Ectopic Expression of BcCUC2 Involved in Sculpting the Leaf Margin Serration in Arabidopsis thaliana

Abstract

Leaf margin serration is a morphological characteristic in plants. The CUC2 (CUP-SHAPED COTYLEDON 2) gene plays an important role in the outgrowth of leaf teeth and enhances leaf serration via suppression of growth in the sinus. In this study, we isolated the BcCUC2 gene from Pak-choi (Brassica rapa ssp. chinensis), which contains a 1104 bp coding sequence, encoding 367 amino acid residues. Multiple sequence alignment exhibited that the BcCUC2 gene has a typical conserved NAC domain, and phylogenetic relationship analysis showed that the BcCUC2 protein has high identity with Cruciferae plants (Brassica oleracea, Arabidopsis thaliana, and Cardamine hirsuta). The tissue-specific expression analysis displayed that the BcCUC2 gene has relatively high transcript abundance in floral organs. Meanwhile, the expression profile of BcCUC2 was relatively higher in the ‘082’ lines with serrate leaf margins than the ‘001’ lines with smooth leaf margins in young leaves, roots, and hypocotyls. In addition, the transcript level of BcCUC2 was up-regulated by IAA and GA3 treatment, especially at 1–3 h. The subcellular localization assay demonstrated that BcCUC2 was a nuclear-target protein. Furthermore, leaf serration occurred, and the number of the inflorescence stem was increased in the transgenic Arabidopsis thaliana plants’ overexpressed BcCUC2 gene. These data illustrated that BcCUC2 is involved in the development of leaf margin serration, lateral branches, and floral organs, contributing to further uncovering and perfecting the regulation mechanism of leaf serration in Pak-choi.

1. Introduction

Leaves are important vegetative organs in plants, and their shape and size directly affect the photosynthesis, transpiration, stress response, and ornamental value of plants. Leaf morphogenesis starts from the flanks of a small group of totipotent stem cells, the shoot apical meristem (SAM) [1]. The development of leaves can be divided into three main stages: (1) the initiation of leaf primordium; (2) the establishment of primary leaf shape, the leaf primordium continuing to grow and differentiate, and the production of secondary structures such as serrate leaves, lobed leaves, and leaflets; (3) the formation of secondary leaf morphology, producing leaf margins, stomata, trichome, and eventually forming mature leaves [2,3].

Plant leaves can be defined as entire leaves (smooth margins), serrate leaves, and lobed leaves according to the margins of the leaf and leaflet blades [4]. Leaf morphology is complex and varied in diverse species, which mainly depends on the regulation of genetic, developmental, and environmental factors [5]. So far, a number of leaf margin regulators have been identified with crucial roles in elaborating leaf shape. The KNAT1, KNAT2, and SHOOT MERISTEMLESS (STM) genes belong to KNOTTED-like homeodomain class I (KNOX1) transcription factors, which are involved in the formation and maintenance of the SAM. The expression level of KNOX1 genes is down-regulated during early leaf initiation, and the overexpression of KNAT1 and KNAT2 leads to occasional ectopic shoots on the adaxial surface of leaves, lobed leaves, and ectopic stipules [6,7,8,9,10]. In Arabidopsis thaliana (A. thaliana) (L.) Heynh., the loss of function of STM is associated with the evolution of the unlobed leaf form; thereby, STM is essential for lobe formation [11].

CUP-SHAPED COTYLEDON (CUC), members of the NAC transcription factors (such as NAM in Petunia hybrida and ATAF1/2 and CUC2 in A. thaliana), which contain a conserved NAC domain at the N-terminus and a highly variable domain in the C-terminal region [12], act as major players in shoot apical meristem (SAM) construction, organ separation, leaf development, and the regulation of the axillary meristem initiation in leaf axils [4,13,14,15,16,17]. In Arabidopsis, the CUC subfamily contains three members, CUC1, CUC2, and CUC3, which act redundantly to regulate cotyledons’ separation, organ boundary specification, and embryonic shoot meristem formation, in part [13,16,18]. The CUC1 and CUC2 genes are necessary for shoot meristem initiation via promoting the transcript level of STM [19]. The cuc1 and cuc2 single mutants display few morphological phenotypes due to their functional redundancy, while the cuc1cuc2 mutant exhibits complete absence of shoot meristem and forms dramatically fused cotyledons [13,18,20]. Mutation of the CUC homologs, the CUPULIFORMIS, NO APICAL MERISTEM (NAM), and GOBLET genes in snapdragon, petunia, and tomato, respectively, results in similar development defects [21,22,23,24], illustrating that these genes share an evolutionarily conserved function in organ separation and SAM development. CUC2 plays an important role in the initiation of leaf serration during the early phase and has a synergistic interaction with CUC3 in the maintenance of leaf serration during the later stage [15]. In addition, MIR164A encodes a microRNA, which is involved in the regulation of leaf margin serration through cooperating with CUC2 in Arabidopsis. The mir164a mutants display significant deep serrate leaf margins compared with the wild type in Arabidopsis [4]. The CUC2 gene is one of the target genes of miR164, and the balance between CUC2 and miR164a has a prominent role in the extent of leaf serrations. In Solanum lycopersicum, loss of GOBLET (GOB), the homologous gene of Arabidopsis CUC2, results in reduced complexity of compound leaves and fruit shape [24], suggesting that the CUC2 gene has diverse functions in different species and plays a crucial role in leaf development.

Pak-choi (Brassica rapa ssp. chinensis) belongs to Brassicaceae crops, is an important leafy vegetable, and is widely cultivated in the middle and lower regions of the Yangtze River. Leaf morphology, as an important agronomic trait, has a direct effect on its yield and ornamental value. In this study, we isolated a CUC2 gene encoding 367 amino acids from Pak-choi, which has three highly conserved CUC2-specific motifs. The expression patterns and biological function of BcCUC2 were systematically investigated.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Pak-choi cultivar ‘001’ with entire leaf margin and ‘082’ with serrate leaf margin were used in this study. Seeds were dispersed on wet filter paper for germination and grown in pots containing humus soil/vermiculite (2:1) mixture in plant artificial climate chamber controlled at 23/17 °C, 16/8 h for light/dark cycle. The illumination intensity was set to 12,000 xl, and the relative humidity was 65–75%. For different tissues, the samples were harvested at seedling, rosette, flowering, and podding stages. Three biological replicates were used for each sample.

A. thaliana wild type (Columbia-0) was used in this study and grown in illumination incubator under the same condition. Four-week-old tobacco (Nicotiana benthamiana) seedlings were used for subcellular localization assay.

2.2. IAA and GA3 Treatments

Seedlings of cultivar ‘001’ with six fully expanded leaves were foliar sprayed with 100 μM GA3 and IAA. The leaves were collected at 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h after treatment (0 h was used as a control) and frozen rapidly in liquid nitrogen, followed by storage at −70 °C in refrigerators for RNA extraction. Three biological repeats were implemented for each sample.

2.3. Cloning and Sequence Analysis

Total RNA was extracted from leaves using RNAprep pure Plant Kit (TIANGEN, Beijing, China), and the first strands of cDNA were synthesized via reverse transcription using PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The coding sequence (CDS) of BcCUC2 was amplified by gene-specific primers (see

Table A1. Primers used in this study.

Primer	Sequence (5′-3′)
Cloning
BcCUC2-F	ATGGACATTCCGTACTACCAC
BcCUC2-R	GTAATTCCATACGCAATCAAG
qRT-PCR
qBcCUC2-F	CGGAGGCTCAGCAGAAGCAA
qBcCUC2-R	GGTGTAGCCGAGGGTTGTGG
qBcActin-F	GTTGCTATCCAGGCTGTTCT
qBcActin-R	AGCGTGAGGAAGAGCATAAC
qAtActin-F	TTGACAATTGATGCAAACAAT
qAtActin-R	CCATTGCTTAATTCCACGGAC
Overexpression
KpnI-BcCUC2-F	GGGGTACCATGGACATTCCGTACTACCAC
BamHI-BcCUC2-R	CGGGATCCGTAATTCCATACGCAATCAAG

) based on the sequence of Bra022685 using homology cloning referred to in our previous report [25]. The resulting fragment was cloned into pEASYBlunt Simple Vector (Transgene, Beijing, China) and sequenced by Genscript Company (Nanjing, China). The physicochemical characteristics of BcCUC2 were predicted using Expasy proteomics server (https://web.expasy.org/protparam/, accessed on 10 July 2020). The secondary structure of BcCUC2 was predicted using PSIPRED 4.0 (http://bioinf.cs.ucl.ac.uk/psipred/, accessed on 10 July 2020).

2.4. Phylogenetic Tree Analysis

The protein sequences of A. thaliana were obtained from TAIR database (http://arabidopsis.org/index.jsp, accessed on 15 July 2020). The protein sequences of Brassica oleracea, Brassica hirsuta, Solanum lycopersicum, Oryza sativa, Zea mays, and Vitis vinifera were downloaded from NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 15 July 2020). Multiple sequence alignment of CUC genes in different species was performed using ClustalW software. The phylogenetic tree was constructed by MEGA 7.0 using neighbor-joining (NJ) method with the bootstrap of 1000 replications. All protein sequences used in this study are listed in

Table A2. The protein sequences of CUC gene used in this study.

Gene Name	GenBank Accession	Plant Species	Protein Sequences
AtCUC1	NM_112380	Arabidopsis thaliana	MDVDVFNGWGRPRFEDESLMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVDLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEAGYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSCWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKSGVVSRETNLISSSSSSAVTGEFSSAGSAIAPIINTFATEHVSCFSNNSAAHTDASFHTFLPAPPPSLPPRQPRHVGDGVAFGQFLDLGSSGQIDFDAAAAAFFPNLPSLPPTVLPPPPSFAMYGGGSPAVSVWPFTL
AtCUC2	NM_124774	Arabidopsis thaliana	MDIPYYHYDHGGDSQYLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEAGYWKATGKDREIFSSKTCALVGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKTTLASTGAVSEGGGGGGATVSVSSGTGPSKKTKVPSTISRNYQEQPSSPSSVSLPPLLDPTTTLGYTDSSCSYDSRSTNTTVTASAITEHVSCFSTVPTTTTALGLDVNSFSRLPPPLGFDFDPFPRFVSRNVSTQSNFRSFQENFNQFPYFGSSSASTMTSAVNLPSFQGGGGVSGMNYWLPATAEENESKVGVLHAGLDCIWNY
AtCUC3	NM_106292	Arabidopsis thaliana	MMLAVEDVLSELAGEERNERGLPPGFRFHPTDEELITFYLASKIFHGGLSGIHISEVDLNRCEPWELPEMAKMGEREWYFYSLRDRKYPTGLRTNRATTAGYWKATGKDKEVFSGGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLENDHSHRHTCKEEWVICRVFNKTGDRKNVGLIHNQISYLHNHSLSTTHHHHHEALPLLIEPSNKTLTNFPSLLYDDPHQNYNNNNFLHGSSGHNIDELKALINPVVSQLNGIIFPSGNNNNDEDDFDFNLGVKTEQSSNGNEIDVRDYLENPLFQEASYGLLGFSSSPGPLHMLLDSPCPLGFQL
ChCUC1	ACL14369.1	Cardamine hirsuta	MDIVVFNGSERPRFEDDTLMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVNLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEAGYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSSWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKSGVVNRETKSISSSTSAAGEFSSPGSTIAPIIDAFASEHVSCFSNDAAHANESFRTTYLPAPPPSLPPRQPRHIGDDVAFGQFMDFGFSGQIHYDAAAFFPNLPSLPPTALPPPPSFAMYGGGSTLSYWPFAL
ChCUC2	ACL14370.1	Cardamine hirsuta	MDIPYYHYDHGGDSQYLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEAGYWKATGKDREIYSSKTCALIGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKTGLLSTGAAGAGAIVSGSNGTGTSKKTKIPSTISRNYQEQPSSPSSVSLPPLLGYTDSSCSYDGHSTNTTVTATGITEHVSCFSTATTTNTTTTDLGLDVNVDSFNHFPPPVFDPLPRFVSRNVSNLSNFRSFQDNQFPYFGSSSSASTMTSSVHLPSSQSGGSGVSGMNYWLQATAEENETKAGVLQAGLDCIWNY
ChCUC3	ACL14365.1	Cardamine hirsuta	MMLAVEDVLSELAGEERNERGLPPGFRFHPTDEELISFYLASKVFDGGLCGIHITEVDLNRCEPWELPEMAKMGEKEWYFYSLRDRKYPTGLRTNRATTAGYWKATGKDKEVFGSGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLETDLSHRHSCKEEWVICRVFNKTGDRKNVGVHSQISCLHNHSLSTYHHHHHETLPPLLEPSKTISNFPSLLYDDHTHQNHNNNLFHGSSGHHHIDELKALINPVVSQLNGIIFSPGNNNNVDDEDDFNLGVKTEPFLNGGSNELDVRDYLENPLFHEVGYGLLGVSSAPGPLHMLLDSPCPLGFQL
OsNAM	EAZ00836.1	Oryza sativa	MERCSVLGLGGGGGGGGRLDGELPPGFRFHPTDEELITYYLLRKVVDGSFNGRAIAEIDLNKCEPWELPEKAKMGEKEWYFYSLRDRKYPTGLRTNRATGAGYWKATGKDREIRSARTGALVGMKKTLVFYRGRAPKGQKTQWVMHEYRLDGTYAYHFLSSSTRDEWVIARIFTKPGVFPVVRKGRLGISGGGGDTSCFSDSTSASVGGGGGTSASSALRAPLAEASLFAAAAAPAVDGADSSNYGGGGGGGSATATANLVTGLELVPCFSTTAHMDASFGTGQYNPAPLAVEPPPPPPAFFPSLRSLQENLQLPLFLSGGMQAGVSSQPLSGSGAFHWQSGMDVKVEGAVGRAPPQMAVGPGQLDGAFAWGF
OsCUC3	NP_001062212.1	Oryza sativa	MPTTEILQHYSVVSQIKSHGKGIASEFPSALASWSADQISTDGAANLAGKLKQARSQIKNWTKNRTSCRFLDNDCKFVIDLFDFLEELRELSAPERLLRQMVQDKFTQYKLMQASYWKQRGKVKKIRLGIDNTHFFKAHATQNHRRTFIRSIKLTDMEVSEHSDKATTMFSYYNSILGASTETSWSFDLHTIYHGCAMANADELVQPFSEQEIFQAIKHMDKNSAPGPDGFGPGFFQAAWAMIKPDILHLLQSFYDETADMERINRAFIVLIPKPGKTNTPDGYRPISLQNCSVKIIAKVMANRMQRQLPPLIDLDQSGFLKREKHLGELHLCKILQARGFPFLWRSWTNRLLQTSKSAIMINGVPGNWINCKRGVRQGDPMSPYLFILVADVLQKLIRHSGEIKHPIYPDQPCATLQYADDTIIICRATEQDLAALKTCLNHFAAATGLHINFSKSTLVTMHVPDEVTTALANILQCKTDSSWGRWIWQEHSGSALFCDNQLGPHWDSLSTLLPILQRLTRVQVGDGTRTSFWHDCWYGSSTFKDRFAPLFSHALNREATVAVFLSKPIEDQFAPRLSSTAETQLARLREMLQNFYLSNSTDLCPSRDAPGILRTKFIYSSTHMGLPLCKNWRFIWDNRAPPRVQFFAWLLAKDRLPTKANLHKKNIVPTAGCIVCNCADETATHLFLQCQFAQEFWRALRTSVVSNVQDLADLVAPCHLPVKHFQVFFLLCFWGLWNHRHDVVFRGLPNSRTRNLQSLKPAESQAAGQPPEVWYACVAWAQLCRDIADNRTKPRSAAFEKSRNQPAIEHACMQGKNGFCVRGTSDSEMHHHSATMGDALWEMLGEEMAAAAAAAGEHGLPPGFRFHPTDEELVTFYLAAKVFNGACCGGVDIAEVDLNRCEPWELPEAARMGEKEWYFFSLRDRKYPTGLRTNRATGAGYWKATGKDREVVAAAAAGGALIGMKKTLVFYKGRAPRGEKTKWVLHEYRLDGDFAAARRSTKEEWVICRIFHKVGDQYSKLMMMKSPASYYLPVSHHHPSSIFHDLPPVPFPNPSLVPFHHDLPTSFHPPLLQHSHANSKNSSSNNGGFVFPNEPNTTNSSDNHISCNGAMAAAAAAAFPSFSCASTVTGKGGPPAQLGVNAGQQEPPPPTWMDAYLQHSGFIYEMGPPAVPRGA
ZmNAM1	CAH56057.1	Zea mays	MERFGLDGGGGGGELPPGFRFHPTDEELITYYLLRKAVDGSFCGRAIAEIDLNKCEPWELPGKAKMGEKEWYFYSLRDRKYPTGLRTNRATVAGYWKATGKDREIRSGRTGALVGMKKTLVFYRGRAPKGQKTHWVMHEYRLEGAYAYHFLPSSTRQDEWVIARVFQKPGEVPPAARKHRLGALSSTTGTAAGDSCFSDSTSASIGGASSSSTPGPLFASAAAAVANAGAADGDTSSYCGGAANHGNLVTGRELVPCFSTATINGPLVAAALGIGQPYNAAPLPFEQQPPPPAFLPSLRSLQDNLQLPPFLSAGGLGGGGALHWLPAGGMEVKVEGRSAPPQMAVGPGQLDGAFGWSF
ZmNAM2	CAH56058.1	Zea mays	MERLGVGVGVGELPPGFRFHPTDEELITYYLLCKAVDGGFCGGRAIAEIDLNKCEPWELPGKAKMGEKEWYFYCLRDRKYPTGLRTNRATAAGYWKATGKDREVRSGRSGALVGMKKTLVFYRGRAPRGQKTRWVMHEYRLDGTYAYHFLPGSTRDEWVIARVFQKPGEVPCGRKHRLGGPSAAAGDSCFSDSTTSASIGGGGGGGASASSRPLLTVTDTSSPSLFVANANAAASNNNGNPVTGRELVPCFSTTASPLEAAALGVVGHPYNAAPLRLGLDFEAPSPGFVVPNLRSLQVQDDGGLPLFLSAAAGGGMSSATLGIMGSLGGSLHCPPHAGMDVVKVEGRAAPPQMAVGPGLLDGAFAWGF
ZmCUC3	CAH56059.1	Zea mays	MAAAGGEHGLPPGFRFHPTDEELVTFYLAAKVFNGACCGIDIAEVDLNRCEPWELPDAARMGEREWYFFSLRDRKYPTGLRTNRATGAGYWKATGKDREVLNAATGALLGMKKTLVFYKGRAPRGEKTKWVLHEYRLDGDFAAARRPCKEEWVICRILHKAGDQYSKLMMVKSPYYLPMAMDPSSFCFQEDPTGHPLPNPSGCTPFHHGHPHHSMQPPPPLPPSNHAGKAVFTGAAAACCMQQEPADGSNSAVLPMPPFPPFTPIVAGKPAAPAPPPQVVNAGPQEPPPPTWLEAYLQHTGGILYEMGPTAAPRGA
SlNAM	ACL14371.1	Solanum lycopersicum	MEIYHQMQFDCGDPHLPPGFRFHPTDEELITYYLLKKVLDCNFTARAIAEVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEAGYWKATGKDREIFSSKTCALVGMKKTLVFYRGRAPKGEKSNWVMHEYRLDGKFAYHYISRSSKDEWVISRVFQKSTGSNGAATSTGGGKKRLSSSINMYQEVSSPSSVSHLPPLLDSSPYSTTATSAAAIVIGDRDRDHSFKKEHVPCFSTTATATITAQSLTFDPTSVFDISSNTLHALQPTPSFASILDSSPSNFTNYTRNSTFPSLRSLHENLQLPLFSGGTSAMHGGFSNPMVNWTVPETQKVEQSELDCMWSY
VvNAM1	XP_002282655.1	Vitis vinifera	MDAYHHFDNSDAHLPPGFRFHPTDEELITYYLIKKVLDSNFTGRAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEAGYWKATGKDREIYSSKTCSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKFAYHYLSRSSKDEWVISRVFQKSGSSGGGGATGGKKARLSSTVNLYPEVSSPSSASLPPLLDVSPYAGTSAAAAVNDRESCSYDGGESSNNSNARDQHVPWFSTIAAAAAAAAANSFNAHHQPPPFDLAPPSIIGSIDPSRFPRNGAVPAFPNLRSLQENLHLPFFFSQVAPPIPSSGDPSTEMGITNSAGNWPAPENQKMDNGRLPMGATELDCMWSY
VvNAM2	XP_002280812.1	Vitis vinifera	MEEERKEETLPPGFRFHPTDEELITCYLINKISDATFTGRAIADVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGVRTNRATNTGYWKTTGKDKEIFNSVTSELVGMKKTLVFYRGRAPRGEKTNWVMHEYRIHSKSAFRTSKDEWVVCRVFQKSAAGKKYPSNQSRGMNPYSLDIGPSVMPPPMLQADSSQFPMGRNYVSNAELAELTRVLRGGSTGGLNLPIQSQLNYPLGGGCFTISGLNLNLGGTSTQPVLRPNSLPQPMQMNQQDHMMTSPMLTSGSIPTDQTGYGAEVNNGNGHNSRFMNMVDHCVDLDNYWPPY
VvNAM3	XP_002276293.2	Vitis vinifera	MQQAANQMHEKMEESLPPGFRFHPTDEELITYYLTPKVSNTNFASRAIADVDLNKCEPWDLPAKASMGEKEWYFFSLRDRKYPTGIRTNRATEAGYWKTTGKDKEIYRAGILVGMKKTLVFYKGRAPKGEKSNWVMHEYRLETKLSFKPKKEEWVVCRVFKKSSAVKKPHQPAPSSLPSLESPCDTNTIVSEFGDIEFPNMNSIANSSSGFSNINSAQSYNTSTDDNLNMNMNMNMNMNWAAAREAASLPSLPWSSSLLSPNLQMNSLLIKALQLGSYRPREATSTENYSFLPQGISNFGTDFISNFQASSSKVLDSLHQQQQQQQQEQPFNLDSINWFGHMSRSNSLNYLGKHSTSRLINSHRTAPQHKQAAGLSSCHMFLTPGPAPFLVNVLKTHGHHGSQQQSILCHTITHTKQLTQLLTSSAALLAACHSGLLPGASTAAPLCSVASTGQWLPSSICSAQPTCRRRQDSAKENPWDGLMGMVGDNGYEEVNRKGHVWPCKGGGDRHEGRASMVMGIRRTMSLAWRLMHGMIPHGA
VvCUC3	XP_002273222.1	Vitis vinifera	MLAMEEVLCELSREDINEQGLPPGFRFHPTDEELITFYLASKVFNGSFCGVEIAEVDLNRCEPWELPDVAKMGEREWYFFSLRDRKYPTGLRTNRATGAGYWKATGKDREVHSASSGALLGMKKTLVFYKGRAPRGEKTKWVMHEYRLDGDFSYRHTCKEEWVICRIFHKTGEKKNPMFQGQAYLLGSSAAAAVATSSLPALLESQTTLLESQSHPTMQGGISSSFLVHHHHDQESNELKALINPVLSQSPLAFPINSGFQSCSFSTTPTTNIPNTNNINSTTGNNNPSTSILFKSLLSHQECSLKEQTTIPKQCKTEANFSHFQLPDANMHWVDRMNSNLHQNPLFFEMDYCSGGVLGFTAATATGGGGGGGGASAAATSAAVASAAETVHEMSTSIAFNRAGFQMMVDFPIRVPGGESWPLDP
BoCUC2	HQ703968	Brassica oleracea	DHGGDSQYLPPGFRFHPTDEELITHYLLRKVIEGCFSSRAIAEVDLNKSEPWQLPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEAGYWKATGKDREIYSSKTCALVGMKKTLVFYKGRAPKGEKSSWVMHEYRLEGKFSYHFISRSSKDEWVISRVFKKTGLATTGASAGASISVSNCTGTSKKTKIPSNISTNYREQPSSPSSVSLPPLLDPTTTLGYTDSSWSYDSRSTNTPVITTAIT
BoCUC3	HQ703970	Brassica oleracea	NDRGLPPGFRFHPTDEELITFYLASKVFHGGLCGIHIAEVDLNRCEPWELPEMAKMGEREWYFYSLRDRKYPTGLRTNRATTAGYWKATGKDKEVFAGGGSGGGALVGMKKTLVFYKGRAPRGLKTKWVMHEYRLETDLSHRHTCKEEWVICRVFNKTGDRKNVGIHNQISYLHNTSLSTTHQQRNHNHYHHLEILPPLLEPSKTLTNFPSLLYDDTHQNYNNNLLHGSSGHNVDEFKTLINPAVSQLNGVIFSPEISNYNNEDDNNFGIKTEQYSNGGNNDLDVRDYLDNPFCQEAGYGLLGLSSSPGPLM

. The conserved motifs distribution was analyzed by the MEME program, and the number of motifs was set as 10 (Figure 1B).

2.5. Subcellular Localization Assay

The full-length CDS of BcCUC2 without stop codon was subcloned into linear pRI101-GFP vector with NdeI and BamHI restriction enzyme to generate the construct 35S:BcCUC2-GFP. For transient expression assay, empty vector (35S:GFP) and recombinant construct were introduced into Agrobacterium tumefaciens GV3101 and injected into tobacco foliar epidermis, respectively. After dark culturing for 24 h, the seedlings were moved to normal growth conditions for 24–36 h, and then fluorescent images were photographed using a confocal laser scanning microscope (Zeiss, LSM780, Jena, Germany).

2.6. Ectopic Expression in Arabidopsis

The coding sequence of BcCUC2 was inserted into the pCAMBIA1301 vector to produce the construct 35S:BcCUC2-GUS; then, the recombinant construct was transformed into Arabidopsis using the floral dipping method via Agrobacterium-mediated transformation [26]. In brief, the 35S:BcCUC2-GUS plasmids were transferred into A. tumefaciens GV3101 and agroinfiltrated into the Arabidopsis flowers for 45–60 s, cultivated in dark conditions for 2 d, and then grown in plant artificial climate chamber. To obtain the positive overexpression lines, seeds of Arabidopsis transgenic plants were surface sterilized and sown on half-strength Murashige and Skoog (1/2 MS) tissue culture plates containing 30 mg/L of hygromycin. The resistant seedlings were further testified using gene-specific primers amplification and GUS staining. Finally, 20 plants for each T3 positive transgenic line were cultivated and used for phenotype analysis.

2.7. Quantitative Real-Time PCR

Total RNAs were extracted using RNAprep pure Plant Kit (TIANGEN, Beijing, China) according to the operation manual, and cDNA was synthetized with PrimeScript™RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China). qRT-PCR was performed with ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TransStart Tip Green qPCR SuperMix (TransGen, Beijing, China). The PCR procedure was carried out with the following parameters: 94 °C for 30 s and 40 cycles of 94 °C for 5 s, 60 °C for 15 s, 72 °C for 10 s. The Actin genes of Pak-choi and Arabidopsis were used as internal control, and the relative expression levels were calculated utilizing the 2^−ΔΔCt method [27]. All primers used in this study are listed in Table A1.

3. Results

3.1. Cloning and Characteristic Analysis of BcCUC2

The full-length fragment of the BcCUC2 gene was amplified from Pak-choi cultivar ‘001’ using primer pair BcCUC2-F and BcCUC2-R. The BcCUC2 protein is an unstable and hydrophilic protein (GRAVY = −0.567), encoding 367 amino acid residues; the theoretical isoelectric point (pI) is 8.47, and the molecular weight is 40.97 KDa via Expasy online prediction. The BcCUC2 protein has a typical conserved NAC domain at the N-terminal (18–144 amino acid sites) through conserved domain search analysis in NCBI, and it belongs to the NAC family of plant-specific transcription factors. Meanwhile, multiple sequence alignment analysis revealed that the CUC protein sequence of different species shares a highly conserved NAC domain at the N-terminal, while the C-terminal is a transcriptional activation region with abundant variation, reflecting the diversity of species, which is also a general structural feature of NAC transcription factors (Figure 1A). Furthermore, multiple sequence alignment implied that the BcCUC2 protein shares 38.61% and 48.5% identity with OsNAM from rice and SlNAM from tomato, while sharing 81.38% and 74.73% identity with AtCUC2 from Arabidopsis and ChCUC2 from Cardamine hirsuta, suggesting the conservation of Cruciferae species in evolution. Meanwhile, the MEME analysis indicated that motifs 1/2/3/6 were extremely conserved sequences in all species, while motifs 7/9/10 were CUC2-specific sequences of Cruciferae plants (Figure 1B). Additionally, the secondary structure analysis showed that the BcCUC2 protein was mainly composed of α helices, random coils, and extended strands (Figure A1).

3.2. Phylogenetic Tree Analysis of BcCUC2

To explore the phylogenetic relationship of CUC genes, an unrooted phylogenetic tree was constructed using MEGA 7.0 software with the neighbor-joining (NJ) method. As shown in the phylogenetic tree (Figure 2), the CUC genes were firstly divided into two clades, the NAM/CUC1/CUC2 clade and the CUC3 clade. Meanwhile, the CUC genes from dicots (A. thaliana, C. hirsuta, S. lycopersicum, V. vinifera, B. oleracea, and Pak-choi) were clustered together and separated from monocots (O. sativa and Z. mays) into two clades, and the dicot–monocot split occurred 150 million years ago (Mya) [28]. Furthermore, BcCUC2 has a higher similarity with the B. oleracea CUC2 gene (Brassica), followed by the A. thaliana and C. hirsuta CUC2 gene (Cruciferae), and Brassica rapa diverged from A. thaliana and B. oleracea at 20 Mya and 8 Mya, respectively [29,30]. The result indicated that the molecular evolutionary relationship of the CUC2 protein between Pak-choi and seven other species is basically consistent with the genetic relationship.

3.3. Expression Pattern Analysis of BcCUC2 in Pak-choi

The NAM/CUC3 subfamily plays an important role in the formation of shoot meristem and boundary, leaf margin serration, compound leaf, and axillary meristem (lateral branch) [4,13,14,15]. To mirror the spatiotemporal expression patterns of BcCUC2 in Pak-choi, quantitative RT-PCR analysis was carried out. As shown in Figure 3, BcCUC2 was expressed relatively higher in petioles at the seedling stage, in stems and roots at the rosette stage, and in floral organs at the flowering and podding stages, while being weakly expressed in leaves and hypocotyls in cultivar ‘001’ with entire leaf margins.

In addition, to investigate the function of BcCUC2 in leaf morphology, we comprehensively examined the transcript levels of the BcCUC2 gene in cultivar ‘082’ with a serrate leaf margin. The result indicated that BcCUC2 has higher expression in leaves, roots, and hypocotyls at the seedling stage, stems at the rosette stage, and pods and floral organs at the flowering and podding stages. The transcript levels of BcCUC2 were significantly higher in the young leaves of cultivar ‘082’ than in cultivar ‘001’, while being decreased in mature leaves, suggesting a possibility that BcCUC2 has a role in the early steps of leaf serration. Therefore, we inferred that BcCUC2 may participate in leaf morphogenesis and flower development.

3.4. Expression Analysis of BcCUC2 Gene under Hormone Treatment

Several studies have shown that auxin and gibberellin have an important role in the regulation of leaf shape development [31,32,33]. To dissect the response of BcCUC2 to auxin and gibberellin, quantitative expression analysis was performed for the BcCUC2 gene under IAA and GA3 treatment. As shown in Figure 4, the BcCUC2 gene was highly expressed under exogenous IAA and GA3 treatment at the initial stage and peaked at the 3 h time point, demonstrating that the BcCUC2 gene may be regulated by IAA and GA3 through the means of promotion.

3.5. Subcellular Localization Analysis of BcCUC2

To confirm whether the BcCUC2 gene, as a putative transcription factor, is localized in the nucleus, the 35S:BcCUC2-GFP fusion protein was constructed and used for transient transformation in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated transfection methodology. The laser confocal scanning microscope images of tobacco epidermal cells showed that the green fluorescence of the 35S:BcCUC2-GFP fusion protein was mainly distributed in the cell nucleus, while the empty vector (35S:GFP protein, negative control) was expressed in the whole cell, illustrating that BcCUC2 is a nuclear-localized protein (Figure 5).

3.6. Ectopic Expression of BcCUC2 Caused Leaf Margin Serration and Increased Lateral Branches in Arabidopsis

In order to explore the potential function of BcCUC2, we first transformed the BcCUC2 gene into Arabidopsis. The positive transgenic plants were authenticated by PCR amplification using specific primers and GUS staining, and the qRT-PCR was used to check the gene expression level for further verification (Figure A2). Finally, thirteen transgenic lines (termed as OE1-OE13) were obtained, and four lines (OE5, OE6, OE9, OE11) with relatively higher expression levels of BcCUC2 were selected for further research (Figure 6B). We observed that leaf margins were modified when the BcCUC2 gene was overexpressed in Arabidopsis. The phenotype of leaf margin serration was significantly presented in transgenic Arabidopsis plants expressing the BcCUC2 gene, which were clearly distinct from the leaves of wild-type Arabidopsis (Figure 6C). In addition, the ectopic expression lines with the BcCUC2 gene displayed a significant increase in the number of inflorescence stems, flowers, and siliques in comparison with the wild type (Figure 6D). These results demonstrated that BcCUC2 plays critical roles in the formation of leaf margin serration and the development of flower and lateral branches, which is consistent with the high expression abundance in stem and floral organs.

4. Discussion

Previous studies have shown that the serrate morphology of deep-lobed leaf blades is conducive to heat dissipation and defense against high-temperature burns, thereby improving the survival probability of plants [34]. The hydraulic efficiency of the deep-lobed leaves is high, which enhances their adaptability in arid environments [35]. Meanwhile, deep-lobed morphology makes the leaves have a larger specific leaf area in space and thus has stronger competition for light resources and higher photosynthesis efficiency than entire leaves [36]. In addition to its adaptive ability in diverse conditions, the degree of dissection of the leaf margins may add to the ornamental value of leaves in different species. The functional analysis of genes associated with leaf serration development contributes to improving the leaf margin traits using biotechnological methods. Leaf shape is a major agronomic trait, and leaf margins can be lobed, serrate, or entire in plants. Pak-choi, as an economically leafy vegetable, has important edible value and is widely cultivated in Asia. However, the candidate genes and molecular mechanism of serrate leaf margins remain to be fully elucidated.

The NAM/CUC subfamily, including CUC1, CUC2, and CUC3, belongs to the NAC (NAM, ATAF1/2, and CUC) transcription factors family, playing a central and redundant role in plant organ development and organ boundary formation, e.g., in floral organs (gynoecium and ovules), leaf serration, and primary and axillary shoots (reviewed in [37]). So far, CUC genes have been widely studied in several species. For example, in Arabidopsis, CUC1 and CUC2 are involved in the formation of carpel margin meristem by controlling shoot meristem activity [38] and regulating carpel margin development through interacting with SPATULA (SPT) [39], and CUC3 has significant roles in regulating organ boundary formation and postembryonic shoot meristem [16]. In addition, CUC2 participates in ovule primordia formation via direct interaction with the DELLA protein GAI [33]. The balance between miR164a and CUC2 is responsible for the extent of leaf serrations [4]. In strawberry, the miR164-CUC2 regulatory module plays conserved and novel roles in specifying leaf and floral organ morphology [40]. In tomato, the GOBLET (GOB) gene, homologous to the CUC2 gene, plays important roles in regulating fruit shape and the complexity of compound leaves [24]. In Liriodendron chinense, the LcCUC2-like (LcCUC2L) gene, homologous to AtCUC2 in sequence, has an important role in controlling cotyledon development and rosette leaf number [32]. According to the above reports, the CUC2 gene has prominent roles in organ boundary formation and organ number.

The functional importance and relationship of the CUC2 gene in Pak-choi, however, is poorly understood. Here, we performed systematic analysis of the characterization and function of BcCUC2 using bioinformatics tools, real-time PCR, and ectopic expression in Arabidopsis. The phylogenetic tree displayed that the BcCUC2 gene was clustered into the NAM/CUC1/CUC2 clade and close to the B. oleracea BoCUC2 and A. thaliana AtCUC2, which all belong to Cruciferae, consistent with their evolutionary history. The tissue-specific expression analysis showed that BcCUC2 has relatively higher transcript levels in stem and floral organs and differential expression in leaves with smooth margins or serrate margins, indicating that BcCUC2 may be involved in the proper control of leaf morphology and the development of floral organs. In addition, the expression abundance of the BcCUC2 gene was observably greater in young leaves with serrate margins (cultivar ‘082’) than smooth margins (cultivar ‘001’), while being markedly decreased in mature leaves, possibly implying that BcCUC2 participated in the formation of serrate margins in early leaf development. In the Pro35:BcCUC2 lines, the transcript levels of BcCUC2 were significantly up-regulated and the phenotype of leaf serration and increased inflorescence stems, flowers, and siliques were found, supporting our hypothesis that BcCUC2 is involved in modulating the development of floral organs and leaf morphology. It is interesting that the phenotype of leaf serration was significant at the seedling stage, while being diminished at mature and flower stages in transgenic lines, which is consistent with the spatiotemporal expression profile of BcCUC2. On the other hand, given that the overexpression of BcCUC2 in Arabidopsis resulted in non-significant enhancement of serration, it is possible that BcCUC2 may be less functional in Arabidopsis than in Pak-choi, or line ‘082’ may have another potential gene to enhance serration.

In plants, phytohormones have a crucial role in the growth and development and cell morphogenesis of diverse tissues. Leaf shape traits are regulated by an intricate regulatory network concerning transcription factors and hormone signaling [4]. For instance, PIN1, as an auxin efflux carrier, plays an essential role in auxin distribution in the placenta, and the expression of PIN1 was up-regulated by CUC genes, which have crucial roles in leaf shape [31]. In L. chinense, LcCUC2L regulates leaf development by regulating the auxin content [32]. Furthermore, the ovule primordia formation was modulated by CUC2’s direct interaction with the gibberellin signaling protein GAI [33]. To investigate the response of BcCUC2 to auxin and gibberellin, the expression patterns of BcCUC2 under IAA and GA3 treatment were analyzed. The result showed that the transcript levels of BcCUC2 were significantly increased at 1–3 h after treatment, indicating that BcCUC2 responds to auxin and gibberellin. Based on the above results, we speculated that BcCUC2 is involved in the formation of serrate leaf margins through the mediation of auxin or gibberellin.

Although the expression patterns and potential function of BcCUC2 have been preliminarily determined based on real-time PCR and overexpression assay, the specific reason for the difference in leaf morphology and BcCUC2 expression profiles between the lines ‘001’ and ‘082’ remains unclear. Several studies have shown that miR164 level or auxin levels have an essential role in the development of leaf morphology [4,31,32,40]. Thus, the promoter sequence of BcCUC2, miR164 level, or auxin levels between the lines ‘001’ and ‘082’ will be analyzed to further elucidate the molecular mechanism of leaf morphology and BcCUC2 expression difference between the lines.

5. Conclusions

In conclusion, we have isolated a CUP-SHAPED COTYLEDON (CUC) gene in Pak-choi named BcCUC2. The phylogenetic reconstruction displayed that the BcCUC2 gene is clustered into the CUC1/CUC2 clade and is close to the B. oleracea BoCUC2 and A. thaliana AtCUC2, which all belong to Cruciferae. The expression patterns of the BcCUC2 gene were significantly different in leaves and petioles between cultivar ‘001’ with entire leaf margins and ‘082’ with serrate leaf margins, while similar in floral organs. In addition, the transcript abundance of BcCUC2 was significantly induced by IAA and GA3 treatment. The heterologous expression of BcCUC2 resulted in leaves with serrate margins and more inflorescence stems.