Functional Classification of Skinning Injury Responsive Genes in Storage Roots of Sweetpotato

Skinning injury in sweetpotato due to loss of skin or periderm which occurred during harvest is inevitable and account for financial loss due to dehydration, pests, and pathogens. Hence, studies on gene expression changed due to skinning injury can provide important information about this protective tissue and for improving the life of storage roots. New candidate genes involved in skinning injury were isolated with an Annealing Control Primer (ACP). Using 20 ACP primers, a total of 103 differentially expressed genes (DEGs) were retrieved. In this study, the functional annotation of these selected 15 up-regulated DEGs (10 contigs and 5 singletons) were characterized. The results showed that these 15 “DEG-unigenes” are mainly associated with defense and stress responses, regulation and signaling, protein synthesis and fate, and metabolism may play an important role in the primary responses to skinning injury in storage roots of sweetpotato. This study showed the importance of defense and stress responses genes to the formation of wound periderm. Furthermore, this results can be used for better understanding of the molecular mechanism of skinning/mechanical injury-related genes in the storage roots of sweetpotato as well as to all stems, fruits, and roots of all plants.


INTRODUCTION
Sweetpotato storage roots are underground storage organs covered by skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Skinning, the loss of the skin from the surface of the storage roots of sweetpotato, is responsible for significant postharvest losses due to mechanical injury, weight loss, sprouting, pests and diseases. Thus, because of its short shelf-life, in developing countries the sweetpotato storage 37  April 2017 roots has to be consumed or marketed within 1 to 2 weeks after harvesting (Rees et al., 2008).
Periderm formation which is characterized by secondary growth are usually found in the plants following wounding under conditions of high temperature and humidity (curing). In order to understand well about wound periderm formation, a study on the molecular mechanisms associated with its formation to extend the storage life of sweetpotato storage roots is needed. Moreover, to understand the molecular mechanisms underlying skinning injury, attempts have turned toward the isolation of genes regulated by skinning injury. This allows insight into their functions and the pathways that lead to their expression. Although several responses of plants to skinning injury, including short term metabolic and physiological changes may not require changes in gene expression, the majority are predicted to rely on alterations in gene expression. For example, wounding alters changes in gene expression in response to mechanical wounding in potato skin using a suppression subtractive hybridization library (Soler et al., 2011). They showed that in response to wounding, the highest mRNA transcripts were dominated by genes involved in stress and defense followed by genes involved in signal transduction and regulations. Furthermore, they also showed the activation of genes involved in suberin and wax and cell wall in response to wounding in potato skin. Effendy et al. (2013) showed that intentional skinning in storage roots of sweetpotato induced the expression of genes involved in abiotic stresses, wounding responses, transcriptional regulation/signaling, and lignin and suberin biosynthesis. Other study by Lulai and Neubauer (2014) in potato skin showed the spatial and temporary wounding induced suberization genes that involved in the closing layer and wound periderm formation.
In order to understand the molecular basis of skinning injury responses, the differentially expressed genes (DEGs) in skinning-treated storage roots must be characterized. Low levels of DEGs and their transcripts are often hard to detect in abundant mRNA within tissues. A highly reliable, accurate, and reproducible specific polymerase chain reaction (PCR) amplification is a prerequisite. A novel annealing control primer (ACP)-based Reverse Transcriptase (RT) PCR technology is based on a unique tripartite structure of a specific oligonucleotide primer 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 highannealing 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. This method specifically targets the template sequence for hybridization through a polydeoxyinosine linker (Hwang et al., 2003). Here, ACP-based RTPCR is performed to identify genes up-regulated in storage roots of sweetpotato using cDNA from skinned storage roots to determine the common genes. Freshly harvested sweetpotatoes were used for this approach. Briefly, this work presents 15 DEGs that could be used to further investigate their role in skinning injury and wound healing responses at the molecular level. The objective of this work was to describe transcriptional changes in LA 07-146 subjected to skinning injury in storage roots of sweetpotato at 2, 4, 8, and 12 h.

Place and Time of the Research
This research was carried out in the Molecular Biology Laboratory, School of Plant, Environmental, and Soil Sciences, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, United States of America from August to October, 2011.

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 as detailed in Effendy et al. (2013).

RNA Isolation, cDNA Preparation and ACP-based Gene-Fishing PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following manufacturer's instructions (Effendy et al., 2013). First-strand cDNA synthesis was performed using a GeneFishing TM DEG premix kit (Seegene, Rockville, MD) as previously described (Seegene, Rockville, MD). Second strand cDNA synthesis and subsequent PCR amplification were performed in a single tube using the protocol as detailed in the GeneFishing TM DEG premix kit manual (Seegene, Rockville, MD).

Cloning and Sequencing of DEGs
Based on intensity or presence/absence) between control and skinning were performed following the manufacturer's instruction (QIAquick ® Gel Extraction Kit Qiagen, Valencia, CA).

Nucleotide and Deduced Amino Acid Sequencing Analyses
The DEGs were cut from the gel and cloned into pGEM ® -T Easy vector (Promega, Madison, WI). Muzuni et al. (2010) has successful using this vector to clone fragment cDNA H + -ATPase from Melastoma malabathricum. The positive colonies from DEGs were confirmed by colony PCR using M13F and M13R primers. Plasmids isolated from these clones were single-pass sequenced with T7 primer in an ABI 3730x1 genetic analyzer.
DNA sequences were processed manually to clean the vector backbone and the poly (A) tail and searched against the non redundant nucleotide and protein database of National Center for Biotechnology Information using BLASTN and BLASTX interference (http://www.ncbi.nlm. nih.gov/BLAST) (Altschul et al., 1997).

Effect of Skinning Treatment on Storage Root RNA Populations
To examine the effect of skinning treatment on storage root RNA populations, freshly harvested storage roots were exposed to skinning treatment. In order to determine how rapidly changes in the mRNA population occurred following the onset of a skinned treatment, time course experiments were carried out. RNA was isolated from sweetpotato storage roots for 0 (control), 2, 4, 8+12 h following the onset of a skinning treatment and used for ACP-based RTPCR. The RNAs results obtained were then used for ACP-based differential display (DD)-RTPCR with 20 ACP primers. Differentially expressed genes were used to examine and compare the patterns of expressed mRNAs present in skinning injury treated storage roots. Subsequently, a number of skinned-induced partial cDNAs were isolated and examined in roots subjected to skinning. The results showed that skinning induced changes in the accumulation of a number of different DEGs (Table 1); indicative of a skinned-induced activation and repression of gene expression. A number of changes in mRNA accumulation that occurred in skinnedtreated roots occurred rapidly within 2 h after subjected to skinning injury. In previous studies, it was observed that a wounding treatment resulted in the altered synthesis and accumulation of a number of genes in roots of potato (Soler et al., 2011;Boher et al., 2013;Effendy et al., 2013;Lulai and Neubauer, 2014). In the present study, skinned-induced changes in the accumulation of a number of different RNAs was demonstrated (Table 1). A substantial proportion of the changes in RNA accumulation that occurred in skinningtreated storage roots were transient in nature and many occurred rapidly within 2 h of skinning (Table 1). The rapid induction of gene expression is consistent with previous studies with the wound-enhanced accumulation of genes expression in roots in which the earliest changes were observed within 2 h after wounding in storage roots of sweetpotato (Effendy et al., 2013).

Cloning and Sequencing of Skinning Injury Responsive DEGs
ACP-based DD-RTPCR clearly demonstrated that changes in mRNA population of storage roots occurred during a period of skinning injury. A total of 70 DEGs of interest were excised and 119 independent clones (from 42 DEGs) were successfully re-amplified and cloned. The majority of these represented DEGs products with and induced or enhanced accumulation in skinning-treated storage roots. The inserts of these clones were sequenced and this revealed that among the 119 cDNA clones, 101 sequences (consisted of 44 singletons and 19 contigs) contained distinct sequences which matched to plant-specific cDNA sequences. Fifteen DEGs (10 contigs and 5 singletons) were further selected to study their functions in plants (Table 1).

Group
Type 0h

Functional Annotation of Selected DEGs
The genes obtained in the storage root ACP Gene-Fishing library were manually sorted into functional categories taking into account the main metabolic and cellular processes in the periderm. Four functional categories: stress and defense, regulation and signaling, protein synthesis and fate, and metabolism were identified.
DEGs accumulation related to wounding responses as a result of skinning injury have the highest DEGs, with 6 DEGs (6 contigs), followed by the accumulation of genes involved in regulation and signaling with 4 DEGs (2 contigs and 2 singletons) ( Table 2). Skinning injury also induced genes involved in general abiotic stress response due to skinning injury with 4 DEGs (1 contigs and 3 singletons) and lignin and suberin 1 DEG (1 contig) ( Table 2). A list of selected genes with their annotation based on similarity to databases is shown in Table 2.

Number of DEGs
Putative  Table 2. Functional annotation of fifteen DEGs in response to skinning injury Note: a Putative description for sweetpotato genes were obtained using Basic Local Alignment Search Tool (Altschul et al., 1997). b Functional classification was done according to the Munich Information Center for Protein Sequences (Schoof et al., 2002). c Functional annotation was confirmed by qRT-PCR (Effendy et al., 2013) IbH34 showed 90% similarity with a translationally controlled tumor protein (TCTP). We also found that TCTP is the only full length gene found using ACP-based DD-RTPCR. Our study also showed that TCTP is the most abundant DEGs constitutes of 13.89% transcripts. Studies by Rho et al. (2011) showed that TCTP has an anti-apoptotic activity and can prevent apoptosis. Furthermore, TCTP is an important component of TOR (target of rapamycin) signaling pathway, a major regulator of cell growth. Berkowitz et al. (2008) showed that silencing TCTP caused slow vegetative growth and reduced leaf expansion. Furthermore they showed that knockout TCTP caused impaired pollen tube growth.
In present study, one DEG IbF61 showed similarity with transaldolase (TAL). Only few studies report on expression of TAL in plants. Plant TAL genes are induced by abiotic stress conditions including wounding (Caillau and Quick, 2005). Caillau and Quick (2005) reported that TAL gene (ToTal1) accumulation has been observed in root, stem and red fruit of tomato. Another study by Yang et al. (2014) showed that rice TAL was expressed in leaf, panicle, stem, knot, and root. A knockdown TAL plant using a genespecific RNA interference (RNAi) construct in rice, showed reduction of TAL transcript in leaves of TAL-RNAi line. They found that knockdown TAL gene leads to changes in number and pattern of vascular bundles (Yang et al., 2014). TAL up-regulation may enhance the flux to shikimate pathway (Misra et al., 2010) from primary to secondary metabolites enabling synthesis of aromatic amino acid, flavonoids, and lignin.
Sporamin, a major storage protein in sweetpotato storage roots belong to soybean trypsin inhibitor superfamily can be grouped into two subfamilies Spor A and Spor B. Tao et al. (2012) showed that Spor B expressed the highest levels in roots and barely detectable in young leaves, mature leaves and stem. On the contrary, Spor A expressed in all tissues measured (young leaves, mature leaves, stem, fibrous roots and storage roots at three developmental stages at 1.5, 3, 5 months after planting). Studies by Rajendran et al. (2014) showed that sporamin expression is induced by biotic and abiotic stresses. They showed that endogenous level of jasmonic acid and salicylic acid can regulate the sporamin expression in response to mechanical wounding or herbivory attack. These studies suggest that sporamin can function as storage proteins as well as signaling pathway when subjected to both stresses.
In the present study, IbF82 showed 98% similarity with cyclophilin (Cyc) from I. batatas and was induced in response to skinning as early as 2 h after skinning treatment. Cyps, ubiquitous proteins of immunophilin superfamily,  Genomic studies by Mainali et al. (2014) identified 62 CYP (Cyclophilin) genes in the soybean genome. Studies have demonstrated the importance of many PPIases in plant biology, but no genome-wide analysis of the CYP gene family has been conducted for a legume species. CYP transcripts are commonly up-regulated in response to salt stress (Ruan et al., 2011), osmotic stress and high light condition (Kim et al., 2012), desiccation and salt stress (Ahn et al., 2010, Trivedi et al., 2012, low temperature, abscisic acid (ABA). Various CYPs have been found in cytosol, nucleus, chloroplast, secretory, and mitochondria (Mainali et al., 2014) suggesting a substantial role in a wide array of plant defense and cellular processes such as protein folding, mRNA processing, protein degradation and signal transduction which are crucial during development and stress response (Laxa et al., 2007).

CONCLUSIONS
In conclusion, ACP-based DD-RTPCR provides an effective way to explore transcript profiles of skinninginjury responsive genes in storage roots of sweetpotato. The DEG data presented here to the best of our knowledge is the first overview of genes that are expressed in response to skinning injury in sweetpotato. Our study is focusing on native storage root intentional skinning and it indicates that, in addition to its function as a protective layer, the skin responds to plant stress and defense responses by altered gene expressions. The presence of these DEGs in this study implied that tolerance of sweetpotato to skinning injury can be achieved by an up-regulated expression of skinning related genes. Periderm formation which is characterized by secondary growth are commonly found in plants following wounding. Periderm formation, development, and anatomy are highly similar in all plant species, therefore the functional characterization of genes that are expressed during storage root skinning injury may have implications for other periderm systems in all plants as well. These upregulated genes identified in this research can be exploited further to unravel regulatory networks involved in skinning injury responses in plants and to develop strategies towards impaired skin healing.