citation
  • Kiribuchi, K., Jikumaru, Y., Kaku, H., Minami, E., Hasegawa, M., Kodama, O., Seto, H., Okada, K., Nojiri, H. & Yamane, H. 2005 Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants Biosci. Biotechnol. Biochem. 69 1042 1044

    • Search Google Scholar
    • Export Citation
  • Kushman, L.J. & Wright, F.S. 1969 Sweetpotato storage. U.S. Dept. Agr. Hdbk. 358. U.S. Dept. Agr., Washington, DC

  • La Bonte, D.R. & Wright, M.E. 1993 Image analysis quantifies reduction in sweetpotato skinning injury by preharvest canopy removal HortScience 28 1201

    • Search Google Scholar
    • Export Citation
  • Lee, S., Rajasekaran, K., Ramanarao, M.V., Bedre, R., Bhatnagar, D. & Baisakh, N. 2012 Identifying cotton (Gossypium hirsutum L.) genes induced in response to Aspergillus flavus infection Physiol. Mol. Plant Pathol. 80 35 40

    • Search Google Scholar
    • Export Citation
  • Lorkovic, Z.J. 2009 Role of plant RNA-binding proteins in development, stress response and genome organization Trends Plant Sci. 14 229 236

  • Maeshima, M., Sasaki, T. & Asahi, T. 1985 Characterization of major proteins in sweet potato tuberous roots Phytochemistry 24 1899 1902

  • Marivet, J., Frendo, P. & Burkard, G. 1992 Effects of abiotic stresses on cyclophilin gene expression in maize and bean and sequence analysis of bean cyclophilin cDNA Plant Sci. 84 171 178

    • Search Google Scholar
    • Export Citation
  • Moehs, C.P., Allen, P.V., Friedman, M. & Belknap, W.R. 1996 Cloning and expression of transaldolase from potato Plant Mol. Biol. 32 447 452

  • Nishiguchi, M., Yoshida, K., Sumizono, T. & Tazaki, K. 2002 A receptor-like protein kinase with a lectin-like domain from Lombardy poplar: Gene expression in response to wounding and characterization of phosphorylation activity Mol. Genet. Genomics 267 506 514

    • Search Google Scholar
    • Export Citation
  • Pinto, F., Berti, M., Olivares, D., Sierralta, W.D., Hinrichsen, P. & Pinto, M. 2011 Leaf development, temperature and light stress control of the expression of early light-inducible proteins (ELIPs) in Vitis vinifera L Environ. Exp. Bot. 72 278 283

    • Search Google Scholar
    • Export Citation
  • Ramanarao, M.V., Weindorf, D., Breitenbeck, G. & Baisakh, N. 2011 Differential expression of the transcripts of Spartina alterniflora Loisel (smooth cordgrass) induced in response to petroleum hydrocarbon Mol. Biotechnol. doi: 10.1007/s12033-011-9436-0

    • Search Google Scholar
    • Export Citation
  • Rees, D., van Oirschot, Q.E.A. & Aked, J. 2008 The role of carbohydrates in wound-healing of sweetpotato roots at low humidity Postharvest Biol. Technol. 50 79 86

    • Search Google Scholar
    • Export Citation
  • Romano, P.G., Horton, P. & Gray, J.E. 2004 The Arabidopsis cyclophilin gene family Plant Physiol. 134 1268 1282

  • Rozen, S. & Skaletsky, H.J. 2000 Primer3 on the WWW for general users and for biologist programmers, p. 365–386. In: Krawetz, S. and E. Misener (eds.). Bioinformatics methods and protocols: Methods in molecular biology. Humana Press, Totowa, NJ

  • Schuler, M. 1996 Plant cytochrome P450 monooxygenases Crit. Rev. Plant Sci. 15 235 284

  • Seo, J.-S., Kim, J.J., Kim, M.-J., Nahm, Y.-K., Song, B.H. & Cheong, S.I. 2011 OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice Plant J. 65 907 921

    • Search Google Scholar
    • Export Citation
  • Shewry, P.R. 2003 Tuber storage proteins Ann. Bot. (Lond.) 91 755 769

  • Solis, J. 2012 Genomic approaches to understand sweetpotato root development in relation to abiotic factors. PhD diss., Louisiana State Univ., Baton Rouge, LA

  • Takabatake, R., Seo, S., Ito, N., Gotoh, Y., Mitsuhara, I. & Ohashi, Y. 2006 Involvement of wound-induced receptor-like protein kinase in wound signal transduction in tobacco plants Plant J. 47 249 257

    • Search Google Scholar
    • Export Citation
  • Tao, L., Zeba, N., Ashrafuzzaman, M. & Hong, C.B. 2011 Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity Environ. Exp. Bot. 72 297 303

    • Search Google Scholar
    • Export Citation
  • van Oirschot, Q.E.A., Rees, D., Aked, J. & Kihurani, A. 2006 Sweetpotato cultivars differ in efficiency of wound healing Postharvest Biol. Technol. 42 65 74

    • Search Google Scholar
    • Export Citation

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Contributor Notes

Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 2013-306-7856.

Supported by the USDA-Borlaug grant to the Louisiana State University Agricultural Center and a USDA-NIFA Research Award Number 2009-51181-06071.

Julio Solis is acknowledged for providing the primers for elongation factor and CCOMT genes of sweetpotato.

Corresponding author. E-mail: nbaisakh@agcenter.lsu.edu.

  • View in gallery

    Representative gels from polymerase chain reaction (PCR) with annealing control primers (ACP) showing differentially expressed genes (DEGs) in sweetpotato storage root at 0, 2, 4, and 8 (+12) h after skinning injury. Upward and downward arrows indicate up-regulated and down-regulated DEGs, respectively; M = 1-kb DNA size marker.

  • View in gallery

    Semiquantitative reverse transcription polymerase chain reaction (PCR) analysis of differentially expressed genes in storage root of sweetpotato at 0, 2, 4, 8, and 12 h after skinning injury. Elongation factor gene (EF) was used as the endogenous reference for cDNA loading control.

  • View in gallery

    Expression of differentially expressed genes in storage root of sweetpotato at 2, 4, 8, and 12 h relative to 0 h after skinning. HRS = hepatocyte growth factor-regulated tyrosine kinase substrate (IbSIn69); Cyc = cyclophilin (IbSIn48); PPI CYP40 = peptidyl-prolyl cis-trans isomerase CYP40 (IbSIn 29); GDC = glutamate decarboxylase (IbSIn61b); TAL = transaldolase (IbSIn46); ELIP3 = early light-inducible protein (IbSIn61a); CCOMT = caffeic acid 3-O-methyl transferase; PAL = phenylalanine ammonia lyase; Ext = extensin, RPK = ribonucleoprotein complex subunit (IbSIn15); SRP = serine-rich protein (IbSIn21); bHLH = basic Helix-loop-helix (IbSIn30a); Cyt P450 = cytochrome P450 76C4 (IbSIn30b); NifU = nitrogen fixation unit (IbSIn40); Spor B = sporamin B (IbSIn60); TCTP = translationally controlled tumor protein (IbSIn59); TH2 = thioredoxin H2 mRNA (IbSIn04); RNP = ribonucleoprotein complex subunit 3 (IbSIn56). The error bars represent se.

  • Alamillo, J.M. & Bartels, D. 2001 Effects of desiccation on photosynthesis pigments and the ELIP-like dsp 22 protein complexes in the resurrection plant Craterostigma plantagineum Plant Sci. 160 1161 1170

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  • Gupta, S., Wang, B.B., Strykera, G.A., Zanetti, M.E. & Lal, S.K. 2005 Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites Biochim. Biophys. Acta 1728 105 114

    • Search Google Scholar
    • Export Citation
  • Guzzardi, P., Genot, G. & Jamet, E. 2004 The Nicotiana sylvestris extensin gene, Ext 1.2A, is expressed in the root transition zone and upon wounding Biochim. Biophys. Acta 1680 83 92

    • Search Google Scholar
    • Export Citation
  • Hattori, T., Yoshida, N. & Nakamura, K. 1989 Structural relationship among the members of multigene family coding for the sweetpotato tuberous roots storage proteins Plant Mol. Biol. 13 563 572

    • Search Google Scholar
    • Export Citation
  • He, G., Tarui, Y. & Iino, M. 2005 A novel receptor kinase involved in jasmonate-mediated wound and phytochrome signaling in maize coleoptiles Plant Cell Physiol. 46 870 883

    • Search Google Scholar
    • Export Citation
  • Helekar, S.A. & Patrick, J. 1997 Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression Proc. Natl. Acad. Sci. USA 94 5432 5437

    • Search Google Scholar
    • Export Citation
  • Hoeflich, K.P. & Ikura, M. 2002 Calmodulin in action: Diversity in target recognition and activation mechanisms Cell 108 739 742

  • Huang, D.-J., Chen, H.-J., Hou, W.-C. & Lin, Y.-H. 2004 Isolation and characterization of thioredoxin h cDNA from sweet potato Plant Sci. 166 515 523

  • Jones, A., Dukes, P.D. & Schalk, J.M. 1986 Sweet potato breeding, p. 1–35. In: Bassett, J.M. (ed.). Breeding vegetable crops. AVI Publ., Westport, CT

  • Kim, Y.J., Kwak, C.I., Gu, Y.Y., Hwang, I.T. & Chun, J.Y. 2004 Annealing control primer system for identification of differentially expressed genes on agarose gels Biotechniques 36 424 434

    • Search Google Scholar
    • Export Citation
  • Kiribuchi, K., Jikumaru, Y., Kaku, H., Minami, E., Hasegawa, M., Kodama, O., Seto, H., Okada, K., Nojiri, H. & Yamane, H. 2005 Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants Biosci. Biotechnol. Biochem. 69 1042 1044

    • Search Google Scholar
    • Export Citation
  • Kushman, L.J. & Wright, F.S. 1969 Sweetpotato storage. U.S. Dept. Agr. Hdbk. 358. U.S. Dept. Agr., Washington, DC

  • La Bonte, D.R. & Wright, M.E. 1993 Image analysis quantifies reduction in sweetpotato skinning injury by preharvest canopy removal HortScience 28 1201

    • Search Google Scholar
    • Export Citation
  • Lee, S., Rajasekaran, K., Ramanarao, M.V., Bedre, R., Bhatnagar, D. & Baisakh, N. 2012 Identifying cotton (Gossypium hirsutum L.) genes induced in response to Aspergillus flavus infection Physiol. Mol. Plant Pathol. 80 35 40

    • Search Google Scholar
    • Export Citation
  • Lorkovic, Z.J. 2009 Role of plant RNA-binding proteins in development, stress response and genome organization Trends Plant Sci. 14 229 236

  • Maeshima, M., Sasaki, T. & Asahi, T. 1985 Characterization of major proteins in sweet potato tuberous roots Phytochemistry 24 1899 1902

  • Marivet, J., Frendo, P. & Burkard, G. 1992 Effects of abiotic stresses on cyclophilin gene expression in maize and bean and sequence analysis of bean cyclophilin cDNA Plant Sci. 84 171 178

    • Search Google Scholar
    • Export Citation
  • Moehs, C.P., Allen, P.V., Friedman, M. & Belknap, W.R. 1996 Cloning and expression of transaldolase from potato Plant Mol. Biol. 32 447 452

  • Nishiguchi, M., Yoshida, K., Sumizono, T. & Tazaki, K. 2002 A receptor-like protein kinase with a lectin-like domain from Lombardy poplar: Gene expression in response to wounding and characterization of phosphorylation activity Mol. Genet. Genomics 267 506 514

    • Search Google Scholar
    • Export Citation
  • Pinto, F., Berti, M., Olivares, D., Sierralta, W.D., Hinrichsen, P. & Pinto, M. 2011 Leaf development, temperature and light stress control of the expression of early light-inducible proteins (ELIPs) in Vitis vinifera L Environ. Exp. Bot. 72 278 283

    • Search Google Scholar
    • Export Citation
  • Ramanarao, M.V., Weindorf, D., Breitenbeck, G. & Baisakh, N. 2011 Differential expression of the transcripts of Spartina alterniflora Loisel (smooth cordgrass) induced in response to petroleum hydrocarbon Mol. Biotechnol. doi: 10.1007/s12033-011-9436-0

    • Search Google Scholar
    • Export Citation
  • Rees, D., van Oirschot, Q.E.A. & Aked, J. 2008 The role of carbohydrates in wound-healing of sweetpotato roots at low humidity Postharvest Biol. Technol. 50 79 86

    • Search Google Scholar
    • Export Citation
  • Romano, P.G., Horton, P. & Gray, J.E. 2004 The Arabidopsis cyclophilin gene family Plant Physiol. 134 1268 1282

  • Rozen, S. & Skaletsky, H.J. 2000 Primer3 on the WWW for general users and for biologist programmers, p. 365–386. In: Krawetz, S. and E. Misener (eds.). Bioinformatics methods and protocols: Methods in molecular biology. Humana Press, Totowa, NJ

  • Schuler, M. 1996 Plant cytochrome P450 monooxygenases Crit. Rev. Plant Sci. 15 235 284

  • Seo, J.-S., Kim, J.J., Kim, M.-J., Nahm, Y.-K., Song, B.H. & Cheong, S.I. 2011 OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice Plant J. 65 907 921

    • Search Google Scholar
    • Export Citation
  • Shewry, P.R. 2003 Tuber storage proteins Ann. Bot. (Lond.) 91 755 769

  • Solis, J. 2012 Genomic approaches to understand sweetpotato root development in relation to abiotic factors. PhD diss., Louisiana State Univ., Baton Rouge, LA

  • Takabatake, R., Seo, S., Ito, N., Gotoh, Y., Mitsuhara, I. & Ohashi, Y. 2006 Involvement of wound-induced receptor-like protein kinase in wound signal transduction in tobacco plants Plant J. 47 249 257

    • Search Google Scholar
    • Export Citation
  • Tao, L., Zeba, N., Ashrafuzzaman, M. & Hong, C.B. 2011 Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity Environ. Exp. Bot. 72 297 303

    • Search Google Scholar
    • Export Citation
  • van Oirschot, Q.E.A., Rees, D., Aked, J. & Kihurani, A. 2006 Sweetpotato cultivars differ in efficiency of wound healing Postharvest Biol. Technol. 42 65 74

    • Search Google Scholar
    • Export Citation
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