horticulturae

Study Protocol Protein Improves the Cryopreservation Efficiency of Hosta capitata by Regulating the Genes Involved in the Low-Temperature Tolerance Mechanism

Phyo Phyo Win Pe 1,2,†, Aung Htay Naing 3,† , Chang Kil Kim 3,* and Kyeung Il Park 1,*

1 Department of Horticulture and Life Science, Yeungnam University, Gyeongsan 38541, Korea; [email protected] 2 Department of Horticulture, Yezin Agricultural University, Nay Pyi Taw 15013, Myanmar 3 Department of Horticulture, Kyungpook National University, Daegu 41566, Korea; [email protected] * Correspondence: [email protected] (C.K.K.); [email protected] (K.I.P.) † Phyo Phyo Win Pe and Aung Htay Naing equally contributed to this work.

Abstract: In this study, whether the addition of antifreeze protein (AFP) to a cryopreservative solution ( vitrification solution 2 (PVS2)) is more effective in reducing injuries in Hosta capitata than PVS2 alone at different cold exposure times (6, 24, and 48 h) is investigated. The upregulation of C-repeat binding factor 1 (CBF1) and 1 (DHN1) in response to low temperature was observed

in shoots. Shoots treated with distilled water (dH2O) strongly triggered gene expression 6 h after  cold exposure, which was higher than those expressed in PVS2 and PVS2+AFP. However, 24 h  after cold exposure, gene expressions detected in dH2O and PVS2 treatments were similar and Citation: Pe, P.P.W.; Naing, A.H.; higher than PVS2 + AFP. The expression was highest in PVS2+AFP when the exposure time was Kim, C.K.; Park, K.I. Antifreeze extended to 48 h. Similarly, nitric reductase activities 1 and 2 (Nia1 and Nia2) genes, which are Protein Improves the responsible for nitric oxide production, were also upregulated in low-temperature-treated shoots, Cryopreservation Efficiency of Hosta as observed for CBF1 and DHN1 expression patterns during cold exposure periods. Based on the capitata by Regulating the Genes gene expression patterns, shoots treated with PVS2+AFP were more likely to resist cold stress, which Involved in the Low-Temperature was also associated with the higher cryopreservation efficiency of PVS2+AFP compared to PVS2 Tolerance Mechanism. Horticulturae alone. This finding suggests that the improvement of cryopreservation efficiency by AFP could be 2021, 7, 82. https://doi.org/10.3390/ due to the transcriptional regulation of CBF1, DHN1, Nia1, and Nia2, which might reduce freezing horticulturae7040082 injuries during cryopreservation. Thus, AFP could be potentially used as a cryoprotectant in the

Academic Editor: Amit Dhingra cryopreservation of rare and commercially important plant germplasm.

Received: 14 March 2021 Keywords: antifreeze protein; cryopreservation; endangered plant germplasm; gene expression; Accepted: 13 April 2021 low-temperature tolerance Published: 15 April 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Hosta species are herbaceous perennial . Their decorative foliage, diverse flower iations. colors, attractive inflorescences, and ability to tolerate shade make them popular as ground- cover ornamental plants in the landscape industry. Korea is one of the countries where some of these species, such as Hosta capitata, Hosta clausa, Hosta longipes, Hosta minor, and Hosta yingeri, are distributed [1]. However, the viral disease Hosta virus X has been found to Copyright: © 2021 by the authors. infect the leaves of ornamental plants cultivated in Korea [2]. In addition, these species face Licensee MDPI, Basel, Switzerland. a steady decline in their natural habitats due to overcollection and global climate change. This article is an open access article This increases the risk of their extinction and potentially harms their use as groundcover distributed under the terms and plants in the landscape industry. conditions of the Creative Commons Cryopreservation has been widely used as a novel tool for both germplasm conser- Attribution (CC BY) license (https:// vation and the production of virus-free plants [3–5]. However, severe intracellular ice creativecommons.org/licenses/by/ formation has been the main challenge for the cryopreservation technique. Antifreeze 4.0/).

Horticulturae 2021, 7, 82. https://doi.org/10.3390/horticulturae7040082 https://www.mdpi.com/journal/horticulturae Horticulturae 2021, 7, 82 2 of 10

protein (AFP) is added into the loading solution or plant vitrification solution 2 (PVS2) before dipping the biological materials into liquid nitrogen (LN) to increase the survival rate and plant regeneration. This intervention can inhibit the formation of ice and protect cells from freezing injury [6]. Despite its application in the cryopreservation of cells [7–9], there have been only a few reports of AFP as a cryoprotectant in the cryopreservation of plant tissues and organs [10–12]. Jeon et al. [11] reported AFP’s inhibitory role in ice formation in the cryopreservation of chrysanthemum shoot tips. Seo et al. [12] reported the transcriptional regulation of cold regulatory genes, such as C-repeat binding factor 1 (CBF1) and dehydrin 1 (DHN1), in cryopreserved potato shoots by AFP. In addition, AFP’s influence in the transcriptional regulation of CBF1 and DHN1 genes in H. capitata seedlings at a low temperature was also reported [13]. Previous reports have indicated the involvement of nitric oxide (NO) in plant accli- mation and freezing tolerance, as NO acts as a signaling molecule in response to abiotic and biotic stresses [14,15]. At a low temperature (4 ◦C), NO production mainly depends on nitric reductase (NR) activity, which is controlled by Nia1 and Nia2 genes; gene knockout in plants reduced NO production and freezing tolerance [16,17]. NO synthesis in response to a low temperature has also been reported in Lotus japonicus and Pisum sativum [18,19]. However, the transcriptional regulation of Nia1 and Nia2 genes by AFP has not been investigated so far. Thus, it is also interesting to examine the genes involved in the cold tolerance mechanism in this study. There have been no studies reporting the successful cryopreservation of Hosta species (H. capitata). Therefore, it is necessary to develop an optimal protocol for the successful cryopreservation of this species for germplasm conservation. It is of interest to determine whether AFP acts as a cryoprotectant in cryopreserved tissues. Hence, before cryopreserva- tion, AFP’s role in the transcriptional regulation of genes involved in the cold tolerance mechanism is first tested. Its beneficial role in improving the cryopreservation efficiency of H. capitata is then investigated.

2. Materials and Methods 2.1. Plant Materials In-vitro-grown H. capitata plants, obtained from a previous study, were kept in a hormone-free Murashige and Skoog (MS) medium [20] and subcultured in the same medium every 4 weeks. Only uniform and healthy plants were selected to obtain meristems (0.5 mm–1.0 cm) used as explants for this experiment.

2.2. AFP Treatment To determine whether AFP can be used as a cryoprotectant in the cryopreservation process, before cryopreservation was started, its involvement in the low-temperature tolerance mechanism was initially accessed. Briefly, meristems were precultured for 3 h in a liquid MS medium (0.3 M sucrose) on a rotary shaker at 60 rpm. Meristems were dipped in a loading solution (LS; liquid MS medium, 0.4 M sucrose, 2.0 M glycerol (pH 5.8)) [21] for 30 min and then in plant vitrification solution 2 (PVS2; liquid MS medium, 3.28 M glycerol, 2.42 M ethylene glycol, 1.9 M dimethyl sulfoxide, 0.4 M sucrose (pH 5.8)) for 30 min at 25 ◦C [22] with or without AFP (100 or 0 ng/mL AFP III; A/F Protein, Inc., Waltham, MA, USA). Meristems were air-dried and cultured in a hormone-free MS medium and then kept at a low temperature (4 ◦C) for 6, 24, and 48 h. Meristems that were not dipped in any solution but kept at room temperature were used as controls. Those dipped in dH2O and placed at 4 ◦C were used as negative controls (Figure1). A treatment contained 15 explants with three replications. After low-temperature treatment, meristems from each treatment were collected, placed in 1.5 mL Falcon tubes, immersed in liquid nitrogen (LN), and stored at −80 ◦C for RNA extraction and gene expression analysis. Horticulturae 2021, 7, x FOR PEER REVIEW 3 of 10

Horticulturae 2021, 7, 82 3 of 10 each treatment were collected, placed in 1.5 mL Falcon tubes, immersed in liquid nitrogen (LN), and stored at −80 °C for RNA extraction and gene expression analysis.

Figure 1. Schematic diagramdiagram showingshowing thethe application application of of antifreeze antifreeze protein protein (AFP) (AFP) as as a cryoprotectanta cryoprotectant in thein the low-temperature low-tempera- turtolerancee tolerance mechanism. mechanism.

2.3. RNA RNA Extraction Extraction and and Gene Gene Expression Expression Analysis RNA was was extracted extracted from from the the meristems meristems of ofthe the treatments treatments mentioned mentioned above above using using the RNeasythe RNeasy Plant Plant Mini Mini Kit Kit (Qiagen, (Qiagen, Hilden, Hilden, Germany), Germany), as as described described by by Naing Naing et et al. al. [ [2323].]. cDNAcDNA was synthesized synthesized from from 1 µgµg total RNA using an oligo(dT)20 primer primer.. The transcript levelslevels of of genes ( CBF1,, DHN1,, Nia1,, and Nia2) involved in the low-temperaturelow-temperature tolerance mechanism were detected using the Step One Plus Real Real-Time-Time Polymerase C Chainhain Reaction (PCR) System (Applied (Applied Biosystems, Foster City, CA,CA, USA). The actin gene (ACT) was used as the reference gene.gene. TheThe primersprimers andand PCR PCR conditions conditions used used for for gene gene detection detection are are listed listed in inTable Table1. Three 1. Three independent independent biological biological samples samples per per treatment treatment were wer usede used for for the the analysis analysis of ofeach each gene. gene.

Table 1.1. Primer sequences and PCR conditions used in this study for quantitative real real-time-time PCR (qRT (qRT-PCR)-PCR) analysis of Hosta capitata.

GenesGenes Primer Primer Sequence Sequence (5′→ (50 →3′) 30)PCR PCR Condition Condition F- AGGGTCAAAGGACACACGTCF- AGGGTCAAAGGACACACGTC 95 °95C (10◦C (10min) min) →→ [95[95 °C◦ C(15 (15 s) s) →→ 5959 °C◦C (30 (30 s)]s)] ×× 40 cycles CBF1CBF1 ◦ ◦ ◦ R- GGGAGCACACGGAGTTTTTGR- GGGAGCACACGGAGTTTTTG → 95→ °95C (15C (15s) → s) →5858 °C C(1(1 min) min) →→ 9595 °CC (15 (15 s)s) F- CAGTCTGACATCACCAGGGTA 95 ◦C (10 min) → [95 ◦C (15 s) → 59 ◦C (30 s)] × 40 cycles DHN1 F- CAGTCTGACATCACCAGGGTA 95 °C (10 min) → [95 °C (15 s) → 59 °C (30 s)] × 40 cycles → DHN1 → ◦ → ◦ → ◦ R- GGAGTTATAGGAGAGACAGATTR- GGAGTTATAGGAGAGACAGATT 95 °C95 (15C s) (15 → s) 57 °57C (1C min) (1 min) → 9595 °C C(15 (15 s) s) F- CCACCAGGAGAAACCGAACA 95 ◦C (10 min) → [95 ◦C (15 s) → 59 ◦C (30 s)] × 40 cycles Nia1 F- CCACCAGGAGAAACCGAACA 95 °C (10 min) → [95 °C (15 s) → 59 °C (30 s)] × 40 cycles → Nia1 R- TTGAAAGACTCGTCCCAGGC → 95 ◦C (15 s) → 55 ◦C (1 min) → 95 ◦C (15 s) R- TTGAAAGACTCGTCCCAGGC 95 °C (15 s) → 55 °C (1 min) → 95 °C (15 s) F- CCTTCTTTGGTAGACGCCGA 95 ◦C (10 min) → [95 ◦C (15 s) → 59 ◦C (30 s)] × 40 cycles Nia2 F- CCTTCTTTGGTAGACGCCGA 95 °C (10 min) → [95 °C (15 s) → 59 °C (30 s)] × 40 cycles → Nia2 R- TCGTGACATGGCGTCGTAAT → 95 ◦C (15 s) → 53 ◦C (1 min) → 95 ◦C (15 s) R- TCGTGACATGGCGTCGTAAT 95 °C (15 s) → 53 °C (1 min) → 95 °C (15 s)

2.4. Cryopreservation Cryopreservation Cryopreservation of of meristems meristems was was performed performed following following Jeon Jeon et et al.’s al.’s [11] [11] protocol, with some some modifications. modifications. Briefly, Briefly, meristems meristems (approximately (approximately 5 mm) 5 mm) were were excised excised from from 4- week4-week-old-old in invitro vitro H.H. capitata capitata. The. The excised excised meristems meristems were were precultured precultured in MS in basal MS basal me- diummedium containing containing 0.3 0.3M Msucrose sucrose for for 3 h 3 hand and then then loaded loaded with with LS LS for for 3030 min. min. The The loaded meristems were were treated treated with with the the PVS2 PVS2 solution solution,, with with or or without without AFP AFP (100 (100 ng/mL) ng/mL),, for for30 ◦ min30 min at 0at °C. 0 Then,C. Then, meristems meristems treated treated with with the the PVS2 PVS2 solution solution and and AFP AFP were were transferred transferred to to cryovials (Thermo Scientific, Waltham, MA, USA) containing fresh PVS2 and AFP; those treated with PVS2 solution only were also transferred to a cryovial containing PVS2 solution only. They were immersed in LN for 30 min. Meristems that were not treated with

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Horticulturae 2021, 7, 82 4 of 10 cryovials (Thermo Scientific, Waltham, MA, USA) containing fresh PVS2 and AFP; those treated with PVS2 solution only were also transferred to a cryovial containing PVS2 solu- tion only. They were immersed in LN for 30 min. Meristems that were not treated with any loading solutionany loading but immersed solution in but LN immersed were used in as LN controls. were used Meristems as controls. immersed Meristems in immersed in ◦ LN were rapidlyLN warmed were rapidly in a water warmed bath inat a38 water °C for bath 3 min at 38andC then for 3 washed min and for then 20 min washed for 20 min ◦ at 25 °C with unloadingat 25 C with solution unloading (MS + solution1.2 M sucrose) (MS + 1.2and M washed sucrose) thrice and washedwith dH thrice2O. with dH2O. (Figure 2). There(Figure were 215). Thereexplants were in a 15 cryovial explants, with in a cryovial,three replications. with three replications.

Figure 2. FigureSchematic 2. Schematic diagram diagram showing showing the steps the involved steps involved in the in cryopreservation the cryopreservation of Hosta of Hosta capitata. capitata.LN stands for liquid nitrogen.LN stands for liquid nitrogen.

2.5. Recovery of Cryopreserved2.5. Recovery ofMeristems Cryopreserved Meristems Cryopreserved Cryopreservedmeristems were meristems then transferred were then to a transferredpostculture to medium a postculture containing medium containing + + ◦ NH4 -free MS forNH 34 days-free in MS the for dark 3 days at 25 in ± the 2 ° darkC. The at recovered 25 ± 2 C. shoots The recovered were then shoots trans- were then trans- ferred to an MSferred basal tomedium an MS basalcontaining medium α-naphthalene containing αacetic-naphthalene acid and aceticbenzyladenine acid and benzyladenine (KIN) for 4 weeks.(KIN) The for survival 4 weeks. and The shoot survival regeneration and shoot rates regeneration, with or without rates, with AFP or, were without AFP, were compared to controlscompared at 4 toweeks controls postculture. at 4 weeks The postculture. total number The of total explants number per oftreatment explants per treatment was 45. Survivalwas rate 45. was Survival defined rate as wasthe percentage defined as theof meristems percentage showing of meristems green showingtissues, green tissues, and shoot regenerationand shoot was regeneration defined as wasthe percentage defined as theof meristems percentage inducing of meristems shoots. inducing shoots.

2.6. Statistical Analysis2.6. Statistical Analysis Data were statisticallyData were analyzed statistically using analyzedanalysis of using variance analysis (ANOVA) of variance with (ANOVA)SPSS ver- with SPSS ver- sion 11.09 (IBMsion Corporation, 11.09 (IBM Armonk, Corporation, NY, USA). Armonk, Data NY, are USA). presented Data as are means presented ± standard as means ± standard errors (SEs). Meanerrors separations (SEs). Mean were separations carried out were using carried the le outast usingsignificant the least difference significant test difference test (LSDT), and significance(LSDT), and was significance determined was at the determined 5% level. at the 5% level. 3. Results 3. Results 3.1. Antifreeze Protein Regulates Genes Involved in the Low Temperature Tolerance Mechanism 3.1. Antifreeze Protein Regulates Genes Involved in the Low Temperature Tolerance Mechanism The transcript levels of CBF1 and DHN1 genes expressed in shoot tips exposed to room The transcripttemperature levels of didCBF1 not and vary DHN1 among genes treatment expressed times in (6, shoot 24, and tips 48 exposed h) and wereto significantly room temperaturelower did than not thosevary among exposed treatment to a low times temperature (6, 24, and (4 ◦ C).48 h) Generally, and were the signifi- expression patterns cantly lower thanof thethose detected exposed genes to a were low similartemperature in trend (4 for°C). this Gene investigation.rally, the expression At the low temperature, patterns of the detected genes were similar in trend for this investigation. At the low tem- shoots treated with dH2O triggered CBF1 and DHN1 gene expression after 6 hr. Similarly, perature, shootsshoots treated treated with dH with2O PVS2 triggered alsotriggered CBF1 and gene DHN1 expression, gene expression but their after levels 6 hr. were significantly Similarly, shootslower treated than with those PVS2 in dH also2O. triggered Gene induction gene expression, was also observedbut their levels in shoots were treated with the significantly lowerAFP-containing than those PVS2 in dH solution,2O. Gene which induction was even was lower also observedthan PVS2 in (Figures shoots3 and4). treated with the AFP-containing PVS2 solution, which was even lower than PVS2 (Figures 3 and 4).

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Figure 3. Transcript levels of CBF1 in meristems of Hosta capitata treated with dH2O and plant vitrification solution 2 (PVS2), with or without AFP (AFP III), at 4 °C for different time intervals ((a): 6 h, (b): 24 h, and (c): 48 h). Plants at 25 °C were used as controls. The actin gene (ACT) was used as the reference gene. Data are presented as the means of three replicates. Error bars indicate standard errors of the means. Means with the same letter are not significantly different by the least significant difference test (LSDT, p < 0.05).

Figure 3. TranscriptTranscript levels levels of ofCBF1 CBF1in in meristems meristems of Hostaof Hosta capitata capitatatreated treated with with dH O dH and2O plant and vitrificationplant vitrification solution solution 2 (PVS2), 2 Higher expression was changed from dH22O to PVS2 when the treatment time was 24 (PVS2), with or without AFP (AFP◦ III), at 4 °C for different time intervals ((a): 6 h, (b): 24 h, and (c): 48 h). Plants◦ at 25 °C with or without AFP (AFP III),h, whereas at 4 C for their different expression time intervals in PVS2+AFP ((a): 6 h, (b) was: 24 h,still and the (c) :lowest. 48 h). Plants When at 25the Ctreatment were used time wereas controls. used as The controls.actin gene The (ACT actin) wasgene used (ACT as) thewas reference used as gene.the reference Data are gene. presented Data asare the presented means of as three the replicates.means of three Error replicates. Error bars indicatewas standard further errors extended of the to means. 48 h, their Means expression with the same significantly letter are notdecreased significantly with differentthe dH2 Oby treat- bars indicate standard errors of the means. Means with the same letter are not significantly different by the least significant the least significant differencement, test (LSDT,but not p with< 0.05). PVS2, whereas CBF1 and DHN1 gene expression sharply increased difference test (LSDT, p < 0.05).with PVS2+AFP and were the highest. Higher expression was changed from dH2O to PVS2 when the treatment time was 24 h, whereas their expression in PVS2+AFP was still the lowest. When the treatment time was further extended to 48 h, their expression significantly decreased with the dH2O treat- ment, but not with PVS2, whereas CBF1 and DHN1 gene expression sharply increased with PVS2+AFP and were the highest.

Figure 4. Transcript levels of DHN1 in meristems of Hosta capitata treated with dHdH2O and plant vitrification vitrification solution 2 (PVS2)(PVS2),, with or wi withoutthout AFP (AFP III),III), at 4 ◦°CC for different time intervals ((((a)a):: 6 h,h, (b)b):: 24 h,h, and (c)c): 48 h).h). Plants at 25 ◦°CC were used as controls. The actin gene (ACT) was used as the reference gene. Data are presented as the means of three were used as controls. The actin gene (ACT) was used as the reference gene. Data are presented as the means of three replicates. Error bars indicate standard errors of the means. Means with the same letter are not significantly different by replicates. Error bars indicate standard errors of the means. Means with the same letter are not significantly different by the the least significant difference test (LSDT, p < 0.05). least significant difference test (LSDT, p < 0.05). Figure 4. Transcript levels of DHN1 in meristems of Hosta capitata treated with dH2O and plant vitrification solution 2 To investigate NO and its involvement in cold acclimation, NR activities that produce (PVS2), with or without AFP (AFPHigher III), at expression4 °C for different was time changed intervals from ((a dH): 6 2hO, (b to): PVS224 h, and when (c): 48 the h). treatment Plants at 25 time °C was were used as controls. The NO24actin h, were gene whereas detected(ACT) their was in expressionused shoots as theat lowreference in PVS2+AFPtemperature gene. Data was (4 are° stillC) presentedand the room lowest. as temperature the When means the of (25 treatmentthree °C) by analyzing the transcript levels of Nia1 and Nia2 genes. As expected, shoots at 4 °C trig- replicates. Error bars indicatetime standard was further errors of extended the means. to 48Means h, their with expression the same letter significantly are not significantly decreased different with the by dH 2O the least significant differencegeredtreatment, test higher(LSDT, but genep not< 0.05). expression with PVS2, than whereas shootsCBF1 at 25and °CDHN1 regardlessgene of expression exposure sharply times. However, increased shootswith PVS2+AFP pretreated and with were PVS2 the and highest. PVS2+AFP induced lower expression levels than those treatedToTo withinvestigate dH2O atNO 6 hand after its cold involvement involvement exposure, in whereas cold cold acclimation, acclimation, those expressed NR activities activities in PVS2+AFP that that produce produce were theNONO lowest.were detected At 24 h in after shoots cold at exposure, low temperature gene expression (4 °◦C) and in dHroom 2O temperature and PVS2 was (25 not °◦C) sig- by nificantlyanalyzinganalyzing dif theferent transcripttranscript but levelshigher levels of ofthanNia1 Nia1 PVS2+AFP.and andNia2 Nia2genes. Interestingly,genes As. As expected, expected, their shoots expression shoots at 4 at◦C 4 triggeredin ° CdH trig-2O startedgeredhigher higher to gene decline gene expression when expression the than exposure than shoots shoots time at 25 at was 25◦C °increased regardlessC regardless to of 48 of exposure hexposure, but they times. times. were However,still However, mod- eratelyshootsshoots pretrea pretreatedexpressedted inwith PVS2 PVS2 and and highly PVS2+AFP expressed induced induced in PVS2+AFP. lower lower expression However, levels the expressionthan those levelstreatedtreated betw with witheen dH dH the2O2O at genes at 6 h 6 after h were after cold slightly cold exposure, exposure, different whereas whereas for those all treatments those expressed expressed and in PVS2+AFP exposure in PVS2+AFP timeswere (Figthewere lowest.ures the 5 lowest. andAt 24 6). h AtThese after 24 h coldresults after exposure, cold suggest exposure, gene that expressionlow gene-temperature expression in dH2-Oinduced in and dH PVS22O upregulation and was PVS2 not wassig- of Nia1nificantlynotsignificantly and Nia2different result different buts in higher NO but production higherthan PVS2+AFP. than in PVS2+AFP. plants Interestingly, at a Interestingly,low temperature; their expression their at expression 6 andin dH 242 inOh exposure,starteddH2O startedto declinethe low to - declinewhentemperature the when exposure-induced the exposure time gene was upregulation time increased was increased to was 48 h lower, but to they48in shoots h, were but pretreated theystill mod- were eratelystill moderately expressed expressed in PVS2 and in PVS2highly and expressed highly in expressed PVS2+AFP. in PVS2+AFP. However, the However, expression the levelsexpression betw levelseen the between genes thewere genes slightly were different slightly different for all treatments for all treatments and exposure and exposure times (Figtimesures (Figures 5 and5 6).and These6 ). These results results suggest suggest that thatlow low-temperature-induced-temperature-induced upregulation upregulation of

Nia1of Nia1 andand Nia2Nia2 resultresultss in inNO NO production production in in plants plants at at a alow low temperature; temperature; at at 6 6 and and 24 24 h exposure, the low-temperature-induced gene upregulation was lower in shoots pretreated

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exposure, the low-temperature-induced gene upregulation was lower in shoots pretreated with PVS2+AFP. TakenTaken together,together, AFP AFP is is likely likely to to be be used used for for the the reduction reduction of of cold cold stress stress in inwiththis this species. PVS2+AFP. species. Taken together, AFP is likely to be used for the reduction of cold stress in this species.

Figure 5. TranscriptTranscript levels of Nia1 inin meristems meristems of of HostaHosta capitata capitata tretreatedated with with dH dH22OO and and plant plant vitrification vitrification solution solution 2 2 (PVS2) (PVS2),, withFigure or 5. withoutwithout Transcript AFPAFP levels (AFP (AFP of III), III) Nia1 at, at 4 in 4◦C meristems°C for for different different of Hosta time time intervalscapitata intervals tre ((a)ated (:(a 6) h,with: 6 ( b)h, dH :( 24b)2:O h,24 and and h, plantand (c): 48( cvitrification) h).: 48 Plants h). Plants at solution 25 at◦C 25 were 2°C (PVS2) were used, usedaswith controls. asor controls.without The actin AFPThe gene actin(AFP gene ( ACTIII), ()atACT was 4 °C) used was for used asdifferent the as reference the time reference intervals gene. gene. Data ((a are)Data: 6 presentedh ,are (b )presented: 24 h as, and the as means(c the): 48 means ofh). three Plants of three replicates. at 25 replicates. °C Errorwere Errorused asbars controls. indicate The standard actin gene errors (ACT of) wasthe means.used as Meansthe reference with the gene. same Data letter are arepresented not significantly as the means different of three by replicates. the least bars indicate standard errors of the means. Means with the same letter are not significantly different by the least significant sErrorignificant bars dindicateifference standard test (LSDT, errors p < of0.05). the means. Means with the same letter are not significantly different by the least difference test (LSDT, p < 0.05). significant difference test (LSDT, p < 0.05).

Figure 6. Transcript levels of Nia2 in meristems of Hosta capitata treated with dH2O and plant vitrification solution (PVS2), 2 withFigure or 6.without Transcript AFP levels (AFP of III) Nia2Nia, at2 in4 °C meristems for different of Hosta time capitata intervals treated ((a): with6 h, ( dHb): 224O and andh, and plant plant (c) vitrification: vitrification 48 h). Plants solution at 25 °C (PVS2) (PVS2), were, withused orasor withoutcontrols.without AFP AFPThe (AFP actin(AFP III),gene III) at, (atACT 4 ◦4C °C) forwas for different useddifferent astime the time reference intervals intervals ((gene.a) (:( 6a )Data h,: 6 ( b)h ,are: ( 24b )presented: h, 24 and h, and (c): as 48(c the) h).: 48 means Plants h). Plants of at 25three at◦C 25 replicates. were °C were used used as controls. The actin gene (ACT) was used as the reference gene. Data are presented as the means of three replicates. Erroras controls. bars indicate The actin standardgene (ACT errors) was of used the means. as the reference Means with gene. the Data same are letter presented are not as thesignificantly means of threedifferent replicates. by the Errorleast sErrorignificant bars dindicateifference standard test (LSDT, errors p < of0.05). the means. Means with the same letter are not significantly different by the least bars indicate standard errors of the means. Means with the same letter are not significantly different by the least significant significant difference test (LSDT, p < 0.05). difference test (LSDT, p < 0.05). 3.2. Influence of Antifree Protein on Cryopreservation Efficiency 3.2. Influence of Antifree Protein on Cryopreservation Efficiency 3.2. InfluenceTo further of clarify Antifree whether Protein onthe Cryopreservation cryoprotective role Efficiency of AFP improves the cryopreserva- tion efficiencyTo further of clarify this species, whether shoots the cryoprotective were loaded with role LSof ,AFP cryoprotected improves thewith cryopreserva- PVS2 (alone To further clarify whether the cryoprotective role of AFP improves the cryopreser- ortion in efficiency combination of this with species, AFP), shootsand then were immersed loaded with in LN. LS The, cryoprotected addition of withAFP PVS2to the ( PVS2alone vation efficiency of this species, shoots were loaded with LS, cryoprotected with PVS2 solutionor in combination improved with the survival AFP), and rate then (46.7 immersed%) and shoot in LN. regeneration The addition (28.9 of AFP%) compared to the PVS2 to (alone or in combination with AFP), and then immersed in LN. The addition of AFP to PVS2solution alone improved (which theshowed survival a survival rate (46.7 rate%) andof 33.3 shoot% a regenerationnd shoot regeneration (28.9%) compared of 17.8% to). the PVS2 solution improved the survival rate (46.7%) and shoot regeneration (28.9%) com- However,PVS2 alone the (which controls showed that werea survival not treated rate of with 33.3 % any and cr yoshootprotectant regeneration solution of but17.8 im-%). pared to PVS2 alone (which showed a survival rate of 33.3% and shoot regeneration of mersedHowever, in LN the did controls not survive that were at all not (Table treated 2). This with result any crsuggestyoprotectants that the solution improvement but im- 17.8%). However, the controls that were not treated with any cryoprotectant solution but ofmersed cryopreservation in LN did not of thissurvive species at all by (Table AFP could 2). This be throughresult suggest the transcriptionals that the improvement regulation immersed in LN did not survive at all (Table2). This result suggests that the improvement of thecryopreservation genes mentioned of this above. species by AFP could be through the transcriptional regulation of cryopreservation of this species by AFP could be through the transcriptional regulation of the genes mentioned above. of the genes mentioned above. Table 2. Effect of antifreeze protein III (AFP III) on the improvement of the cryopreservation effi- ciencyTable 2.of EffectHosta ofcapitata antifreeze. protein III (AFP III) on the improvement of the cryopreservation effi- ciency of Hosta capitata. Treatment Survival Rate (%) Shoot Regeneration Rate (%) TreatmentControl Survival0c Rate (%) Shoot Regeneration0c Rate (%) ControlPVS2 33.3b0c 17.8b0c PVS2+PVS2AFP 46.7a33.3b 28.9a17.8b PVS2+AFP 46.7a 28.9a

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Table 2. Effect of antifreeze protein III (AFP III) on the improvement of the cryopreservation efficiency of Hosta capitata.

Treatment Survival Rate (%) Shoot Regeneration Rate (%) Control 0c 0c PVS2 33.3b 17.8b PVS2+AFP 46.7a 28.9a Data represent the means of three replicates. Means marked with the same letter are not significantly different by the least significant difference test (LSDT, p < 0.05). Survival rate % = the number of meristems showing green tissues/total number of meristems × 100. Shoot regeneration rate % = the number of meristems inducing shoots/total number of meristems × 100.

4. Discussion H. capitata, a popular groundcover plant in the landscape industry, has been facing a steady disappearance in natural habitats due to the overcollection of its germplasm and global climate change, which does not favor its natural growth in habitats. These factors increase the risk of extinction and potentially harm its use as a groundcover plant in the landscape industry. Unfortunately, to maintain its germplasm for long periods, an effective storage method for this species is not yet available. The cryopreservation tech- nique has been widely used as a promising tool for the long-term preservation of genetic resources of various plants, including fruit trees, landscape ornamental plants, and cut flowers [11,24–27], due to reduced labor and space required for long-term storage. How- ever, intracellular ice formation that occurs during cryopreservation remains a challenge. According to the results of preliminary works (unpublished data), cryopreservation of this species was unsuccessful, although several attempts have been made. Many studies have recently reported the cryoprotective role of AFP in cryopreservation and low-temperature treatment studies of horticultural plants [1,11,12,28]. Moreover, Cutler et al. [29] earlier reported that AFP’s vacuum infiltration into potato leaves also caused them to show in- creased cold tolerance compared to those infiltrated with water (control). Therefore, it was hypothesized that the cryopreservation of this species would be successful using AFP III in the cryopreservation process. Hence, it is of interest to apply AFP to the cryopreservation of H. capitata. The mechanism underlying AFP’s role as a cryoprotectant, by transcriptional analysis of genes involved in the cold tolerance mechanism, was investigated. Before cryopreservation was started, to investigate whether the addition of AFP to the PVS2 solution would reduce freezing damage compared to PVS2 alone in the cryop- reservation process, shoots were treated with PVS2 and PVS2+AFP, whereas shoots treated ◦ with dH2O were used as negative controls. Shoots were placed at 4 C for 6, 24, and 48 h, respectively; untreated shoots placed at 25 ◦C were used as controls. Transcriptional analysis of CBF1, DHN1, Nia1, and Nia2 in shoots involved in the cold tolerance mechanism was then performed. Compared to 25 ◦C, upregulation of CBF1 and DHN1 at a low tem- perature was observed in this study, regardless of exposure times, as reported in previous studies [12,13,30–37]. Thus, it is suggested that CBF1 and DHN1 are low-temperature- inducible genes involved in the cold acclimation process of this Hosta species. Despite exposure to a low temperature for 6 h, higher gene expression was observed in dH2O compared to PVS2 and PVS2+AFP. It could be that shoots treated with dH2O were more responsive to cold than those treated with PVS2 and PVS2+AFP, which have cryoprotective activities. Therefore, the former triggered higher gene expression levels to adapt to the cold condition. Transcriptional control of genes by AFP in potato and the same species at a low temperature was recently reported [12,13]. When the exposure time was increased to 24 h, gene expression in dH2O and PVS2 was similar and higher than PVS2+AFP. It seemed that the presence of AFP in PVS2 is not required to trigger the genes as much as dH2O and PVS2 to adapt to the cold. However, dH2O treatment could not highly trigger the genes at 48 h after cold treatment, whereas PVS2 still triggered higher expression. This can be explained by the fact that the dH2O treatment was no longer able to resist the cold with increasing exposure times. However, the presence of AFP in the PVS2 solution was more likely to tolerate the cold than PVS2 alone because PVS2+AFP started to trigger the genes Horticulturae 2021, 7, 82 8 of 10

at 48 h, with higher expression compared to PVS2. Taken together, shoots treated with the AFP-containing PVS2 solution could reduce freezing injuries by enhancing the genes’ transcript levels. Previous studies have indicated increased NO production in plants in response to a low temperature [14,15]. In this study, the upregulation of Nia1 and Nia2 genes that indirectly favor NO production in shoots at a low temperature was noticed. Shoots treated with dH2O triggered higher gene expression than those treated with PVS2 and PVS2+AFP at 6 h after cold exposure. It was likely that the former was more sensitive to the cold than the latter, thus immediately producing a higher amount of NO via gene upregulation to be used as signaling molecules in the early cold acclimation process. The latter’s lower sensitivity to cold stress could be due to the cryoprotective effect of PVS2 and AFP. Thus, they did not immediately need to trigger a higher gene expression level for NO production. The authors of [17] also observed NO production in Arabidopsis within 1 to 4 h after cold exposure, whereas NO production was positively associated with expression levels of Nia1 and Nia2 genes, and plants with a knockout of these genes (nia1/nia2 mutants) could not sufficiently produce NO at a low temperature. Increased NO production was likely to continue in shoots treated with dH2O until 24 h after cold exposure because gene expression levels did not significantly decline compared to 6 h. In contrast to 6 h, PVS2 treatment triggered higher expression at 24 h, suggesting a need for high content NO to adapt to cold stress when the exposure time was extended. At 48 h after cold exposure, a higher decline of the expression levels in dH2O than in PVS2 treatments could be due to the no-longer-high productivity of NO by shoots treated with dH2O at long exposure, compared to PVS2, which has cryoprotective activity. The presence of AFP in PVS2 was likely to have more resistance to cold stress than PVS2 alone, as the expression levels induced by AFP were more significant than those induced by PVS2 alone despite long exposure to cold. Despite NO production by cold exposure for 24 or 48 h [16,19], this is the first report of AFP controlling NO production at a low temperature. In this study, the expression patterns of CBF1, DHN1, Nia1, and Nia2 in response to a low temperature were similar. Cantrel et al. [17] also observed that CBF1 expression was positively associated with those of Nia1/Nia2, whereas CBF1 was greatly reduced in nia1/nia2 mutant plants at a low temperature compared to wild-type plants. These results further confirm the evidence for AFP’s potential role in reducing chilling injuries in the cold acclimation process. When AFP was used as a cryoprotectant in the cryopreservation of this species, the presence of AFP in PVS2 gave higher cryopreservation efficiency results than PVS2 alone, suggesting its beneficial role as a cryoprotectant in the cryopreservation of H. capitata.

5. Conclusions The upregulation of CBF1, DHNI, Nia1, and Nia2 genes in H. capitata shoots in response to a low temperature was observed, demonstrating an important role in the cold stress tolerance mechanism. The gene expression patterns in shoots varied depending on the type of treatments and cold exposure times. Shoots treated with PVS2+AFP were likely insensitive to cold stress because they did not strongly trigger the genes at 6 and 24 h after cold exposure, whereas those treated with dH2O and PVS2 did so. Therefore, based on the gene expression patterns, the addition of AFP to PVS2 could be more effective in cold protection than PVS2 alone, as also confirmed by the higher cryopreservation efficiency of PVS2+AFP compared to PVS2 alone. This study suggests that AFP can be a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasm.

Author Contributions: Conceptualization, A.H.N.; methodology, P.P.W.P.; data curation, P.P.W.P.; writing—original draft preparation, P.P.W.P.; writing—review and editing, A.H.N.; project adminis- tration, K.I.P. and C.K.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a 2021 Yeungnam University Research Grant. Horticulturae 2021, 7, 82 9 of 10

Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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