J Plant Biotechnol (2023) 50:163-168
Published online September 22, 2023
https://doi.org/10.5010/JPB.2023.50.020.163
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: cefle@gnu.ac.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sweetpotato (Ipomoea batatas [L.]) is a globally important root crop cultivated for food and industrial processes. The crop is susceptible to the root-knot nematode (RKN) Meloidogyne incognita, a major plant-parasitic RKN that reduces the yield and quality of sweetpotato. Previous transcriptomic and proteomic analyses identified several genes that displayed differential expression patterns in susceptible and resistant cultivars in response to M. incognita infection. Among these, several sporamin genes were identified for RKN resilience. Sporamin is a storage protein primarily found in sweetpotato and morning glory (Ipomoea nil). In this study, transcriptional analysis was employed to investigate the role of sporamin genes in the defense response of sweetpotato against RKN infection in three susceptible and three resistant cultivars. Twenty-three sporamin genes were identified in sweetpotato and classified as group A or group B sporamin genes based on comparisons with characterized sweetpotato and Japanese morning glory sporamins. Two group A sporamin genes showed significantly elevated levels of expression in resistant but not in susceptible cultivars. These results suggest that the elevated expression of specific sporamin genes may play a crucial role in protecting sweetpotato roots from RKN infection.
Keywords defense signaling, root-knot nematodes, sweetpotato, sporamin, transcriptome
Sweetpotato [
Sporamin is the major storage protein in sweetpotato storage roots, accounting for 60-80% of the total soluble protein in the storage roots (Yeh et al. 1997a). Tissue-specific expression of sporamin genes is mainly observed in storage roots, with minimal or no expression in stems and leaves under normal conditions (Hattori et al. 1990). Sporamin proteins are encoded by various genes, classified into sporamin A and B groups according to their sequence similarities (Hattori et al. 1989). Recent studies showed that expression of sporamin genes was induced by wounding, pathogen exposure, and nematode infection, as well as treatment with plant hormones and sugars (Cai et al. 2003; Senthilkumar and Yeh 2012). This suggests that sporamin expression is closely related to defense responses in sweetpotato storage roots. Supporting this, a protective role for sporamin against herbivorous damage was observed in transgenic tobacco overexpressing the sporamin gene
Our previous research reported the results of proteome and transcriptome analysis of susceptible and resistant
Six sweetpotato cultivars (
Identification and similarity of sequences were determined using NCBI BLAST multiple sequence alignments performed using the BioEdit program. Phylogenetic analysis was performed using the Maximum Likelihood method in the Molecular Evolutionary Genetics Analysis program (MEGA11) (Tamura et al. 2013).
Total RNA was isolated from RKN-treated fibrous roots of sweetpotato samples using TRIzol reagent (Invitrogen) and treated with RNase-free DNaseI to remove genomic DNA contamination. Real-time reverse-transcriptase PCR analysis was performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad) with EvaGreen fluorescent dye according to the manufacturer’s instructions. Linear data were normalized to the average threshold cycle (Ct) of the ADP-RIBOSYLATION FACTOR (ARF) reference gene (Park et al. 2012). Gene-specific primers are listed in Table 1.
Table 1 Oligonucleotide primers used for qRT-PCR analysis
Transcript ID | Primer sequence (5’-3’) | PCR product (bp) | Sporamin group | |
---|---|---|---|---|
G13675|TU22356 | Forward primer | CATCTGCCACCATGAAAGCC | 189 | A |
Reverse primer | CTATGTAGTAGTTCCCGCCGG | |||
G34382|TU56396 | Forward primer | CCCCAACCCAACTCATTCCA | 200 | A |
Reverse primer | CGCATTCGTTCGAGGAGGAA |
Data were analyzed by one-way analysis of variance (ANOVA). The subsequent multiple comparisons were examined based on the least significant difference (LSD) and Duncan’s multiple range test. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 12), and statistical significance was set at P < 0.05.
Transcriptome analysis was previously conducted on
To investigate RC-specific and SC-specific expression patterns, transcription of sweetpotato sporamin genes was examined in SCs (DHM, SHM, and YM) and RCs (DJM, PWM, and JHM) during RKN infection (Fig. 3). Of the 23 candidate sporamin genes, two genes (G13675|TU22356 and G34367|TU56356), both in the sporamin A group, exhibited higher expression in RCs than in SCs.
Transcriptional changes of G13675|TU22356 and G34367| TU56356, which were elevated in RCs but not SCs during infection, were examined during infection progression in YM (susceptible) and JHM (resistant) cultivars using quantitative RT-PCR analysis (Fig. 4). In both infected plants and uninfected controls, both genes had higher expression levels in JHM than in YM until 4 weeks after infection, after which expression levels decreased to very low levels in both YM and JHM by 8 weeks post-infection. In JHM, in both treated and untreated groups, expression of both genes was highest 1 week after infection, dropping by about half by 4 weeks post-infection. Expression levels were similar between RKN-treated and untreated plants. In YM, expression of both genes was lower in RKN-treated plants than in untreated controls at both 1 week and 4 weeks after infection.
The role of sporamin genes in resistance to infection with the RKN
Tubers and storage root crops contain abundant storage proteins (Shewry 2003). Storage proteins such as patatin from potato, sporamin from sweetpotato, and dioscorin from yam display enzymatic activity in response to external pathogen infection. A study of purified enzymes from potato tubers revealed that patatin displayed enzymatic activity that catalyzed the deacylation of several lipid substrates (Galliard 1971). Subsequent studies demonstrated that the acyl hydrolase activity was due to patatin, which also acted as an esterase (Racusen 1986). The specificity of acyl hydrolases was later studied in more detail (Anderson et al. 2002), especially their activity as phospholipases for phospholipid and lysophospholipid substrates (Hirschberg et al. 2001; Senda et al. 1996). Another type of hydrolytic activity for patatin, as acidic β-1,3-glucanase (Tonon et al. 2001), was recently described. β-1,3-glucanases are thought to contribute to plant defense against fungal pathogens by digesting β-1,3-glycans in the hyphal cell wall, forming part of a pathogenesis-relevant (PR) protein response (Van Loon and van Strien 1999), suggesting that patatin might play a role in defending potato tubers. Sporamin from sweetpotato also displayed enzymatic activity, catalyzing the activation of trypsin inhibitor (Yeh et al. 1997a). Hou and Lin (1997) reported that sporamin also possessed antioxidant activity through acting as a dehydroascorbate reductase and as a monodehydroascorbate reductase associated with intermolecular thiol/disulfide exchange. Sporamin can also scavenge both 1,1-diphenyl-2 picrylhydrazl radicals and hydroxyl radicals (Hou et al. 2001). The biological significance of these observations is not clear. An in vivo role in regulating protease activity is suggested by the observation that sporamin inhibits endogenous serine proteinases in sweetpotato storage roots (Hou and Lin 2002). In particular, a role for sporamin in the resistance response, which protects plants from damage by herbivores and parasitic nematodes, was suggested by an increase in trypsin inhibitor activity when sporamin was overexpressed during attack by tobacco cutworm larvae (
This study describes the first transcriptome-based analysis of the sporamin gene family in sweetpotato, a genetically complex and agronomically important food crop. Sporamin genes were identified in sweetpotato and their expression profiles were compared between RKN-resistant and -susceptible sweetpotato cultivars. These results increase our understanding of the role of sporamin in plant defense, particularly in response to RKN infection.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2021R1A2C400188711), and the project PJ009250072013 of the National Institute of Crop Science, Rural Development Administration, Republic of Korea.
J Plant Biotechnol 2023; 50(1): 163-168
Published online September 22, 2023 https://doi.org/10.5010/JPB.2023.50.020.163
Copyright © The Korean Society of Plant Biotechnology.
Jung-Wook Yang ・Yun-Hee Kim
Department of Crop Cultivation & Environment, Research National Institute of Crop Science, RDA, Suwon, Republic of Korea
Department of Biology Education, Gyeongsang National University, Jinju, Republic of Korea
Correspondence to:e-mail: cefle@gnu.ac.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sweetpotato (Ipomoea batatas [L.]) is a globally important root crop cultivated for food and industrial processes. The crop is susceptible to the root-knot nematode (RKN) Meloidogyne incognita, a major plant-parasitic RKN that reduces the yield and quality of sweetpotato. Previous transcriptomic and proteomic analyses identified several genes that displayed differential expression patterns in susceptible and resistant cultivars in response to M. incognita infection. Among these, several sporamin genes were identified for RKN resilience. Sporamin is a storage protein primarily found in sweetpotato and morning glory (Ipomoea nil). In this study, transcriptional analysis was employed to investigate the role of sporamin genes in the defense response of sweetpotato against RKN infection in three susceptible and three resistant cultivars. Twenty-three sporamin genes were identified in sweetpotato and classified as group A or group B sporamin genes based on comparisons with characterized sweetpotato and Japanese morning glory sporamins. Two group A sporamin genes showed significantly elevated levels of expression in resistant but not in susceptible cultivars. These results suggest that the elevated expression of specific sporamin genes may play a crucial role in protecting sweetpotato roots from RKN infection.
Keywords: defense signaling, root-knot nematodes, sweetpotato, sporamin, transcriptome
Sweetpotato [
Sporamin is the major storage protein in sweetpotato storage roots, accounting for 60-80% of the total soluble protein in the storage roots (Yeh et al. 1997a). Tissue-specific expression of sporamin genes is mainly observed in storage roots, with minimal or no expression in stems and leaves under normal conditions (Hattori et al. 1990). Sporamin proteins are encoded by various genes, classified into sporamin A and B groups according to their sequence similarities (Hattori et al. 1989). Recent studies showed that expression of sporamin genes was induced by wounding, pathogen exposure, and nematode infection, as well as treatment with plant hormones and sugars (Cai et al. 2003; Senthilkumar and Yeh 2012). This suggests that sporamin expression is closely related to defense responses in sweetpotato storage roots. Supporting this, a protective role for sporamin against herbivorous damage was observed in transgenic tobacco overexpressing the sporamin gene
Our previous research reported the results of proteome and transcriptome analysis of susceptible and resistant
Six sweetpotato cultivars (
Identification and similarity of sequences were determined using NCBI BLAST multiple sequence alignments performed using the BioEdit program. Phylogenetic analysis was performed using the Maximum Likelihood method in the Molecular Evolutionary Genetics Analysis program (MEGA11) (Tamura et al. 2013).
Total RNA was isolated from RKN-treated fibrous roots of sweetpotato samples using TRIzol reagent (Invitrogen) and treated with RNase-free DNaseI to remove genomic DNA contamination. Real-time reverse-transcriptase PCR analysis was performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad) with EvaGreen fluorescent dye according to the manufacturer’s instructions. Linear data were normalized to the average threshold cycle (Ct) of the ADP-RIBOSYLATION FACTOR (ARF) reference gene (Park et al. 2012). Gene-specific primers are listed in Table 1.
Table 1 . Oligonucleotide primers used for qRT-PCR analysis.
Transcript ID | Primer sequence (5’-3’) | PCR product (bp) | Sporamin group | |
---|---|---|---|---|
G13675|TU22356 | Forward primer | CATCTGCCACCATGAAAGCC | 189 | A |
Reverse primer | CTATGTAGTAGTTCCCGCCGG | |||
G34382|TU56396 | Forward primer | CCCCAACCCAACTCATTCCA | 200 | A |
Reverse primer | CGCATTCGTTCGAGGAGGAA |
Data were analyzed by one-way analysis of variance (ANOVA). The subsequent multiple comparisons were examined based on the least significant difference (LSD) and Duncan’s multiple range test. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 12), and statistical significance was set at P < 0.05.
Transcriptome analysis was previously conducted on
To investigate RC-specific and SC-specific expression patterns, transcription of sweetpotato sporamin genes was examined in SCs (DHM, SHM, and YM) and RCs (DJM, PWM, and JHM) during RKN infection (Fig. 3). Of the 23 candidate sporamin genes, two genes (G13675|TU22356 and G34367|TU56356), both in the sporamin A group, exhibited higher expression in RCs than in SCs.
Transcriptional changes of G13675|TU22356 and G34367| TU56356, which were elevated in RCs but not SCs during infection, were examined during infection progression in YM (susceptible) and JHM (resistant) cultivars using quantitative RT-PCR analysis (Fig. 4). In both infected plants and uninfected controls, both genes had higher expression levels in JHM than in YM until 4 weeks after infection, after which expression levels decreased to very low levels in both YM and JHM by 8 weeks post-infection. In JHM, in both treated and untreated groups, expression of both genes was highest 1 week after infection, dropping by about half by 4 weeks post-infection. Expression levels were similar between RKN-treated and untreated plants. In YM, expression of both genes was lower in RKN-treated plants than in untreated controls at both 1 week and 4 weeks after infection.
The role of sporamin genes in resistance to infection with the RKN
Tubers and storage root crops contain abundant storage proteins (Shewry 2003). Storage proteins such as patatin from potato, sporamin from sweetpotato, and dioscorin from yam display enzymatic activity in response to external pathogen infection. A study of purified enzymes from potato tubers revealed that patatin displayed enzymatic activity that catalyzed the deacylation of several lipid substrates (Galliard 1971). Subsequent studies demonstrated that the acyl hydrolase activity was due to patatin, which also acted as an esterase (Racusen 1986). The specificity of acyl hydrolases was later studied in more detail (Anderson et al. 2002), especially their activity as phospholipases for phospholipid and lysophospholipid substrates (Hirschberg et al. 2001; Senda et al. 1996). Another type of hydrolytic activity for patatin, as acidic β-1,3-glucanase (Tonon et al. 2001), was recently described. β-1,3-glucanases are thought to contribute to plant defense against fungal pathogens by digesting β-1,3-glycans in the hyphal cell wall, forming part of a pathogenesis-relevant (PR) protein response (Van Loon and van Strien 1999), suggesting that patatin might play a role in defending potato tubers. Sporamin from sweetpotato also displayed enzymatic activity, catalyzing the activation of trypsin inhibitor (Yeh et al. 1997a). Hou and Lin (1997) reported that sporamin also possessed antioxidant activity through acting as a dehydroascorbate reductase and as a monodehydroascorbate reductase associated with intermolecular thiol/disulfide exchange. Sporamin can also scavenge both 1,1-diphenyl-2 picrylhydrazl radicals and hydroxyl radicals (Hou et al. 2001). The biological significance of these observations is not clear. An in vivo role in regulating protease activity is suggested by the observation that sporamin inhibits endogenous serine proteinases in sweetpotato storage roots (Hou and Lin 2002). In particular, a role for sporamin in the resistance response, which protects plants from damage by herbivores and parasitic nematodes, was suggested by an increase in trypsin inhibitor activity when sporamin was overexpressed during attack by tobacco cutworm larvae (
This study describes the first transcriptome-based analysis of the sporamin gene family in sweetpotato, a genetically complex and agronomically important food crop. Sporamin genes were identified in sweetpotato and their expression profiles were compared between RKN-resistant and -susceptible sweetpotato cultivars. These results increase our understanding of the role of sporamin in plant defense, particularly in response to RKN infection.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2021R1A2C400188711), and the project PJ009250072013 of the National Institute of Crop Science, Rural Development Administration, Republic of Korea.
Table 1 . Oligonucleotide primers used for qRT-PCR analysis.
Transcript ID | Primer sequence (5’-3’) | PCR product (bp) | Sporamin group | |
---|---|---|---|---|
G13675|TU22356 | Forward primer | CATCTGCCACCATGAAAGCC | 189 | A |
Reverse primer | CTATGTAGTAGTTCCCGCCGG | |||
G34382|TU56396 | Forward primer | CCCCAACCCAACTCATTCCA | 200 | A |
Reverse primer | CGCATTCGTTCGAGGAGGAA |
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