Isolation of EST clones for low temperature inducible genes and their categorization based on nucleotide sequence
An SSH-EST library, which was enriched with cDNAs corresponding to low temperature inducible genes, was constructed. Two hundred sixty clones were obtained as EST clones. All clones were sequenced and searched for homologous genes. Out of them, 192 clones showed similarity to the genes registered in databases and were categorized into 62 distinct groups based on the functions of the proteins, which were encoded by the clones, according to the classification method of Bevan et al. (1998).
In order to study the involvement of 62 genes corresponding to the categorized EST clones in freezing tolerance of lettuce, the expression levels of the genes during low temperature treatment were investigated with RT-qPCR. Out of them, 45 genes were confirmed to be low temperature inducible genes, although their expression patterns were diverse (Fig. 1). On the other hand, relative expression levels of other 17 genes were not enhanced (data not shown), so the possibilities of their low-temperature-inducibilities and their involvement in the freezing tolerance of lettuce plants were considered low. The nucleotide sequences of the ESTs corresponding to genes that were confirmed to be low temperature inducible were deposited to DDBJ with accession numbers shown in Table 1.
The clones, which were confirmed to be low temperature inducible, were categorized into 13 intracellular functions as follows: metabolism, energy, cell growth/division, transcription, protein destination and storage, transporters, intracellular traffic, cell structure, signal transduction, disease/defense, unclear classification, secondary metabolism based on their putative functions (Bevan et al., 1998). In particular, the “disease/defense” category contained 13 distinct deduced proteins. The second most abundant category was “signal transduction” and “transporters” which respectively contained five distinct deduced proteins. Other categories were as follows: “protein destination and storage” (four distinct deduced proteins), “transcription” (four distinct deduced proteins), “intracellular traffic” (four distinct deduced proteins), “metabolism” (three distinct deduced proteins), “secondary metabolism” (two distinct deduced proteins), “cell growth/division” (two distinct deduced proteins), “energy” (one deduced protein), “cell structure” (one deduced protein), “unclear classification” (one deduced protein), and “unknown”. The involvement of freezing tolerance of the categorized genes were discussed as follows.
Effects of duration of low temperature treatment on expression levels of the genes
As shown in Fig. 1, expression patterns of genes were diverse. By using a statistical method with Jonckheere-Terpstra Tests, effects of duration of low temperature treatment on expression levels of the corresponding genes were investigated. Some genes were transiently induced during low temperature treatment (P > 0.05) and the other most genes were continuously along with duration of the treatment (P < 0.05). Expression levels of some genes, encoding such as 3-ketoacyl CoA thiolase, gigantea-like protein, delta 12 fatty acid desaturase etc. in treated samples gradually increased but did not show any significant differences to those in the non-treated sample.
Involvement of “metabolism”-categorized genes in freezing tolerance
In the category of “metabolism”, six ESTs individually encoded a part of three proteins, whose amino acid sequences were respectively similar to amino acid sequence of inositol-3-phosphate synthase (myo-inositol phosphate synthase; 7 clones), 3-ketoacyl-CoA thiolase (four clones), acyl-CoA-binding domain-containing protein (one clone), or delta 12 fatty acid desaturase (FAD2; one clone).
Inositol-3-phosphate synthase (myo-inositol phosphate synthase: MIPS) is the key enzyme of myo-inositol synthesis (Tan et al., 2013b). Myo-inositol may function as a compatible solute for protection against abiotic stress and can also be converted to other compatible solutes (Tan et al., 2013b). Furthermore, myo-inositol induces the expression of galactinol synthase gene of Medicago falcata (Zhuo et al., 2013). As we previously showed, low temperature enhanced the expression levels of two GolS genes (Honjoh et al., 2018). Thus, MIPS would play an important role for enhancement of freezing tolerance in lettuce.
3-ketoacyl-CoA thiolase (KAT) is an important enzyme involved in fatty acid degradation and is positively involved in abscisic acid (ABA) synthesis via ß-oxidation of fatty acids (Jiang et al., 2011). ABA is well known to play an important role in plant development and stress adaptation as a plant hormone. In the present study, the KAT gene was up-regulated by low temperature treatment, suggesting that ABA-regulated genes would be induced in lettuce.
Overexpression of one type of acyl-CoA-binding protein (ACBP6) was reported to enhance freezing tolerance of Arabidopsis (Chen et al., 2008; Liao et al., 2014). ACBP6-mediated freezing tolerance was accompanied by increased phospholipase D (PLDd) geneexpression, decreased phosphatidyl choline (PC) content, and increased phosphatidic acid (PA) content (Chen et al., 2008). Qiao et al. (2018) suggest that PLDd convert PC into phosphatidyl glycerol (PG), phosphatidyl serine (PS), and phosphatidyl ethanolamine (PE), all of which stabilize the cell membrane and the membrane skeleton, conferring tolerance to various abiotic stress.
FAD2 is delta 12 fatty acid desaturase localized in endoplasmic reticulum and was also shown to be induced by low temperature in several plants including Chlorella (Suga et al., 2002) and cotton (Kargiotidou et al., 2008). As generally well known, desaturation of membrane lead to increase in fluidity of membrane at low temperature, and to cold acclimation of plant.
Involvement of “energy”-categorized genes in freezing tolerance
In the category of “energy”, one EST encoded parts of a protein, whose amino acid sequence was similar to amino acid sequences of phosphoglycerate kinase.
Phosphoglycerate kinase (PGK) was shown to be induced by low temperature in Arabidopsis (Bae et al., 2003). Overexpression of phosphoglycerate kinase-2 in tobacco plants improved salinity stress tolerance by higher chlorophyll retention and enhanced proline accumulation, besides maintaining better ion homeostasis (Joshi et al., 2016). There are several isotype genes encoding phosphoglycerate kinase in plants, so chilling tolerant plant might have an isotype PGK gene, which are induced during low temperature treatment. In the present study, this enzyme would be at least involved in chilling tolerance of lettuce plants.
Involvement of “cell growth/division”-categorized genes in freezing tolerance
In the category of “cell growth/division”, four ESTs individually encoded a part of two proteins, whose amino acid sequences were, respectively, similar to amino acid sequence of adagio protein (1 clone) or that of gigantea-like protein (3 clones).
According to UniProt database (https://www.uniprot.org), adagio protein has alternative name, ZTL protein, and a component of E3 ubiquitin ligase complex involved in the regulation of circadian clock-dependent processes including the transition of flowering time, hypocotyl elongation, cotyledons and leaf movement rhythms (https://www.uniprot.org/uniprot/Q94BT6). Furthermore, Norén et al. (2016) proposed a model that low levels of ZTL protein would result in increased protein levels of two transcriptional factors, long hypocotyl5 (HY5) and pseudo-response regulator5 (PRR5), and in a repression of C-repeat binding factors (CBFs), which are important transcriptional factors for cold tolerance. HY5 and PRR5 act in concert to repress CBF3, and PPR5 represses CBF1 and CBF2. Thus, cold-induction of ZTL (adagio) protein might negatively affect the expression of CBF genesand other cold-responsive genes.
Gigantea (GI) protein is known as flowering time regulator which connects networks involved in developmental stage transitions and environmental stress responses, furthermore repression of one GI protein led to enhancement of salt stress tolerance in poplar (Ke et al., 2017). Fornara et al. (2015) and Xie et al. (2015) respectively reported that loss of GI function led to increase in freezing tolerance of Arabidopsis and Brassica rapa. Furthermore, GI and ZTL are likely to be closely related for adaptation to environmental stress (Gil and Park, 2019; Kim et al., 2007). In the present paper, EST clones encoding GI and ZTL homologous proteins were identified at the same time. Therefore, considering the negative involvement of the two proteins in stress tolerance in previous reports described above and the fact that they were induced at low temperature in the present study, it is considered that they might have a negative effect on the low-temperature tolerance of lettuce.
Involvement of “transcription”-categorized genes in freezing tolerance
In the category of “transcription”, eight ESTs individually encoded a part of four corresponding proteins, whose amino acid sequences were respectively similar to amino acid sequences of multiprotein-bridging factor 1c (MBF1c; 5 clones), transcription factor UNE-10 (1 clone), DEAD-box ATP-dependent RNA helicase (1 clone), and G-patch domain containing protein (1 clone).
Overexpression of MBF1c gene from antarctic moss, Polytrichastrum alpinum, in Arabidopsis seems to enhance tolerances against salt, osmotic, cold, and heat stress (Alavilli et al., 2017). Furthermore, the overexpression of the gene up-regulated the expression of 10 salt-stress inducible genes without salt treatment in Arabidopsis. On the other hand, overexpression of MBF1c gene from Capsicum annuum in Arabidopsis reduced abiotic stress tolerance, accompanying reduced expression levels of stress tolerant genes compared to those in wild type Arabidopsis (Guo et al., 2014). P. alpinum is antarctic moss, which lives under severe cold condition, so it is strong against cold. On the other hand, C. annuum is likely to be weak against cold. Thus, even both the genes encode same MBF1c proteins, the protein encoded by a gene from non-cold tolerant plant might not function to enhance the cold tolerance. In the present paper, the MBF1c gene was up-regulated by low temperature treatment in lettuce and encoded a transcriptional regulator protein. It would be necessary for the protein to investigate whether this protein induces other stress responsive genes and functions for acquisition of freezing tolerance in lettuce as a transcriptional factor.
Although several transcriptional factors are reported to be involved in response to cold stress in plants (Yamaguchi-Shinozaki and Shinozaki, 2006), there are little information regarding to transcription factor UNE10-like protein except for one report regarding to response to light (Jaspers et al., 2009). Thus, it was difficult to discuss the involvement of UNE10-like protein in cold stress tolerance.
DEAD-box ATP-dependent RNA helicase has been reported to be involved in chilling and freezing tolerance of Arabidopsis as a regulator of CBF genes (Gong et al., 2002). In cyanobacteria, a cold-induced DEAD-box RNA helicase was suggested to unwind cold-stabilized secondary structure in the 5’-untranslated region of RNA during cold stress (Yu and Owttrim, 2000). Lu et al. (2020) showed that DEAD-box RNA helicase 42 plays a critical role in pre-mRNA splicing for adaptation to cold stress in rice. Furthermore, as Liu et al. (2016) shows, this enzyme would be an important role in growth and development of plant under low temperature. So, the enzyme coded by this clone would be necessary for cold acclimation of lettuce.
Information of proteins with G-patch (glycine-rich motif) domains, whose roles are predicted as RNA binding or RNA processing (Aravind and Koonin, 1999), are very rare in plant kingdoms. Zhang et al. (2005) suggested that MOS2, a protein containing G-patch and KOW(Kyprides, Ouzounis, Woese) motifs, is essential for innate immunity in Arabidopsis thaliana. The KOW motif seems to be found in a variety of ribosomal proteins. By searching Pfam database, the G-patch domain containing protein encoded by a gene, to which the isolated EST clone in the present paper showed similarity, does not contain a KOW motif, but it contains RNA binding and zing-finger motifs, suggesting this G-patch domain containing protein might regulate translation of transcripts of some genes for stress response in lettuce.
Involvement of “protein destination and storage”-categorized genes in freezing tolerance
In the category of “protein destination and storage”, 25 ESTs individually encoded a part of four proteins, whose amino acid sequences were respectively similar to amino acid sequence of BAG family molecular chaperone regulator 4 (BAG4; 1 clone), probable inactive ATP-dependent zinc metalloprotease FTSHI 3 (1 clone), oligopeptidase A (1 clone), or vacuolar processing enzyme (22 clones).
BAG (Bcl-2-associated athanogene) protein is a ubiquitous family of chaperone regulators of apotosis by interacting with Hsp70 and Hsc70 and stress tolerance of transgenic tobacco plants with different expression levels of BAG4 gene from Arabidopsis were investigated (Doukhanina et al., 2006). Tobacco plant or Arabidopsis plant with low expression level of AtBAG4 reduced chlorosis by abiotic stress such as UV-, oxidative-, drought-, salt- and cold stresses and showed enhancement of stress tolerance compared to other transgenic plants and wild-type plants. Thus, BAG4 protein at low expression level was considered to play an important role for regulating apotosis, which is induced by stress, in plants. Thus, in the present paper, the expression level of this bag4 gene in lettuce would be probably important for cold tolerance of lettuce.
Probably inactive ATP-dependent zinc metalloprotease FTSHI 3 gene was likely to be induced by low temperature in lettuce. FTSH means filamentation-temperature-sensitive protein H and additional “I” means protease-inactive (Mishra et al., 2019). Five kinds of Arabidopsis FTSHIs are localized in the chloroplast envelope and, out of them, FtsHi1 is reported to be involved in biogenesis and division of chloroplast (Kadirjan-Kalbach et al., 2012). It is difficult to discuss the role of this gene in lettuce, however, this gene was highly induced by low temperature.
There are few information regarding to low-temperature-inducible oligopeptidase. However, overexpression of a gene encoding prolyl oligopeptidase from rice was reported to confer abiotic stress tolerance to Escherichia coli (Tan et al., 2013a), suggesting that oligopeptidase is likely contribute to enhancement of stress tolerance of organisms including plants.
Vacuolar processing enzyme (VPE) is a vacuole-localized cysteine proteinase responsible for the maturation and activation of vacuolar proteins, which are synthesized on the endoplasmic reticulum (ER) as a proprotein precursor and are then transported to vacuoles (Hatsugai et al., 2015). The enzyme has a caspase-1-like activity which is related with program cell death (PCD) (Hara-Nishimura and Hatsugai 2011). Under heat stress condition, gVPE deficiency suppressed vacuolar disruption and led to enhancement of heat tolerance in Arabidopsis (Li et al., 2012). Koukalová et al. (1997) reported that cold stress induced PCD of plant cells. Qiao et al. (2002) reported that overproduction of animal cell death suppressors, Bcl-xL and Ced-9, in tobacco cells enhanced resistance to salt, cold, and wound stress. Furthermore, Bcl-2 protein was suggested to suppress hydrogen peroxide-induced PCD by suppressing the expression of OsVPE2 and OsVPE3 in rice (Deng et al., 2011).Thus, the induction of VPE gene was considered to lead to promotion of PCD and play a negative role in acquiring cold tolerance.
In the present papers, several types of proteases or peptidases were identified as low-temperature-inducible. These proteases might quickly and properly degrade denatured proteins for utilization as parts of newly synthesized protein. It is necessary to make clear the involvement in response to low temperature by investigating individual target protein or localization of them in future.
Involvement of “transporters”-categorized genes in freezing tolerance
In the category of “transporters”, eight ESTs individually encoded five proteins, whose amino acid sequences were respectively similar to amino acid sequence of sucrose transporter (1 clone), monosaccharide-sensing protein 2-like (2 clones), early nodulin-like protein (2 clones), metal-nicotianamine transporter (1 clone), or kinesin-like protein KIN-7K (2 clones).
Involvement of sucrose transporter in cold tolerance of plants was reported by several researchers (Jia et al., 2015; Yue et al., 2015). By searching WoLF PSORT database, the identified protein in the present paper was predicted to localize in plastid (data not shown). Furthermore, as Patzke et al. (2019) reported, plastidic sucrose transporter of Arabidopsis is likely to be involved in freezing tolerance. Then, sucrose transporter is also likely to function for development of cold tolerance in lettuce.
There are few papers regarding to monosaccharide-sensing protein like-2. However, deduced amino acid sequence of monosaccharide-sensing protein 2-like in the present paper is likely to be similar to that of tonoplast monosaccharide transporter 2. According to Klemens et al. (2014), sugar accumulation in vacuole seems important for freezing tolerance. Induction of tonoplast monosaccharide transporter by low temperature treatment might be important for enhancement of freezing tolerance in lettuce.
An early nodulin-like protein is reported as a cold-responsive protein in cypress (Pedron et al., 2009). However, as original function of this protein remains unclear, it is difficult to discuss its role in lettuce.
Nicotianamine forms a complex with a metal ion and metal-nicotianamine transporter YSL6 is involved in transportation of metal movement within the plant and functions for homeostasis in plants (Conte et al., 2013). AtYSL6 are likely to function in the influx from the acidic vacuole to the cytoplasm in Arabidopsis. OsYSL6 was constitutively expressed in rice plants (Koike et al., 2004). Although it is difficult to discuss why this gene was induced by low temperature in lettuce, this protein would be involved in cold acclimation.
Kinesin superfamily proteins are important microtubule-based motor proteins with a kinesin motor domain that is conserved among all eukaryotic organisms (Li et al., 2012). Gao et al.(2017b) showed that kinesin-like protein KCA2 was phosphorylated by cold stress in cold tolerant banana cultivar not in cold sensitive cultivar, suggesting that KCA2 plays important role in development of cold tolerance of banana. The protein was also reported to play critical roles in mitosis, morphogenesis, signal transduction, and regulating gibberellin biosynthesis and cell growth by transcriptionally activation (Li et al., 2012). Thus, this protein might play a role in cold tolerance of lettuce.
Involvement of “intracellular traffic”-categorized genes in freezing tolerance
In the category of “intracellular traffic”, five ESTs individually encoded four proteins, whose amino acid sequences were respectively similar to amino acid sequence of BTB/POZ domain-containing protein (2 clones), IST1-like protein (1 clone), phosphatidylinositol/phosphatidylcholine transfer protein (1 clone), or vacuolar sorting-associated protein 32 homolog 2 (1 clone).
BTB/POZ domain-containing protein contains a Broad-complex Tramtrack and Bric-a-brac (BTB) or POX virus and Zinc finger (POZ) domain. This protein was reported to participate in plant responses to biotic and abiotic stress (He et al., 2019). The protein in pepper plant was cold inducible and suggested to interact with DREB2A, an important transcription factor in abiotic stress, resulting in regulation of plant stress response (He et al., 2019). However, reason of the expression of the protein in response to cold stress is unclear.
Endosomal Sorting Complex Required for Transport (ESCRT) pathway is composed of increased salt tolerance 1 (IST1) protein, LIP5, charged multivesicular body protein (CHMP) 1, and SKD1 (Buono et al., 2016). ATPase activity of SKD1 and endosomal trafficking by ESCRT system are regulated by LIP5 and IST1. In Arabidopsis, 12 types of IST1-like (ISTL1) proteins were identified (Buono et al., 2016). Because interaction of one of ISTL1 proteins with LIP5 is likely to be essential for normal plant growth and repression of spontaneous cell death by regulating an important ATP enzyme, SKD1, for endosomal recruitment (Buono et al., 2016), induction of ISTL1 is also probably important for repression of cell death in lettuce plants under low temperature.
Vacuolar sorting-associated protein 32 (VPS32) homolog 2 is also recognized as CHMP2-1 or sucrose non-fermenting (SNF) 7.1 (https://www.uniprot.org/uniprot/Q9SZE4). This protein is also one of core components of ESCRT III (Ibl et al., 2012) and is considered isotype of CHMP1 described paragraph of IST1-like protein. It is also reported to play a role of MVB biogenesis, endosomal sorting or viral replication (Gao et al., 2017a). To our knowledge, induction of VPS32 or IST1-like protein by low temperature has not been reported. In the present paper, identified homologs to VPS32 or IST1 would be different from normal components of ESCRT III.
Phosphatidylinositol/phosphatidylcholine transfer protein was reported to be homologous to Sec14 protein of yeast (Mo et al., 2007). Wang et al. (2016) reported that ZmSEC14p, Sec14 protein from maize was a cold responsive protein and overexpression of the protein in transgenic Arabidopsis conferred tolerance to cold stress. Thus, clarification of the function of the protein is expected in future.
Involvement of “cell structure”-categorized genes in freezing tolerance
In the category of “cell structure”, two ESTs individually encoded one protein, whose amino acid sequence was similar to amino acid sequence of arabinogalactan protein 2 (2 clones).
Arabinogalactan protein is grouped into a superfamily of highly glycosylated hydroxyproline-rich proteins (Gong et al., 2012). Gong et al. (2012) reported the involvement of an arabinogalactan protein, GhAGP31, in cold stress tolerance of cotton and the enhancement of freezing tolerance of yeast and transgenic Arabidopsis by overexpression of its encoding gene. They also suggested that GhAGP31 might interact with pectin to form protein-carbonhydrate linkage within cell walls, which contribute to the stability of the cell wall. In lettuce plants, induced arabinogalactan protein might contribute to enhancement of freezing tolerance.
Involvement of “signal transduction”-categorized genes in freezing tolerance
In the category of “signal transduction”, 8 ESTs individually encoded five proteins, whose amino acid sequences were respectively similar to amino acid sequence of Rho GTPase-activating protein (1 clone), elongation factor 2 (2 clones), serine/threonine-protein kinase (4 clones), or salicylic acid-binding protein (1 clone).
Rho GTPase-activating protein was reported to be involved in the regulation of tolerance to dehydration stress in barley (Suprunova et al., 2007). Although very little information is available on this protein in plants, Rho GTPase-activating protein in yeast has been reported to be an important regulator of multiple biological process including stress resistance and so on (Rahim et al., 2017). This protein might play an important role for response to low temperature stress in lettuce.
Elongation factor 2 (EF-2) protein is also well reported as cold responsive proteins for cold tolerance and plays an important role in protein synthesis (Shi et al., 2019). Shi et al. (2019) reported that overexpression of EF-2 gene enhanced freezing tolerance in tobacco plants by regulating hundreds of protein synthesis under low temperature conditions. In the present paper, this protein is thought to play an important function.
Some of serine/threonine-protein (STP) kinase is also known as cold inducible in Arabidopsis (Mizoguchi et al., 1995). A serine/threonine-protein kinase, OST1, acts upstream of CBF genes to positively regulate freezing tolerance (Ding et al., 2015). Identified STP kinase in the present paper seems to play an important role in signal transduction for cold response in lettuce.
One type of salicylic acid-binding protein (SABP2) possesses methyl salicylate (MeSA) esterase activity, thus it catalyzes the conversion of MeSA to salicylic acid (SA) (Li et al., 2019). Exogenic SA seems to induce freezing tolerance in wheat via hydrogen peroxide and abscisic acid (Wang et al., 2018a). Furthermore, endogenous SA is also likely to be important for chilling tolerance in maize seedlings (Wang et al., 2018b). Although chilling tolerance is different from freezing tolerance, SA seems to influence on tolerance to low temperature stress.
Involvement of “disease/defense”-categorized genes
In the category of “disease/defense”, 34 ESTs individually encoded 13 proteins, whose amino acid sequences were respectively similar to amino acid sequence of 11 kDa late embryogenesis abundant (LEA; 2 clones), LEA (3 clones), cold shock protein (1 clone), dehydrin Xero (10 clones), dehydrin DHN1 (2 clones), catalase (2 clones), early light inducible protein (5 clones), HVA22-like protein (1 clone), low sulfur responsive protein (1 clone), nodulin-related protein (4 clones), pathogenesis-related protein (1 clone), plastid-lipid-associated protein 11 (1 clone), or plastid-lipid-associated protein 6 (1 clone).
LEA proteins are one of probably most well-known stress responsive proteins in plants. In the present paper, 4 kinds of LEA (11-kDa LEA, LEA, dehydrin Xero, dehydrin DHN1) proteins were identified. For classification of LEA proteins, family domain search database, Pfam (https://pfam.xfam.org), is well used (Battaglia et al., 2008; Hundertmark and Hincha, 2008). By using the Pfam database, 11-kDa LEA and another LEA was grouped into LEA group1 and group 4, respectively (data not shown). Cold shock protein was also categorized into dehydrin by using Pham database (data not shown). These proteins would play important roles in freezing tolerance of lettuce.
Catalase is also well known as one of antioxidative enzymes for development of stress tolerance of plants (Saker and Oba, 2018). Feki et al. (2015) suggested that TdCAT1, a catalase gene from durum wheat, is a promising candidate gene for the development of crops with multiple stress tolerances. and transgenic approach expressing gene encoding catalase led to enhancement stress tolerance
Early light-inducible protein is also known as cold inducible under light condition (Adamska and Kloppstech, 1994; Hayami et al., 2015). This protein is likely to play a role in the protection of photosynthetic apparatus from excess light under cold condition (Arora and Rowland, 2011). Thus, the protein would probably play the same role in lettuce.
HVA22-like protein is likely to be localized in ER and Golgi membranes (Guo and Ho, 2008). In particular, HVA22 proteinswere estimated to be involved in vesicular traffic in stressed cells of citrus and one of them is likely to be involved in dehydration tolerance and oxidative stress reduction (Ferreira et al., 2019). The identified HVA22-like protein would also function vesicular traffic in lettuce plant, leading to low temperature stress.
Although information regarding to low sulfur responsive protein is not so much, a plant-specific low-sulfur responsive gene is reported to be responsive to a several stresses including cold stress (Tombuloğlu et al., 2016). Investigation of role of this protein in cold acclimation would be necessary.
Information of regarding to nodulin-related protein is not found. However, the deduced protein assigned to nodulin-related protein also showed sequence-similarity to the drought-induced unknown protein of sunflower (Ouvrard et al., 1996).
According to Van Loon and Van Strien (1999), there are several type proteins known as pathogenesis-related proteins, including chitinase, endoproteinase, peroxidase, defensin, and so on. This gene whose part was identified in the present paper as an EST clone was categorized into genes encoding cysteine-rich secretory protein family by using Pfam database (https://pfam.xfam.org).
Twelve sub-families of fibrillin were reported and some fibrillins categorized in group 1 were likely to be induced by cold (Singh and McNellis, 2011). Furthermore, Arabidopsis knock out mutant of fibrillin 5 was sensitive to cold stress (Kim et al., 2015). Thus, some of fibrillins are likely to be involved in cold tolerance.
Involvement of “secondary metabolism” and “unclear classification”-categorized genes in freezing tolerance
In the category of “secondary metabolism”, 10 ESTs individually encoded two proteins, whose amino acid sequences were respectively similar to amino acid sequence of chalcone synthase (CHS; 9 clones) or chalcone-flavone isomerase (CHI; 1 clone).
CHS is one of the key and rate-limiting enzymes of anthocyanine pathway and converts p-coumaroyl CoA and malonyl CoA to naringenin chalcone (Christie et al., 1994). Then, CHI converts naringenin chalcone to naringenin, which is one of polyphenols during flavonoids or anthocyanin biosynthesis (Christie et al., 1994). In Arabidopsis, mRNA of CHS is induced by cold stress under light (Leyva et al., 1995). Leyva et al. (1995) concluded that CHS induction is light-dependent and Arabidopsis acquire freezing tolerance with exposure to low temperature without light.
An EST clone which encodes root UVB sensitive (RUS) protein 5 is categorized in unclear classification and this encoded protein is one of RUS family. There are very few regarding the functions of RUSproteins. Recently, Rus5 knockout mutant of Arabidopsis was reported to show no visible phenotypic difference to wild type under normal growth conditions (Perry et al., 2021). At present, function of lettuce RUS5 is also not estimated under low temperature. Further research using knockout rus5 plant will be expected under low temperature condition.