The Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers Sprouting Through Modulating Lignin Biosynthesis and H2O2 Accumulation in Potato

International Journal of Molecular Sciences

Article The Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers Sprouting through Modulating Lignin Biosynthesis and H2O2 Accumulation in Potato

Mengsheng Deng †, Jie Peng †, Jie Zhang †, Shuang Ran, Chengcheng Cai, Liping Yu, Su Ni, Xueli Huang, Liqin Li * and Xiyao Wang *

College of Agronomy, Sichuan Agriculture University, Chengdu 611130, China; [email protected] (M.D.); [email protected] (J.P.); [email protected] (J.Z.); [email protected] (S.R.); [email protected] (C.C.); [email protected] (L.Y.); [email protected] (S.N.); [email protected] (X.H.) * Correspondence: [email protected] (L.L.); [email protected] (X.W.); Tel.: +86-28-8629-0867 (L.L. & X.W.) † These authors contributed equally to this work.

Abstract: Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants’ defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression Citation: Deng, M.; Peng, J.; Zhang, level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly J.; Ran, S.; Cai, C.; Yu, L.; Ni, S.; down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin Huang, X.; Li, L.; Wang, X. The content was documented, a finding which was consistent with the precursors’ level. Subsequently, Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase Sprouting through Modulating (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated

Lignin Biosynthesis and H2O2 in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Accumulation in Potato. Int. J. Mol. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation

Sci. 2021, 22, 2287. https://doi.org/ and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content 10.3390/ijms22052287 and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that

StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the Academic Editor: Richard R.-C. Wang sprouting of potato tubers.

Received: 13 January 2021 Keywords: potato tuber; Snakin-2; dormancy; sprouting; lignin; hydrogen peroxide Accepted: 11 February 2021 Published: 25 February 2021

Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Potato (Solanum tuberosum L.) is the third most important food and vegetable crop in iations. the world, with wide adaptability, high yield and rich nutrition [1]. In 2018, the world’s total potato output exceeded 368 million tons, which meets the annual demand for planting, eating and processing. However, the potato industry is facing a serious problem regarding the inappropriate transition from dormancy to sprouting, which leads to the decline of tuber quality and marketability, and seriously affects the production, consumption and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sales [1–3]. Tuber dormancy is established at the beginning of tuber formation and lasts for This article is an open access article a relatively fixed period, and the length of the dormancy period will not be changed even distributed under the terms and under a suitable condition for sprouting [4]. Tuber dormancy is controlled by genotype conditions of the Creative Commons and storage conditions pre- and post-harvest, but the specific mechanism of maintaining Attribution (CC BY) license (https:// and releasing tuber dormancy is still unclear [2,4,5]. Therefore, to reveal the mechanism creativecommons.org/licenses/by/ of tuber dormancy release is a very important issue in potato production, storage and 4.0/). processing.

Int. J. Mol. Sci. 2021, 22, 2287. https://doi.org/10.3390/ijms22052287 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2287 2 of 16

Tuber dormancy release is a complex process that is known to involve several physiological and biochemical changes, mainly including hormonal signaling, carbohydrate metabolism and oxidation-reduction [4,6–8]. It is worth noting that, unlike rice and wheat seeds, tubers had a high moisture content (approximately 80%) and developed sprouts after a few months under normal conditions. Generally, cold storage at 2–4 ◦C is ideal for storing the seed potatoes but not for the potatoes for eating and processing. Worldwide, potatoes are being stored at 8–12 ◦C, and if treated with chlorpropham, camphor and/or essential oil at the same time, they will last up to 9 months [5,6,9]. However, the cost of cold storage is high, and CIPC has also been in control with a maximum residue limit of 10 mg·kg−1. In general, hormonal regulation and carbohydrate metabolism are thought to be the important factors controlling the dormancy release process in potato [4]. Very few studies have focused on the potential role of the periderm’s structure and components during this important physiological process. The complex periderm structure and its variable composition play a critical role in potato dormancy. The outermost shell consists of several strata of dead cells and forms the first natural line regulating dehydration and respiration. Noteworthily, the complex polyphenolic (lignin-like) and polyaliphatic in the phellem cell wall affected the tuber cortex and ultimately resulted in the change of dehydration and respiration [10]. In transgenic tubers with down-regulated expression of FHT, the skin appeared tawny and cracked, which contributed to the respiration with a 14-fold increase [11]. Moreover, the alteration in FHT level affected the content of ferulic acid, fatty acid and primary alcohol, and especially the ratio of guaiacyl/syringyl in tuber wound healing [10]. Similarly, the knockdown of SlDCR resulted in the disrupted epidermal layer of tomato with changes of lignin, suberin, phenylpropanoids and fatty acids [12,13]. Many investigations indicated that polyphenolic exhibited important roles in regulating fruit skin cracking and ensuring superior quality and freshness [12,14]. Vulavala et al. revealed that lignin and suberin are involved in potato periderm formation and maturation, and that the phellogen may affect the incomplete skin-set and skin injuries [15]. Additionally, in Arabidopsis, the decrease of lignin in the testa inhibited seed germination [16,17]. In short, lignin may regulate tubers’ respiration, freshness and dormancy in potato. A previous study has found that StSN2, a member of the Snakin/GASA family, is positively correlated with tuber dormancy, and that StSN2 expression level decreased more slowly in the long-dormant cultivars, suggesting that StSN2 might maintain tuber dormancy [6]. Snakin/GASA members are widely known to be involved in seed dormancy, leaf morphology, flowering and biotic and abiotic stresses [18–25]. Nahirñak et al. found that silencing SN1 shortened plant height and altered the leaf shape in potato, which was accompanied by an alteration of cell wall composition and primary metabolism associated with cell division, whereas a non-significant difference in overexpressing lines was found, affecting reactive oxygen (ROS) and hormone balance including gibberellic acid (GA), brassinosteriod (BR) and salicylic acid (SA) [20,24]. It was reported that AtGASA6 served as an integrator of gibberellic acid and abscisic acid (ABA), and stimulated germination through EXPA1 by promoting cell elongation and increasing the hypocotyls’ length [22]. Similarly, AtGASA4 promoted GA-induced germination, but AtGASA5 showed oppo- site effects [19,26]. Therefore, Snakin/GASA exhibited flexible functions in growth and development. Potato dormancy is regulated by multiple genes, which are mainly related to hormone and sugar metabolism, while the effects of other aspects on tuber dormancy are more interesting and need to be further explored. The present study reported the function of plant antimicrobial peptide StSN2 in maintaining tuber dormancy. Transgenic tubers with a dormant period of a different length were obtained by changing the expression level of StSN2. We analyzed the glossiness and integrity of the tuber surface and the morphology and arrangement of periderm cells. A comprehensive analysis suggested that the significant physiological changes occurred in tubers at ~45 days, and at this stage, tubers were subjected to proteomic and metabolomic analysis. The results showed that silencing Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 16

Int. J. Mol. Sci. 2021, 22, 2287 3 of 16 significant physiological changes occurred in tubers at ~45 days, and at this stage, tubers were subjected to proteomic and metabolomic analysis. The results showed that silencing SN2 promoted the accumulation of lignin precursors and induced lignin-related protein expressiSN2 promotedon in the the tuber accumulation periderm. T ofhe lignin lignin precursors synthase, andcinnamyl induced alcohol lignin-related dehydrogenase protein expression in the tuber periderm. The lignin synthase, cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT1), peroxidase (Prx) and Cinnamoyl-CoA (CAD), caffeic acid O-methyltransferase (COMT1), peroxidase (Prx) and Cinnamoyl-CoA reductase (CCR) significantly increased in the silencing periderm at the transcriptional reductase (CCR) signiﬁcantly increased in the silencing periderm at the transcriptional level and enzyme activity, which finally led to the accumulation of lignin [27–31]. The level and enzyme activity, which ﬁnally led to the accumulation of lignin [27–31]. The crack crack and respiration resulted from much lignin and stimulated the release of tuber dor- and respiration resulted from much lignin and stimulated the release of tuber dormancy. mancy. This research will contribute to a better understanding of the effect of StSN2 on This research will contribute to a better understanding of the effect of StSN2 on lignin lignin synthesis and tuber dormancy. synthesis and tuber dormancy.

2.2. Results Results 2.1.2.1. StSN2 StSN2 Expression Expression Lev Levelel and and Phenotypic Phenotypic Characterization Characterization of of StSN2 StSN2 Transgenic Transgenic Lines Lines InIn our our previous previous studies studies,, transcriptome transcriptome and and proteome proteome analyses analyses indicated indicated that that the the level level ofof SN2 SN2 is isclosely closely related related to to the the maintenance maintenance of of tuber tuber dormancy. dormancy. Further, Further, qRT qRT-PCR-PCR analysis analysis confirmedconfirmed that that the the expression expression of of SN2 SN2 is is positively positively related related to to tuber tuber dormancy. dormancy. Therefore, Therefore, toto explore explore the the function function of of SN2 SN2 in in regulating regulating tuber tuber dormancy, dormancy, different different SN2SN2 constructsconstructs werewere developed developed to to change change the the expression expression of of SN2SN2 inin tubers, tubers, and and 23 23 RNAi RNAi lines lines and and 27 27 over over-- expressionexpression lines lines (OE) (OE) were were obtained obtained.. RNAi RNAi lines lines 7 7and and 8, 8, and and OE OE lines lines 11 11 and and 27 27 were were randomlyrandomly selected selected for for the the following following experiments experiments.. Firstly,Firstly, the the dormancy dormancy characteristic of differentdifferent transgenictransgenic potatoes potatoes were were analyzed analyzed by bycomparing comparing them them with with the the control control (wild-type (wild-type and and empty empty vector vector transformants transformants with with same sametraits). traits). No sprout No sprout was observedwas observed in any in tuberany tuber within within 30 d. 30 However, d. However there, there were significantwere sig- nificantdifferences differences in the dormancyin the dormancy period period of the threeof the groupsthree groups (OE, ~105 (OE, d, ~105 WT, d, ~90 WT, d, ~ RNAi,90 d, RNAi,~75 d). ~75 During d). During 30–75 30 d,– the75 d two, the over-expression two over-expression lines showed lines showed slow sprouting: slow sprouting the highest: the highesproutingst sprouting rate of rate overexpressing of overexpressing line 27 line was 27 only was 41.55% only 41.55% at 75 d,at while75 d, w 87.44%hile 87.44% of RNAi of RNAiline 7line have 7 have sprouted sprouted at the at samethe same time time (Figure (Figure1A). 1A). Photographs Photographs of the of sproutingthe sprouting in the in thepotatoes potatoes were were taken taken after after 15 15 and and 75 75 d (Figured (Figure1B). 1B 105). 105 days days later, later, the the sprouting sprouting rates rates of oftwo two over-expression over-expression tubers tubers with with a longa long dormancy dormancy period period were were still still the the lowest low comparedest com- paredwith RNAiwith RNAitubers tubers and wild-type, and wild-type and, were and were 79.70% 79.7 and0% 82.90%, and 82.9 respectively.0%, respectively Conversely,. Con- versely,the RNAi the lines RNAi and line wild-types and wild tubers-type sprouted tubers sprouted completely completely at 105 d (Figure at 1051 dA). (Figure The results 1A). Thesuggested results suggested that overexpressing that overexpressing SN2 could SN2 significantly could significantly inhibit tuber inhibit sprouting. tuber sprouting.

A 120 RNAi7 RNAi8 WT OE11

100 OE27 ）

% 80 （

40 Sprouting rate Sprouting 20

0 0 15 30 45 60 75 90 105 120 Storage time (d)

B 15 d 75 d

RNAi 7

OE 27

2 cm

FigureFigure 1. 1. SproutingSprouting rate rate and and phenotypes phenotypes of ofStSN2StSN2 transgenictransgenic tubers tubers and and control controlss during during differ differentent storage times. (A) Statistical results of sprouting rate. (B) Sprout phenotypes on the 15th and 75th storage times. (A) Statistical results of sprouting rate. (B) Sprout phenotypes on the 15th and 75th day of storage. Data are means ± SD of three biological replicates. Scale bar = 1 cm. day of storage. Data are means ± SD of three biological replicates. Scale bar = 1 cm.

Int. J. Mol. Sci. 2021, 22, 2287 4 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 16

TheThe SN2 SN2 expression expression level level was was detected detected in inan an apical apical sprout sprout by by using using qRT qRT-PCR.-PCR. In In overover-expressed-expressed tubers tubers,, the the level level was was significantly signiﬁcantly increased, increased, and and became became 3.63 3.63–4.71–4.71 times times higherhigher than than that that of ofthe the control control.. Moreover, Moreover, the the high high level level was was completely completely consistent consistent with with theirtheir long long dormancy dormancy period. period. For For example, example, ov overexpressingerexpressing tubers tubers with with high high StSN2StSN2 levellevel showedshowed that that only only 95.21% 95.21% of ofthe the tubes tubes sprouted sprouted as aslate late as as130 130 d. d.To To analyze analyze the the expression expression strengthstrength of ofSN2SN2 in inRNAi RNAi lines, lines, we we measured measured the the SN2SN2 expressionexpression level level of oflines lines 7 and 7 and 8 as 8 as 30.08%30.08% and and 20.79% 20.79%,, respectively, respectively, in in comparison comparison with with the the control control (Figure (Figure 2A).2A). In addition, a similar tendency was obtained at the protein level (Figure2B). a similar tendency was obtained at the protein level (Figure 2B).

A 6 a 5 b 4 3 2 elative expression elative c R 1 d d 0 RNAi7 RNAi8 WT OE11 OE27

B SN2

Proteins

FigureFigure 2. Comparison 2. Comparison of gene of gene and andprotein protein expression expression levels levels in sprouts in sprouts of wild of-type wild-type (WT) with (WT) with transgenictransgenic plants plants either either down down-regulating-regulating (RNAi) (RNAi) or oroverexpressing overexpressing (OE) (OE) the the StSN2StSN2 gene.gene. (A) ( AGene) Gene expression levels in transgenic lines. Data are means ± SD of three biological replicates. Different expression levels in transgenic lines. Data are means ± SD of three biological replicates. Different letters indicate significant differences at p < 0.05. (B) Protein expression levels measured by west- letters indicate signiﬁcant differences at p < 0.05. (B) Protein expression levels measured by western ern blot (WB). The wild-type (WT), potato cultivar Chuanyu 10, was used as the control. blot (WB). The wild-type (WT), potato cultivar Chuanyu 10, was used as the control.

2.2.2.2. Effect Effect of StSN2 of StSN2 on onCracking Cracking and and Cell Cell Morphology Morphology of ofPeriderm Periderm and and Water Water Loss Loss DuringDuring 120 120 d storage, d storage, the the weight weight of ofthe the tubers tubers in ineach each line line decreased decreased gradually gradually,, but but thethe reduction reduction rates rates were were significantly signiﬁcantly different different.. The The weight weight of ofthe the overexpress overexpressinging tubers tubers alwaysalways remained remained at atthe the highest highest level level comparing comparing with with other other groups groups,, amounting amounting to to52.4 52.4 g g andand 55.6 55.6 g at g at120 120 d, d,respectively. respectively. However, However, the the silencing silencing StSN2StSN2 tubers,tubers, in inRNAi RNAi line line 7 and 7 and RNAiRNAi line line 8, 8,the the suffered suffered a rapid a rapid weight weight loss loss at at0– 0–6060 d, d,and and the the loss loss rate rate slowed slowed down down afterafter sprouting sprouting (60 (60–120–120 d). d). In Inthe the end, end, the the weights weights were were only only 19.7 19.7 g and g and 23.3 23.3 g at g at120 120 d d respectivelyrespectively (Figure (Figure 3A).3A). ComparedCompared with with the the overexpression overexpression and and wild wild-type,-type, the the silencing silencing tubers tubers’’ skin skin was was mostlymostly cracked, cracked, rough rough and and brownish, brownish, and and was was photographed photographed at 30 at days 30 days (Figure (Figures 3B3,B,C).C). MicroscopyMicroscopy analyses analyses of ofthe the tuber tuber skin skin cells cells revealed revealed that that the the cell cellss of ofRNAi RNAi line line 7 were 7 were deformeddeformed and and wrapped wrapped in the in necrotic the necrotic cellular cellular debris debris,, especially especially at the junction at the junction with cells, with whichcells, was which possibl wasy possiblycaused by caused the excessive by the excessivewater loss water of the losstubers of the(Figure tubers 3D). (Figure Consis3t-D). ently,Consistently, at the 45th at day the of 45th storage, day ofa significant storage, a decrease signiﬁcant in decreasethe thick inof the phellem thick of thecell phellemlayers wascell observed layers was in the observed silencing in theline, silencing and the line,outer and 3–4 thecell outer layers 3–4 ha celld completely layers had collapse completelyd. Whereas,collapsed. like Whereas, the fresh like tubers, the freshthe phellem tubers, thecells phellem in overexpressing cells in overexpressing tubers were tubers more or- were derlymore and orderly turgid and due turgid to having due toenough having water enough (Figure water 3E). (Figure 3E).

Int. J. Mol. Sci. 2021, 22, 2287 5 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 16

120 A RNAi7 RNAi8 WT OE11 OE27 100

Tubers weight (g) weight Tubers 20

0 15 30 60 90 120 Storage time (d)

B RNAi 7 WT OE 27

C RNAi 7 WT OE 27

D RNAi 7 WT OE 27

E RNAi 7 WT

pl pl pg pg pd pd OE 27

pg pd Figure 3. Morphological alterations in transgenic tubers. (A) Tuber weight of transgenic lines and Figure 3. Morphological alterations in transgenic tubers. (A) Tuber weight of transgenic lines and wild-type (WT). (B) Tuber phenotype of RNAi line 7, OE line 27 and wild-type (WT) plants. Scale wild-type (WT). (B) Tuber phenotype of RNAi line 7, OE line 27 and wild-type (WT) plants. Scale bar = 5 mm. (C) Scanning electron microscopy (SEM) micrograph of the skin surface from a wild- bartype = 5 mmand.( transgenicC) Scanning tuber. electron Scale microscopy bar = 500 μ (SEM)m. (D) micrograph Micrograph of of the different skin surface tuber from skin as. wild-type Scale bar = and100 transgenic μm. (E) tuber.Microscopy Scale barof cross = 500 sectionsµm. (D) of Micrograph transgenic of tuber different and wild tuber-type skins. (WT). Scale pl, bar phellem; = 100 µ m.pg, (E)phellogen; Microscopy pd, of phelloderm. cross sections Scale of transgenic bar = 100 tuberμm. and wild-type (WT). pl, phellem; pg, phellogen; pd, phelloderm. Scale bar = 100 µm. 2.3. Skin Metabolome in StSN2 Transgenic Potato 2.3. Skin Metabolome in StSN2 Transgenic Potato Potato tubers as storage organs necessitate mechanisms to reduce water loss and reg- Potato tubers as storage organs necessitate mechanisms to reduce water loss and ulate transpiration and respiration, and secondary metabolites such as lignin form an im- regulate transpiration and respiration, and secondary metabolites such as lignin form an portant barrier for these physiological processes. Different skins from RNAi, wild-type important barrier for these physiological processes. Different skins from RNAi, wild-type and overexpressing tubers were subjected to LC-MS, and 371 differentially accumulated and overexpressing tubers were subjected to LC-MS, and 371 differentially accumulated metabolites (DAMs) were identified, including amino acids, organic acids, phenols and

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 16

Int. J. Mol. Sci. 2021, 22, 2287 6 of 16

other secondary metabolites (Table S2). Further analysis revealed that, among the DAMs, 30 lignin-related metabolites were screened and mainly classified into “Phenylpropanoid metabolitesbiosynthesis (DAMs)”, “Phenylalanine, were identified, tyrosine including and amino tryptophan acids, organic biosynthesis acids, phenols” and and“Phenylala- other secondary metabolites (Table S2). Further analysis revealed that, among the DAMs, 30nine lignin-related metabolism metabolites” by Kyoto were Encyclopedia screened and of mainly Genes classified and Genomes into “Phenylpropanoid (KEGG) analysis. Based biosynthesis”,on the change “Phenylalanine, intensity, a hierarchical tyrosine and tryptophanclustering analysis biosynthesis” divided and “Phenylalaninethe candidate metabo- metabolism”lites into four by Kyotogroups Encyclopedia (Figure 4A). of GenesThe contents and Genomes of nine (KEGG) DAMs analysis. (Class BasedI) were on significantly the changehigher intensity, in the silencing a hierarchical line than clustering in the analysis overexpressing divided the line candidate and WT, metabolites while the into contents of fourtwo groups DAMs (Figure (Class4A). IV) The were contents lowest of ninein the DAMs silencing (Class line. I) were To significantlyour surprise, higher the inmajority of theDAMs silencing (Class line II than and in Class the overexpressing III) shared the line same and WT, tendency while the in contents the transgenic of two DAMs lines (Figure (Class4A). IV)Nevertheless, were lowest the in the key silencing metabolites line. To for our p surprise,henylpropanoid the majority biosynthesis of DAMs (Class, such II as 4-hy- anddroxycinnamic Class III) shared acid, the ferulic same tendency acid and in the the monolignols transgenic lines coniferyl (Figure4 alcoholA). Nevertheless, and sinapyl alco- the key metabolites for phenylpropanoid biosynthesis, such as 4-hydroxycinnamic acid, hol, were the most abundant in the RNAi line, providing the sources of lignin synthesis ferulic acid and the monolignols coniferyl alcohol and sinapyl alcohol, were the most abundantand accumulation in the RNAi (F line,igure providing 4B–E). Furthermore, the sources of silencing lignin synthesis StSN2 andresulted accumulation in a 40.45% and a (Figure15.51%4B–E). increase Furthermore, in lignin silencing content StSN2in tworesulted transgenic in a 40.45%lines, respectively, and a 15.51% increasewhile in in the over- ligninexpressed content phellem in two transgenic the lignin lines, content respectively, was only while 24.27 in– the30.94% overexpressed of that in phellem the wild the-type (Fig- ligninure 4F). content was only 24.27–30.94% of that in the wild-type (Figure4F).

1.00 A 4-Coumaric acid B 4-Coumaric acid C Ferulic acid Ferulic Acid 2 2 a Coniferyl Alcohol 0.00 a Phenethylamine I Methyleugenol 1.5 1.5 trans-P-Coumaric acid -1.00 3-Dehydroshikimic acid b b 3,5-Dimethoxy-4-Hydroxycinnamic Acid 1 1 Benzoylformic Acid c c Psoralen 0.5 0.5 Caffeic Acid Shikimic acid Sinapyl Alcohol 0 0 Vanillin RNAi-SN2-#7RNAi 7 WT OE-SN2-#27 OE 27 RNAi-SN2-#7RNAi 7 WT WT OE-SN2-#27 OE 27 II Salicylic acid Coniferin Xanthotoxol p-Coumaric acid Coniferyl alcohol Sinapyl alcohol 2 2 Benzoic acid D E a Pyruvic Acid 4-Methyumbelliferone 1.5 a 1.5 Spermidine L-Phenylalanine b b b Fumaric Acid 1 1 III Phosphopyruvic acid L-Tyrosine c 3,4-Dihydro Coumarin 0.5 0.5 Quinic acid Hydrocinnamic Acid IV P-Coumaryl Alcohol 0 0 RNAi-SN2-#7RNAi 7 WT OE-SN2-#27 OE 27 RNAi-SN2-#7RNAi 7 WT OE-SN2-#27 OE 27

F 80

) a

1 - 60 b c

40 d d

20 Lignin g (mg Lignin content

0 RNAi 7 RNAi 8 WT OE 11 OE 27

FigureFigure 4. 4.Lignin-related Lignin-related metabolites metabolites of control of control and StSN2 and transgenicStSN2 transgenic tuber periderm. tuber periderm. (A) Heat maps (A) Heat ofmaps differentially of differentially accumulated accumulated metabolites metabolites (DAMs) related (DAMs) to lignin related synthesis. to lignin Relative synthesis contents. Relative of con- fourtents key of precursors, four key precursors 4-Coumaric, acid4-Coumaric (B), Ferulic acid acid (B (),C ),Ferulic Coniferyl acid alcohol (C), Coniferyl (D) and Sinapyl alcohol alcohol (D) and Sin- (Eapyl), for alcohol lignin synthesis (E), for lignin were identified synthesis and were quantified identified (LC-MS/MS). and quantified (F) Lignin (LC content-MS/MS in) the. (F RNAi) Lignin con- line,tent OE in linethe andRNAi wild-type line, OE (WT) line periderm. and wild The-type data (WT) are meansperiderm.± SD The of four data biological are means experiments ± SD of four bio- withlogical triplicate experiments measurements with triplicate in each experiment. measurements Different in letterseach experiment. indicate a significant Different difference letters indicate at a psignificant< 0.05 among difference wild-type, at overexpression p < 0.05 among and wild RNAi-type, potato overexpression plants. and RNAi potato plants.

2.4.2.4. Proteomics Proteomics Analysis Analysis of Lignin of Lignin Biosynthesis Biosynthesis ToTo better better understand understand the molecularthe molecular mechanisms mechanisms of StSN2 of StSN2 regulating regulat lignining synthesis, lignin synthesis, a protein expression proﬁle was obtained from transgenic periderm by LC-MS/MS analysis a protein expression profile was obtained from transgenic periderm by LC-MS/MS analy- at the 45th day of storage (Table S3). 382 differentially expressed proteins (DEPs) were th identiﬁedsis at the based 45 day on the of thresholdsstorage (Table (fold changeS3). 382 >1.2 differentially or <0.83). The expressed KEGG pathway proteins annota- (DEPs) were tionidentified analysis based of DEPs on from the eachthresholds comparison (fold showed change that >1.2 they or < were0.83). enriched The KEGG in a number pathway anno- oftation metabolic analysis pathways, of DEPs including from each carbohydrate comparison metabolism, showed aminothat they acid were metabolism enriched and in a num- otherber of secondary metabolic metabolism pathways, (Table including S3). Subsequently, carbohydrate we metabolism, focused on theamino pathway acid en-metabolism and other secondary metabolism (Table S3). Subsequently, we focused on the pathway enrichment analysis of the DEPs in the three comparison groups (RNAi7 vs. WT, RNAi7 vs. OE27 and OE27 vs. WT), and the results indicated that 15 DEPs between RNAi line 7

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 16

and WT were enriched in “Phenylpropanoid biosynthesis”, “Phenylalanine, tyrosine and tryptophan biosynthesis” and “Phenylalanine metabolism”, and 11 DEPs from RNAi line 7 vs. OE line 27 and 4 DEPs from OE line 27 vs. WT were enriched in “Phenylpropanoid biosynthesis” (Figure 5A–C). Moreover, the critical pathway for lignin synthesis “Phe- nylpropanoid biosynthesis”, including peroxidases, CAD and COMT, was up-regulated in RNAi line 7 by comparison with overexpressed and wild-type tubers (Table S3). It is worth noting that a total of nine peroxidases were identified, and the levels of five proteins (Prx1, Prx2, Prx4, Prx5, Prx6) were positively regulated by StSN2, while those peroxidases, from seven to ten, were highly expressing in silencing periderm. The rest of the proteins Int. J. Mol.altered Sci. 2021, 22irregularly, 2287 (Table S4). 7 of 16 To determine whether the StSN2-altered expression affected the transcription and enzyme activities of lignin biosynthesis key genes, the following steps were taken. Firstly, the qRT-PCR analysisrichment indicated analysis that of the the DEPs expression in the three level comparison of COMT groups and (RNAi7 CAD vs. was WT, in- RNAi7 vs. OE27 and OE27 vs. WT), and the results indicated that 15 DEPs between RNAi line 7 creased by ~5 to 10and-fold WT in were the enrichedperiderm in “Phenylpropanoidof silencing lines biosynthesis”, compared with “Phenylalanine, that of the tyrosine wild- and type, but there wastryptophan a slight biosynthesis”change in overexpressing and “Phenylalanine lines. metabolism”, Strikingly and, 11the DEPs expression from RNAi of line 7 Prx10 was significantlyvs. OE different line 27 and among 4 DEPs fromall lines, OE line and 27 vs.the WT highest were enriched one was in “PhenylpropanoidRNAi line 7 (Figure 5D). Finally,biosynthesis” we focused (Figure on 5theA–C). changes Moreover, in theenzyme critical activities pathway for and lignin found synthesis that “Phenyl- the propanoid biosynthesis”, including peroxidases, CAD and COMT, was up-regulated in enzyme activities of peroxidases, COMT and CAD in silencing tubers were significantly RNAi line 7 by comparison with overexpressed and wild-type tubers (Table S3). It is worth higher than that ofnoting the wild that- atype, total ofbut nine decreased peroxidases in were overexpression identiﬁed, and tub the levelsers except of ﬁve for proteins CAD (Prx1, (Figure 5E). SimilarlyPrx2,, we Prx4, found Prx5, Prx6)the inhibitory were positively effect regulated of StSN2 by StSN2, on another while those lignin peroxidases, biosyn- from thesis enzyme CCR.seven In summary, to ten, were highlythese expressingresults suggested in silencing that periderm. StSN2 The inhibited rest of the lignin proteins bio- altered synthesis by mainlyirregularly regulating (Table the S4). expression and activities of several key enzymes.

A RNAi 7 vs WT B RNAi 7 vs OE 27 C OE 27 vs WT

a D 15 15 E 9,000 a 90 a

COMT CAD ) a -1

a ) a -1 10 10 b 6,000 60 b b c activity (U g (U activity c b b

5 5 3,000 30 b

elative expression elative R elative expression elative c c c

c c c COMT

CAD activity (U g (U activity CAD R 0 0 0 0

WT WT WT WT OE 11 OE 27 OE 11 OE 27 OE 11 OE 27 OE 11 OE 27 RNAi 7 RNAi 8 RNAi 7 RNAi 8 RNAi 7 RNAi 8 RNAi 7 RNAi 8

OE-SN2-#11OE-SN2-#27 OE-SN2-#11OE-SN2-#27 Anti-SN2-#7Anti-SN2-#8 OE-SN2-#11OE-SN2-#27 OE-SN2-#11OE-SN2-#27 RNAi-SN2-#7RNAi-SN2-#8 RNAi-SN2-#7RNAi-SN2-#8 RNAi-SN2-#7RNAi-SN2-#8 4.5 450 Prx10 30 a CCR 3,900

) )

a -1

a -1 b b a 3.0 20 2,600 300 b c c b c d d 1.5 c 10 1,300 150

d g (U activity

lative expression lative

rx elative expression elative

e P

R c CCR activity (U g (U activity CCR Re c c 0.0 0 0 0

WT WT WT WT OE 11 OE 27 OE 11 OE 27 OE 11 OE 27 OE 11 OE 27 RNAi 7 RNAi 8 RNAi 7 RNAi 8 RNAi 7 RNAi 8 RNAi 7 RNAi 8

OE-SN2-#11OE-SN2-#27 OE-SN2-#11OE-SN2-#27 OE-SN2-#11OE-SN2-#27 Anti-SN2-#7Anti-SN2-#8 OE-SN2-#11OE-SN2-#27 RNAi-SN2-#7RNAi-SN2-#8 RNAi-SN2-#7RNAi-SN2-#8 RNAi-SN2-#7RNAi-SN2-#8 FigureFigure 5. KEGG 5. KEGG enrichment enrichment and analysis and of analysis lignin biosynthesis of lignin genes biosynthesis expression g andene enzymes expression activities. and KEGG enzyme enrichment ac- analysestivities. of potato KEGG skins enrichment in three comparisons, analyses of RNAi potato 7 vs. skins WT (A in), RNAi7three comparisons, vs. OE27 (B) and RNAi OE27 vs.7 vs WT. WT (C), ( atA the), 45th dayRNAi7 of storage. vs.The OE27 red ( rectangleB) and OE27 highlights vs. theWT lignin (C), metabolismat the 45th pathway. day of Genestorage. expression The red (D) rectangle and enzyme highlights activities (E ) of COMT,the lignin CAD, metabolism Prx and CCR pathway. were investigated Gene byexpression qPCR assay (D and) and enzyme enzyme activities activities assay kits, (E) respectively,of COMT, atCAD, the 45th day.Prx The and data CCR are means were± investigatedSD of four biological by qPCR experiments assay and with enzyme triplicate activities measurements assay in eachkits, experiment.respectively Different, at p lettersthe indicate 45th day. signiﬁcant The data differences are means at < ± 0.05. SD of four biological experiments with triplicate measurements in each experiment. DifferentTo determine letters indicate whether significant the StSN2-altered differences expression at p < 0.05 affected. the transcription and enzyme activities of lignin biosynthesis key genes, the following steps were taken. Firstly, the qRT-PCR analysis indicated that the expression level of COMT and CAD was increased by ~5 to 10-fold in the periderm of silencing lines compared with that of the wild-type, but there was a slight change in overexpressing lines. Strikingly, the expression of Prx10 was

Int. J. Mol. Sci. 2021, 22, 2287 8 of 16

significantly different among all lines, and the highest one was RNAi line 7 (Figure5D ). Finally, we focused on the changes in enzyme activities and found that the enzyme activities of peroxidases, COMT and CAD in silencing tubers were significantly higher than that Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 16 of the wild-type, but decreased in overexpression tubers except for CAD (Figure5E). Similarly, we found the inhibitory effect of StSN2 on another lignin biosynthesis enzyme CCR. In summary, these results suggested that StSN2 inhibited lignin biosynthesis by mainly2.5. StSN2 regulating Interact thes expressionwith Three and Class activities III Peroxidases of several key enzymes. 2.5. StSN2Preliminary Interacts with studies Three indicated Class III Peroxidases that GASAs were involved in regulating plant growth andPreliminary development studies mainly indicated through that GASAs interacti wereng involved with protein in regulatings. For plant example, growth StSN1 was andfound development to interact mainly with through DIM/DWF1 interacting and withSUT1 proteins. [24,32] For. Therefore, example, StSN1co-immunoprecipitation was found to(Co interact-IP) and with mass DIM/DWF1 spectrum and SUT1(MS) [w24ere,32]. applied Therefore, to co-immunoprecipitationexplore the proteins interacting (Co-IP) with andStSN2 mass and spectrum regulating (MS) werelignin applied synthesis to explore in RNAi the line proteins 7 and interacting OE line with27, and StSN2 the results andshowed regulating that three lignin class synthesis III peroxidases in RNAi line, including 7 and OE Prx2, line 27, Prx and 9 and the Prx results 10, showedcan interact with that three class III peroxidases, including Prx2, Prx 9 and Prx 10, can interact with StSN2 StSN2 in vitro, which catalyzed the oxidation and polymerization of lignin (Figure S2, in vitro, which catalyzed the oxidation and polymerization of lignin (Figure S2, Table S2). Furthermore,Table S2). Furthermore, a yeast two-hybrid a yeast assay two confirmed-hybrid assay that StSN2 confirmed interacted that with StS Prx2,N2 interacted Prx 9 with andPrx2, Prx Prx 10 (Figure 9 and6 ).Prx Interestingly, 10 (Figure the 6). same Interestingly, expression the tendency same ofexpression these three tendency proteins of these wasthree detected proteins in proteome was detected and CoIP-MS in proteome data, and and the CoIP high- levelMS data, of these and peroxidases the high maylevel of these contributeperoxidase to lignins may synthesis contribute (Tables to lignin S3 and S4).synthesis The results (Table indicateds S3 and that S4) StSN2. The physically results indicated interactedthat StSN2 with physically peroxidases interact to regulateed with lignin peroxidases synthesis. to regulate lignin synthesis.

SN2+pGAD SN2+Prx2 SN2+Prx9 SN2+Prx10 TOPP4-PIN1

SD/-Trp-Leu

SD/-Trp-Leu-His-Ade

SD/-Trp-Leu-His-Ade +X-gal

FigureFigure 6. 6.StSN2 StSN2 physically physically interacts interacts with with peroxidases peroxidases in a in yeast a yeast two-hybrid two-hybrid system. system. The threeThe three pe- peroxidasesroxidases fused toto pGADT7pGADT7 are are Prx2 Prx2 (M1AU65), (M1AU65), Prx9 Prx9 (M1A251) (M1A251) and and Prx10 Prx10 (M1CCJ9). (M1CCJ9). TOPP4, TOPP4, type-onetype-one protein protein phosphatase phosphatase 4 in Arabidopsis; 4 in Arabidopsis; PIN1, pin-formed1 PIN1, pin-formed1 in Arabidopsis. in Arabidopsis. X-gal, 5-Bromo-4- X-gal, 5- chloro-3-indolyl-b-D-galactopyranosideBromo-4-chloro-3-indolyl-b-D-galactopyranoside acid. The experiments acid. The were experiments repeated three were times. repeated three times. 2.6. StSN2 Altered H2O2 Content and the Activities of Superoxide Dismutase (SOD) and Catalase (CAT) 2.6. StSN2 Altered H2O2 Content and the Activities of Superoxide Dismutase (SOD) and Cata- laseHydrogen (CAT) peroxide is an important condition for lignin synthesis and promotes potato tuber sprouting [33–35]. The level of H2O2 in the bud was analyzed, and the results Hydrogen peroxide is an important condition for lignin synthesis and promotes po- showed that StSN2 signiﬁcantly inhibited the accumulation of H2O2, except for OE line 11 (Figuretato tuber7A ). Moreover,sprouting this [33 difference–35]. The waslevel also of supportedH2O2 in the by bud the results was analyzed, of SOD and and CAT the results activityshowed analysis, that StSN2 and especially significantly the decreaseinhibited of the CAT accumulation activity in RNAi of H line2O2 7,, except which for may OE line 11 play(Figure an important 7A). Moreover, role in maintaining this difference a high was level also of Hsupported2O2 in the budby the (Figure results7B,C). of SOD and CAT activity analysis, and especially the decrease of CAT activity in RNAi line 7, which may play an important role in maintaining a high level of H2O2 in the bud (Figure 7B,C).

Int. J. Mol. Sci. 2021, 22, 2287 9 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 16

A 0.8 B 40 a

) 1

- a ）

a 1 b 0.6 - 30

mol·g c μ b

( ( b 2 0.4

O 20 2 c d 0.2 10

atalase activity(U·g atalase

C ontent of H of ontent

C 0 0

C 200 a b 150

） c c 1 - d

U·g 100 （

activity Superoxide dismutase Superoxide 0

Figure 7. Effect of StSN2 on H2O2 content (A), superoxide dismutase (SOD) activity (B) and cata- Figure 7. Effect of StSN2 on H2O2 content (A), superoxide dismutase (SOD) activity (B) and catalase (CAT) activity (C) in the three periderms.lase (CAT Data) activity are means (C) in± theSD ofthree three periderm biologicals. Data replicates. are means Different ± SD of lowercase three biological letters indicatereplicates. a significant Different lowercase letters indicate a significant difference at p < 0.05 by Duncan’s test analysis. difference at p < 0.05 by Duncan’s test analysis. 3. Discussion 3. Discussion In the potato life cycle, tuber dormancy and its release are the key physiological pro- In the potato life cycle, tuber dormancy and its release are the key physiological cesses and are under genetic and environmental control [8]. However, unlike seeds such processes and are under genetic and environmental control [8]. However, unlike seeds such as rice and corn, potato with high water content sprout easily under right conditions [1]. as rice and corn, potato with high water content sprout easily under right conditions [1]. Perhaps the storage of potato is more similar to that of fresh fruits with buds, so we need Perhaps the storage of potato is more similar to that of fresh fruits with buds, so we need to to consider both controlling sprouting and keeping them fresh. Previously, we screened consider both controlling sprouting and keeping them fresh. Previously, we screened the the SN2 gene, which was positively related to dormancy through proteome and transcrip- SN2 gene, which was positively related to dormancy through proteome and transcriptome tome analysis, and a qRT-PCR analysis also showed that SN2 was highly expressed in analysis, and a qRT-PCR analysis also showed that SN2 was highly expressed in deep deep dormandormancycy tubers tubers[6]. Previous [6]. Previous reports reports indicated indicated that Snakin/GASA that Snakin/GASA played played an im- an important portant role rolein regulating in regulating seed seeddormancy dormancy and germination, and germination, and were and wereinvolved involved in cell indi- cell division, vision, hormonehormone balance, balance, ROS ROS and andother other processes processes [20,22,24,26,3 [20,22,24,266–,3639–].39 It]. was It was also also found, found, in potato, in potato, thatthat StSN1 StSN1 played played a arole role in in cell cell elongation elongation and and hormonal hormonal signaling, signaling, although although there is no there is no directdirect evidence to supportsupport SN2SN2 maintaining maintaining dormancy dormancy [20 [20,,2424].]. Surprisingly, Surprisingly, the expression the expressionlevel level of SN2of SN2in tubersin tubers was was found found to to cause cause a significanta significant difference difference in in the the length of the length of thedormancy dormancy period period of of tubers tubers (Figure (Figure1 and1 and Figure Figure S1). S1) In. In addition, addition, qRT-PCR qRT-PCR and western and westernblot blot analysis analysis suggested suggested that that SN2 SN2 is is mainly mainly expressed expressed in in tubers tubers rather rather than than in in the stem and the stem andthe the leaf leaf (Figure (Figure S2), S2) and, and SN2 SN2 also also played played an an important important role role in in the the process process of of tuber wound tuber woundhealing healing (Figure (Figure4A 4A and and Figure Figure S2). S2) The. The results results showed showed that SN2that hadSN2important had im- biological portant biologicalfunctions functions in regulating in regulating dormancy dorman in tubers.cy in tubers. Most Snakin/GASAMost Snakin/GASA genes have been genes reported have been to be reported induced to by be hormones. induced by In hormones. Ara- In Ara- bidopsis, GASA4bidopsis, and GASA4GASA6 andwereGASA6 inducedwere in general induced by in GA general and BR by GAand andrepressed BR and by repressed by ABA, and GASA6ABA, andparticipated GASA6 participatedin the antagonistic in the antagonisticregulation of regulation seed germination of seed germination by GA by GA and ABA, andand stimulated ABA, and EXP1 stimulated expressing EXP1 in expressing cell walls in[22,23]. cell walls Sun [et22 al,23. ].proposed Sun et al. that proposed that GASA14 wasGASA14 essentialwas for essentialGA-induced for GA-induced germination germinationand inhibited and by inhibitedGAI and byRGL GAI [21]. and RGL [21]. However, otherHowever, members other of membersSnakin/GASA of Snakin/GASA exhibited different exhibited modes different and functions. modes and The functions. The previous studyprevious suggested study that suggested StSN2 thatis inducedStSN2 is by induced ABA but by inhibited ABA but inhibitedby GA [4 by0]. GASimi- [40]. Similarly, larly, in potatoin potato,, the same the tendency same tendency was found was found in the inSN2 the expressionSN2 expression level (Figure level (Figure S2D), S2D), and it and it demonstratdemonstrateded that SN2 that SN2was waslocally locally up-regulated up-regulated in ABA in ABA-induced-induced wounding wounding sig- signaling [41]. naling [41]. Further, in potato dormancy, dormancy, the the level level of of SN2SN2 waswas significantly significantly induced induced by by ABA, but ABA, but repressed by GA and BR, indicating that SN2 may also be involved in the maintenance of tuber dormancy mediated by hormones (Figure S2).

Int. J. Mol. Sci. 2021, 22, 2287 10 of 16

repressed by GA and BR, indicating that SN2 may also be involved in the maintenance of tuber dormancy mediated by hormones (Figure S2). On the other hand, Snakin/GASA participated in the cross-talk of hormones including ABA, GA, BR and SA. OsGSR1 was found to be involved in the crosstalk of GA and BR and to interact with DIM/DWF1 in rice [42]. And in potato, SN1 affected hormone balance by interacting with DIM/DWF1 and decreasing the SA and GA content [24]. Moreover, under the treatment with GA and BR, the expressions of SN2 were all down-regulated, indicating that SN2 may negatively participate in the synergism of GA and BR in sprouting (Figure S2). However, the interaction between Snakin/GASA and hormonal proteins was found in BR signaling rather than in GA and ABA signaling. Subsequently, the yeast two- hybrid assay was applied and demonstrated that SN2 did not interact with DIM/DWF1. Conversely, we identified the physical interaction between SN2 and ABA signaling factor SnRK2.4 by CoIP-MS analysis (Figure S2, Table S5), which was combined with the finding that overexpressing SN2 enhanced the level of SnRK2.4 and the same variation in ABI3 and ABI5 (Table S4, Figure S2). The results showed that SN2 played a positive role in the maintenance of tuber dormancy by ABA. The Snakin/GASA family, including GIP2, GASA5 and GASA14, has a conservative domain with 12 cysteine amino acids, which are involved in redox homeostasis [40]. In potato, Nahirñak et al. detected that SN1-silenced inhibited ROS accumulation in leaves and reduced the content of ascorbic acid [24]. Similarly, SN2 included highly conserved amino acid sequences (CX3CX3CX8CX3CX2CCX2CXC X11CXCX12CX) (Figure S1) and down-regulated the level of H2O2 with the change of Prx, CAT and SOD activities in the bud (Figure7). It is well known that exogenous H 2O2 application induces the breaking of dormancy, which has been identified both in plant seeds and in vegetative buds [34,35]. Liu et al. reported that potato dormancy break needs rely on ROS, and the NADPH oxidase inhibitor (DPI) significantly affected the sprouting rate and down-regulated the level of ROS [4].Consistently, the qPCR assay revealed that RobhA and RobhB, the key genes that catalyzed the production of ROS, were up-regulated in the silencing bud. As mentioned above, SN2 probably has an important role in potato dormancy and its release. In our study, a SN2-regulated high level of H2O2 stimulated the total peroxidases activities (EC 1.11.1.7), but, in fact, not all of the eight differential peroxidases increased in silencing buds (Figure7). In addition, peroxidases can not only catalyze the production of ROS, but also of scavenging reactive oxygen species, so this contradiction mechanism is still unclear, and peroxidases have functional redundancy in regulating plant physiology. However, the important point we focus on is that peroxidases catalyzed the oxidative polymerization of lignin, and H2O2 as oxidant is essential for it. The periderm is the natural barrier for potato development, and its composition and structure are very important for transpiration and respiration during potato dormancy [43]. A set of transgenic tubers with rough skin was developed and exhibited decreased levels of SN2 expression (Figures1 and2). Preliminary results demonstrated a clear effect of the manipulation of SN2 expression on the skin and periderm. It has been confirmed that Snakin/GASA is involved in cell division, elongation and cell wall formation. In potato, silencing StSN1 inhibited cell division and growth in leaves by changing the cell wall composition [24]. On the contrary, overexpressing AtGASA6 enhanced the expression of expansins, promoted hypocotyls cell elongation and stimulated seed germination [22]. In general, cell wall-related Snakin/GASA are located on the cell wall, such as AtGASA10 and FaGAST1, while SN1 is located on the plasma membranes rather than the cell wall [20,44,45]. Although we have not analyzed the location of StSN2, we have found that the effect of StSN2 on periderm cells is manifested in the cell’s arrangement and shape, which seemed to have a weak effect on cell division and elongation, but we still observed the existence of cells with different sizes (Figure3). In previous studies, the skin of fruits became rough and cracked, which was related to aromatic compounds, phenylpropanoids, lignin and fatty acids. As in the case of RNAi- DCR tomato and “Rugiada” apple fruit, the surface of RNAi-StSN2 potatoes appeared Int. J. Mol. Sci. 2021, 22, 2287 11 of 16

brown, rough and cracked [12]. We subsequently performed a characterization of the cracked RNAi-StSN2 potato (Figure3). The results showed a significant increment in the majority of the quantified lignin monomers, notably a massive increase in the contents of coniferyl alcohol and sinapyl alcohol (Figure4). In follow-up experiments, the single most striking observation to emerge from the data comparison was that the expression and activity of key enzymes of lignin synthesis, CAD, Prx and COMT, increased with the down-regulation of StSN2 expression level, and the effect of StSN2 silencing on them was more significant than that of the overexpressing lines (Figure5). Similarly, the alteration of StSN2 level resulted in significant changes in the content of lignin and its precursors, and association analysis revealed that two highly correlated groups were “COMT-caffeic acid, ferulic acid” and “Prx-Coniferyl Alcohol, Sinapyl Alcohol”, respectively (Tables S2 and S3). Furthermore, in Arabidopsis, Liang et al. mutated laccase 15 to change the content of lignin in coat, and found that the low level of lignin contributed to seed germination [16]. It was also found that the germination rate of prx2prx25 mutant seeds is higher than that of wild-type seeds in Arabidopsis, and mainly because peroxidase affects the polymerization and the level of lignin [17]. Meanwhile, in Citrus sinensis, the overexpression of CsPrx25 enhanced H2O2 levels and cell wall lignification [46]. In SN2-RNAi potato, the high lignin level resulted in periderm collapse and was more conducive to tuber sprouting, and CoIP- MS and yeast two-hybrid analysis showed a strong interaction between peroxidases and StSN2, indicating that StSN2 regulated lignin synthesis by interacting with peroxidases (Figure6, Table S5). Therefore, one important aspect of lignin mediated by StSN2 is that it plays a vital role in potato cracking and preservation. In conclusion, we identified a pivotal gene, StSN2, related to dormancy, which maintains tuber dormancy mainly in two patterns, as follows. Firstly, StSN2 inhibited the accumulation of hydrogen peroxide in bud tissue and negatively regulated its effect of stim- ulating tuber sprouting. Secondly, on the basis of inhibiting the accumulation of hydrogen peroxide, StSN2 further affected the biosynthesis of lignin and tuber skin, and regulated the water loss and respiration, finally achieved in the control of sprouting and preservation. The empirical findings in this study provide a novel insight into the mechanism whereby StSN2 maintains dormancy.

4. Materials and Methods 4.1. Plant Material and Growth Conditions The aseptic plantlets were propagated in 60 mm glass bottles containing Murashige and Skoog with 15 g/L sucrose and 7 g/L agar at 20 ± 1 ◦C, 16 h light, 8 h dark, light intensity 100 µmol m−2 s−1. Transgenic plantlets were selected for the resistance to 50 mg/mL kanamycin. Two weeks later, the robust tissue culture plantlets, with a height of ~5 cm, were transplanted into 18 cm pots containing peat soil, under the condition of natural light and 16 h light (21 ◦C) and 8 h dark (18 ◦C). The leaves were sprayed with Hogland nutrient solution at 3 and 6 weeks. When seedlings turned yellow, at about 10 weeks, the harvested tubers with the same maturity were kept in darkness at 13–15 ◦C for 10-day wound healing, then used for morphological observation and subsequent index determination.

4.2. Generation of StSN2 Transgenic Potato Lines The dormancy gene SN2 (Soltu.DM.01G050660.1), a member of the Snakin/GASA family, was screened from transcriptome and proteome data [6]. To generate StSN2 con- structs for overexpressing transgenic potato lines, primers were designed to amplify a 315 bp ORF (open read frame) from tuber cDNA prepared from potato cultivar Chuanyu 10. XbaI and SmaI sites were engineered at the start and the termini, respectively. The recombinant vector pBI121-SN2 was constructed under T4 ligase, then digested with restriction enzymes as described above to check the accuracy. Similarly, a 315 bp StSN2-speciﬁc fragment was ampliﬁed by PCR and used in a restriction-ligation reaction for insertion into the binary vector pBI121. Details of the primer sequences used for cloning are provided in Supplementary Table S1. The recombinant expression vectors were transformed into Int. J. Mol. Sci. 2021, 22, 2287 12 of 16

Agrobacterium strain GV3101 by the frozen-thawed method. The stem segments of the 7-day in vitro culture were infected and transformed as described previously [47]. Finally, the aseptic plantlets with different levels of StSN2 were obtained.

4.3. Western Blot and Quantitative Real-Time PCR Before Western blot, the antibodies from rabbit serum were purified using the respec- tive oligopeptides as the affinity column tag [8]. Western blot was performed to detect protein levels in potato bud eyes. Approximately 10 µg protein was loaded per lane, then 5% non-fat milk was used to block non-specific protein binding, and the nitrocellulose membrane was incubated with StSN2. For the quantitative real-time PCR (qPCR) analysis, the total tuber RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. qPCR was then performed on a 7500 Real Time PCR System (Life Technologies) according to the manufacturer’s instructions. The 2−∆∆Ct method was used for relative quantification. Elongation factor 1 alpha-like (EF1αL) expression was used as an internal control. Three biological replicates and three technical replicates were performed for all experiments.

4.4. Assessment of Sprout Growth, Periderm Morphology and Tuber Weight Tubers from multiple transgenic lines were assessed for tuber sprout growth. Once harvested, the healthy tubers were placed under scattered light at room temperature for wound healing and then transferred to a dark and relatively closed carton and stored at 15 ± 2 ◦C and a relative humidity of 65% ± 5%. The sprouting rate and weight of each transgenic line were measured every 15 d, and photos were taken. The changes in the tuber periderm morphology (such as color and glossiness) were observed periodically under the prompt microscope, and the thickness, cell arrangement and cell size of different transgenic tubers were monitored by light microscopy. The tubers ﬁxed above 24 h were dehydrated with alcohol with different concentrations. After being embedded, periderms about 4 µm thick were observed under the light microscope. For the scanning electron microscopy (SEM), small fragments of tuber periderm were ﬁxed with 1% osmic acid in PBS (pH 7.4) at room temperature for 1–2 h. Fragments were dehydrated with an increasing ethanol concentration series, exchanged through isoamyl acetate and critical point-dried. The fragments were attached to the conductive carbon double-sided adhesive and coated with gold for 30 s. Specimens were observed using the Hitachi SU8100 SEM.

4.5. Targeted Metabolomic Analysis on Metabolites in Potato Skin Tissues (100 mg) were resuspended with 80% methanol and 0.1% formic acid by well vortexing. Some of the supernatant was diluted to a final concentration containing 53 % methanol by LC-MS grade water. After centrifuged, the supernatant was injected into the LC-MS/MS system analysis. LC-MS/MS analyses were performed using an ExionLC™AD system (SCIEX, Shanghai, China) coupled with a QTRAP®6500+ mass spectrometer (SCIEX, Shanghai, China). The samples were injected onto aHSS T3 Column (100 mm × 2.1 mm) using a 25-min linear gradient at a flow rate of 0.35 mL/min for the negative polarity mode. Next, metabolites were identified and quantified using MRM (Multiple Reaction Monitoring).

4.6. Identification and Quantification of Proteins The total proteins were extracted from WT, RNAi line 7 and OE line 27. The concentration of the total protein was determined by a Bradford protein quantitative kit, and the quality of the proteins was assessed by SDS-PAGE. Then, 3 µL 1 µg/µL trypsin (Promega, Madison, WI, USA) and a 500 µL 50 mM TEAB buffer (Triethyl ammonium bicarbonate) were used to digest the proteins of each sample overnight at 37 ◦C, and the products purified by the C18 desalination column were labeled with TMT. Next, we used an L-3000 HPLC system to gradient elute the solution containing protein powder, and 10 fractions were obtained. The chromatographic column was Waters BEH C18 (4.6 × 250 mm, 5 µm), Int. J. Mol. Sci. 2021, 22, 2287 13 of 16

and the column temperature was set at 50 ◦C. For transition library construction, shot- gun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with an Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. A 1-µg sample was injected into a home-made C18 Nano-Trap column (2 cm × 75 µm, 3 µm). Peptides were separated in a home-made analytical column (15 cm × 150 µm, 1.9 µm), using a linear gradient elution. The separated peptides were analyzed by a Q Exactive HF-X mass spectrometer (Thermo Fisher). The resulting MS/MS data were processed using Proteome Discoverer 2.2 (PD 2.2, Thermo).

4.7. CoIP-MS Assays An anti-StSN2 antibody was prepared by polypeptides (SIQTDQVTSNAISEA) in a rabbit, and used to co-immunoprecipitate SN2-interacting proteins [48]. For in-gel tryptic digestion, gel pieces were digested with trypsin at 37 ◦C overnight after destained and dehydrated [49]. Then, peptides were extracted with 50% acetonitrile/5% formic acid, followed by 100% acetonitrile. The peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid, whereafter these unknown peptides were subjected to LC-MS/MS analysis to identify candidate proteins’ name and function.

4.8. Yeast Two-Hybrid Assay The full-length of SN2 was fused to the bait vector pGBKT7, and the AD vector (pGADT7) was used to express peroxidases. Then, both pairs of plasmids (SN2-BD/Prxs- AD) were co-transformed into the yeast strain AH109 (Coolaber). The co-transformation colonies were selected on SD-Trp-Leu plates. Positive clones were transferred and grown on SD/-Leu-Trp-His plates, and the β-Galactosidase activity was measured. SN2-BD and pGADT7 were used as negative controls. AtTOPP4 and AtPIN1 were used as positive controls [50]. The primers used for generating various clones in this study are listed in Supplementary Table S1.

4.9. Measurement of Lignin Content and Enzyme Activities

The extraction and quantiﬁcation of lignin and H2O2 were performed based on the procedure described in the manufacturer’s directions (Solarbio, China). The activities of Prx, SOD, CAT and CAD were measured separately by using a PRX assay kit (Cat. BC0095), SOD assay kit (Cat. BC0175), CAT assay kit (Cat. BC0205) and CAD assay kit (Cat. BC4170) produced by Solarbio life science. All samples were prepared for enzyme activity by homogenizing 0.1 g of tuber in a solution of 0.01 mM PH 7.2 phosphate buffer saline. The homogenate was centrifuged at 12 000 rpm for 10 min at 4 ◦C. The activity of CCR and COMT were determined by the assay kit of Bioroyee (Beijing, China).

4.10. Statistical Analysis For all generated data, at least three biological replicates were performed for each sample. The data were subjected to unpaired Student’s t-tests with p ≤ 0.01 and p ≤ 0.05. Data are shown as the mean ± SE (n = 3), and n represents the biological replicates. Excel 2019 (Microsoft Corporation, Redmond, WA, USA) and the SPSS 14.0 software (IBM, New York, NY, USA) were used for statistical analysis. The statistical results were reported as the mean ± SD.

Supplementary Materials: Supplementary materials can be found in https://www.mdpi.com/1422 -0067/22/5/2287/s1, Supplementary Figures S1–S4 and Tables S1–S5. Author Contributions: L.L. and X.W. conceived and designed the experiments. M.D., J.P., J.Z., S.R., C.C., S.N., X.H. and L.Y. performed the experiments. M.D., J.P. and J.Z. analyzed the data. M.D. wrote the manuscript. L.L. and X.W. directed the study and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Int. J. Mol. Sci. 2021, 22, 2287 14 of 16

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: This research was financially supported by The Sichuan Potato Innovation Team Program of the Chinese Modern Agricultural Industrial Technology System (Agricultural Department of Sichuan Province Document (2019) No. 59). Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References 1. Alamar, M.C.; Tosetti, R.; Landahl, S.; Bermejo, A.; Terry, L.A. Assuring potato tuber quality during storage: A future perspective. Front. Plant Sci. 2017, 8, 2034. [CrossRef][PubMed] 2. Morris, W.L.; Alamar, M.C.; Lopez-Cobollo, R.M.; Cañete, J.C.; Bennett, M.; Van Der Kaay, J.; Stevens, J.; Sharma, S.K.; McLean, K.; Thompson, A.J.; et al. A member of the terminal FLOWER 1/CENTRORADIALIS gene family controls sprout growth in potato tubers. J. Exp. Bot. 2018, 70, 835–843. [CrossRef][PubMed] 3. Sonnewald, S.; Sonnewald, U. Regulation of potato tuber sprouting. Planta 2013, 239, 27–38. [CrossRef] 4. Liu, B.; Zhao, S.; Tan, F.; Zhao, H.; Wang, D.; Si, H.; Chen, Q. Changes in ROS production and antioxidant capacity during tuber sprouting in potato. Food Chem. 2017, 237, 205–213. [CrossRef][PubMed] 5. Shukla, S.; Pandey, S.S.; Chandra, M.; Pandey, A.; Bharti, N.; Barnawal, D.; Chanotiya, C.S.; Tandon, S.; Darokar, M.P.; Kalra, A.; et al. Application of essential oils as a natural and alternate method for inhibiting and inducing the sprouting of potato tubers. Food Chem. 2019, 284, 171–179. [CrossRef] 6. Li, L.-Q.; Zou, X.; Deng, M.-S.; Peng, J.; Huang, X.-L.; Lu, X.; Fang, C.-C.; Wang, X.-Y. Comparative morphology, transcription, and proteomics study revealing the key molecular mechanism of camphor on the potato tuber sprouting effect. Int. J. Mol. Sci. 2017, 18, 2280. [CrossRef] 7. Hou, J.; Liu, T.; Reid, S.; Zhang, H.; Peng, X.; Sun, K.; Du, J.; Sonnewald, U.; Song, B. Silencing of α-amylase StAmy23 in potato tuber leads to delayed sprouting. Plant Physiol. Biochem. 2019, 139, 411–418. [CrossRef][PubMed] 8. Li, L.; Deng, M.; Lyu, C.; Zhang, J.; Peng, J.; Cai, C.; Yang, S.; Lu, L.; Ni, S.; Liu, F.; et al. Quantitative phosphoproteomics analysis reveals that protein modiﬁcation and sugar metabolism contribute to sprouting in potato after BR treatment. Food Chem. 2020, 325, 126875. [CrossRef] 9. Paul, V.; Ezekiel, R.; Pandey, R. Sprout suppression on potato: Need to look beyond CIPC for more effective and safer alternatives. J. Food Sci. Technol. 2015, 53, 1–18. [CrossRef] 10. Jin, L.; Cai, Q.; Huang, W.; Dastmalchi, K.; Rigau, J.; Molinas, M.; Figueras, M.; Serra, O.; Stark, R.E. Potato native and wound periderms are differently affected by down-regulation of FHT, a suberin feruloyl transferase. Phytochemistry 2018, 147, 30–48. [CrossRef] 11. Serra, O.; Hohn, C.; Franke, R.; Prat, S.; Molinas, M.; Figueras, M. A feruloyl transferase involved in the biosynthesis of suberin and suberin-associated wax is required for maturation and sealing properties of potato periderm. Plant J. 2010, 62, 277–290. [CrossRef] 12. Lashbrooke, J.; Cohen, H.; Levy-Samocha, D.; Tzfadia, O.; Panizel, I.; Zeisler, V.; Massalha, H.; Stern, A.; Trainotti, L.; Schreiber, L.; et al. MYB107 and MYB9 homologs regulate suberin deposition in angiosperms. Plant Cell 2016, 28, 2097–2116. [CrossRef] 13. Panikashvili, D.; Shi, J.X.; Schreiber, L.; Aharoni, A. The arabidopsis DCR encoding a soluble BAHD acyltransferase is required for cutin polyester formation and seed hydration properties. Plant Physiol. 2009, 151, 1773–1789. [CrossRef][PubMed] 14. Liao, N.; Hu, Z.; Li, Y.; Hao, J.; Chen, S.; Xue, Q.; Ma, Y.; Zhang, K.; Mahmoud, A.; Ali, A.; et al. Ethylene-responsive factor 4 is associated with the desirable rind hardness trait conferring cracking resistance in fresh fruits of watermelon. Plant Biotechnol. J. 2020, 18, 1066–1077. [CrossRef] 15. Vulavala, V.K.R.; Fogelman, E.; Faigenboim, A.; Shoseyov, O.; Ginzberg, I. The transcriptome of potato tuber phellogen reveals cellular functions of cork cambium and genes involved in periderm formation and maturation. Sci. Rep. 2019, 9, 10216. [CrossRef] 16. Liang, M.; Davis, E.; Gardner, D.; Cai, X.; Wu, Y. Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 2006, 224, 1185–1196. [CrossRef][PubMed] 17. Renard, J.; Martínez-Almonacid, I.; Sonntag, A.; Molina, I.; Moya-Cuevas, J.; Bissoli, G.; Muñoz-Bertomeu, J.; Faus, I.; Niñoles, R.; Shigeto, J.; et al. PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis. Plant Cell Environ. 2019, 43, 315–326. [CrossRef][PubMed] 18. Zhang, S.; Yang, C.; Peng, J.; Sun, S.; Wang, X. GASA5, a regulator of ﬂowering time and stem growth in Arabidopsis thaliana. Plant Mol. Biol. 2009, 69, 745–759. [CrossRef] 19. Rubinovich, L.; Weiss, D. The arabidopsis cysteine-rich protein GASA4 promotes GA responses and exhibits redox activity in bacteria and in planta. Plant J. 2010, 64, 1018–1027. [CrossRef] Int. J. Mol. Sci. 2021, 22, 2287 15 of 16

20. Nahirñak, V.; Almasia, N.I.; Fernandez, P.V.; Hopp, H.E.; Estevez, J.M.; Carrari, F.; Vazquez-Rovere, C. Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition. Plant Physiol. 2011, 158, 252–263. [CrossRef] 21. Sun, S.; Wang, H.; Yu, H.; Zhong, C.; Zhang, X.; Peng, J.; Wang, X. GASA14 regulates leaf expansion and abiotic stress resistance by modulating reactive oxygen species accumulation. J. Exp. Bot. 2013, 64, 1637–1647. [CrossRef] 22. Zhong, C.; Xu, H.; Ye, S.; Wang, S.; Li, L.; Zhang, S.; Wang, X. Gibberellic acid-stimulated arabidopsis6 serves as an integrator of gibberellin, abscisic acid, and glucose signaling during seed germination in arabidopsis. Plant Physiol. 2015, 169, 2288–2303. [CrossRef] 23. Qu, J.; Kang, S.G.; Hah, C.; Jang, J.-C. Molecular and cellular characterization of GA-stimulated transcripts GASA4 and GASA6 in Arabidopsis thaliana. Plant Sci. 2016, 246, 1–10. [CrossRef] 24. Nahirñak, V.; Rivarola, M.; Almasia, N.I.; Barón, M.P.B.; Hopp, H.E.; Vile, D.; Paniego, N.; Rovere, C.V. Snakin-1 affects reactive oxygen species and ascorbic acid levels and hormone balance in potato. PLoS ONE 2019, 14, e0214165. [CrossRef][PubMed] 25. Tianli, W.; Chunzhen, C.; Yun, Z.; Yuanda, L.; Yanyan, M.; Guangyan, Z. Molecular characterization of the gibberellin-stimulated transcript of GASA4 in Citrus. Plant Growth Regul. 2020, 91, 89–99. 26. Rubinovich, L.; Ruthstein, S.; Weiss, D. The arabidopsis cysteine-rich GASA5 is a redox-active metalloprotein that suppresses gibberellin responses. Mol. Plant 2014, 7, 244–247. [CrossRef][PubMed] 27. Liu, W.; Jiang, Y.; Wang, C.; Zhao, L.; Jin, Y.; Xing, Q.; Li, M.; Lv, T.; Qi, H. Lignin synthesized by CmCAD2 and CmCAD3 in oriental melon (Cucumis melo L.) seedlings contributes to drought tolerance. Plant Mol. Biol. 2020, 103, 689–704. [CrossRef] [PubMed] 28. Ho-Yue-Kuang, S.; Alvarado, C.; Antelme, S.; Bouchet, B.; Cézard, L.; Le Bris, P.; Légée, F.; Maia-Grondard, A.; Yoshinaga, A.; Saulnier, L.; et al. Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality. J. Exp. Bot. 2015, 67, 227–237. [CrossRef] 29. Fernández-Pérez, F.; Vivar, T.; Pomar, F.; Pedreño, M.A.; Novo-Uzal, E. Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana. J. Plant Physiol. 2015, 175, 86–94. [CrossRef] 30. Tamasloukht, B.; Wong, Q.L.M.; Martinez, Y.; Tozo, K.; Barbier, O.; Jourda, C.; Jauneau, A.; Borderies, G.; Balzergue, S.; Renou, J.P.; et al. Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: Effects on lignification, fibre development, and global gene expression. J. Exp. Bot. 2011, 62, 3837–3848. [CrossRef] 31. Su, X.; Zhao, Y.; Wang, H.; Li, G.; Cheng, X.; Jin, Q.; Cai, Y. Transcriptomic analysis of early fruit development in Chinese white pear (Pyrus bretschneideri Rehd.) and functional identification of PbCCR1 in lignin biosynthesis. BMC Plant Biol. 2019, 19, 417. [CrossRef] 32. Li, J.; Wu, L.; Foster, R.; Ruan, Y.-L. Molecular regulation of sucrose catabolism and sugar transport for development, defence and phloem function. J. Integr. Plant Biol. 2017, 59, 322–335. [CrossRef] 33. Su, G.; An, Z.; Zhang, W.; Liu, Y. Light promotes the synthesis of lignin through the production of H2O2 mediated by diamine oxidases in soybean hypocotyls. J. Plant Physiol. 2005, 162, 1297–1303. [CrossRef][PubMed] 34. Bajji, M.; M’Hamdi, M.; Gastiny, F.; Rojas-Beltran, J.A.; du Jardin, P. Catalase inhibition accelerates dormancy release and sprouting in potato (Solanum tuberosum L.) tubers. Biotechnol. Agron. Soc. 2007, 2, 121–131. 35. Wojtyla, Ł.; Lechowska, K.; Kubala, S.; Garnczarska, M. Different modes of hydrogen peroxide action during seed germination. Front. Plant Sci. 2016, 7, 66. [CrossRef] 36. Wigoda, N.; Ben-Nissan, G.; Granot, D.; Schwartz, A.; Weiss, D. The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity. Plant J. 2006, 48, 796–805. [CrossRef][PubMed] 37. Ben-Nissan, G.; Lee, J.-Y.; Borohov, A.; Weiss, D. GIP, a Petunia hybrida GA-induced cysteine-rich protein: A possible role in shoot elongation and transition to flowering. Plant J. 2003, 37, 229–238. [CrossRef][PubMed] 38. Li, X.; Shi, S.; Tao, Q.; Tao, Y.; Miao, J.; Peng, X.; Li, C.; Yang, Z.; Zhou, Y.; Liang, G. OsGASR9 positively regulates grain size and yield in rice (Oryza sativa). Plant Sci. 2019, 286, 17–27. [CrossRef] 39. Liu, Z.H.; Zhu, L.; Shi, H.Y.; Chen, Y.; Zhang, J.M.; Zheng, Y.; Li, X.B. Cotton GASL genes encoding putative gibberellin-regulated proteins are involved in response to GA signaling in fiber development. Mol. Biol. Rep. 2013, 40, 4561–4570. [CrossRef] 40. Oliveira-Lima, M.; Benko-Iseppon, A.M.; Neto, J.R.C.F.; Rodriguez-Decuadro, S.; Kido, E.A.; Crovella, S.; Pandolfi, V. Snakin: Structure, roles and applications of a plant antimicrobial peptide. Curr. Protein Pept. Sci. 2017, 18, 368–374. [CrossRef][PubMed] 41. Berrocal-Lobo, M.; Segura, A.; Moreno, M.; López, G.; García-Olmedo, F.; Molina, A. Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiol. 2002, 128, 951–961. [CrossRef][PubMed] 42. Wang, L.; Wang, Z.; Xu, Y.; Joo, S.-H.; Kim, S.-K.; Xue, Z.; Xu, Z.; Wang, Z.; Chong, K. OsGSR1is involved in crosstalk between gibberellins and brassinosteroids in rice. Plant J. 2009, 57, 498–510. [CrossRef] 43. Verdaguer, R.; Soler, M.; Serra, O.; Garrote, A.; Fernandez, S.; Company-Arumi, D.; Antico, E.; Molinas, M.; Figueras, M. Silencing of the potato StNAC103 gene enhances the accumulation of suberin polyester and associated wax in tuber skin. J. Exp. Bot. 2016, 67, 5415–5427. [CrossRef][PubMed] 44. De La Fuente, J.I.; Amaya, I.; Castillejo, C.; Sánchez-Sevilla, J.F.; Quesada, M.A.; Botella, M.Á.; Valpuesta, V. The strawberry gene FaGAST affects plant growth through inhibition of cell elongation. J. Exp. Bot. 2006, 57, 2401–2411. [CrossRef][PubMed] 45. Trapalis, M.; Li, S.F.; Parish, R.W. The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds. Plant Sci. 2017, 260, 71–79. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 2287 16 of 16

46. Li, Q.; Qin, X.; Qi, J.; Dou, W.; Dunand, C.; Chen, S.; He, Y. CsPrx25, a class III peroxidase in Citrus sinensis, confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification. Hortic. Res. 2020, 7, 192. [CrossRef][PubMed] 47. Wang, X.; Li, J.; Zou, X.; Lu, L.; Li, L.; Ni, S.; Liu, F. Ectopic expression of AtCIPK23 enhances tolerance against Low-K+ stress in transgenic potato. Am. J. Potato Res. 2010, 88, 153–159. [CrossRef] 48. Hu, Y.; Yu, D. BRASSINOSTEROID INSENSITIVE2 interacts with ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis. Plant Cell 2014, 26, 4394–4408. [CrossRef] 49. Chen, R.; Xiao, M.; Gao, H.; Chen, Y.; Li, Y.; Liu, Y.; Zhang, N. Identification of a novel mitochondrial interacting protein of C1QBP using subcellular fractionation coupled with CoIP-MS. Anal. Bioanal. Chem. 2016, 408, 1557–1564. [CrossRef] 50. Guo, X.; Qin, Q.; Yan, J.; Niu, Y.; Huang, B.; Guan, L.; Li, S.-S.; Ren, D.; Liu, Y.; Hou, S. TYPE-ONE PROTEIN PHOSPHATASE4 regulates pavement cell interdigitation by modulating PIN-FORMED1 polarity and trafficking in Arabidopsis. Plant Physiol. 2015, 167, 1058–1075. [CrossRef]