1. Introduction
Ornamental kale (
Brassica oleracea var.
acephala) is an ideal landscape plant suitable for flowerbeds and flower seas by virtue of its cold tolerance and attractive leaf coloration. It is becoming increasingly renowned for a wide range of color patterns in the inner leaves under lower temperatures, mainly including red, purple, pink and white. Currently, most research focuses on the color variation and the genetic mechanism of red and purple inner leaves [
1,
2,
3,
4,
5], while little is known about the occurrence of variegated leaves in kale [
6]. Leaf variegation refers to different-colored patches developed on the same leaf tissue, usually consisting of green and white (or yellow) sectors [
7]. In our previous study, a variegated leaf kale showing green margins and white centers, which looks like a lotus and is of unique ornamental characteristics, was created by isolated microspore culture. Although the color transition of kale leaves from green to white could occur naturally, accompanied by some variegated leaves produced, this phenotype would fade away after a brief period. Therefore, such a distinctive and long-lasting green-white variegated leaf mutant is of great value for cultivar breeding and product application in ornamental kale, and provides a favorable tool for dissecting the formation mechanism underlying leaf variegation pattern.
For the variegated leaves, the green sectors possess morphologically normal chloroplasts, whereas the lighter colored sectors mostly contain aberrant chloroplasts, deficient in chlorophyll and carotenoid pigment contents [
8]. Efforts have been made to explore the genetic model of variegated leaves since the early 20th century, while most of the traits in earlier research did not follow Mendelian inheritance, whose phenotypic formations were closely associated with plastid dysfunction. In recent years, with extensive investigations conducted into variegated leaf mutants of the model plant
Arabidopsis thaliana, many nuclear genes modulating leaf variegation have been characterized.
VARIEGATED 2 and
VARIEGATED 3 determine the variegated leaves formation through regulating chloroplast development [
9,
10], while
IMMUTANS, MUTATOR,
CLOROPLASTOS ALTERADOS,
ATASE 2-DEFICIENT,
GERANYLGERANYL DIPHOSPHATE SYNTHASE (
GGPS)
1 and
CRUMPLED LEAF (
CRL) control leaf variegation through regulating chlorophyll and biosynthesis [
11,
12,
13,
14,
15,
16].
Among these mutants, two representative mutants
immutans (
im) and
variegated 2 (
var2) were characterized more fully and provided a theoretical basis for dissecting variegated leaf formation. The
im mutant was first found by Rédei (1963), in which green sectors on the leaves contain normal cell wall and chloroplast, whereas the white sectors contain vacuolated plastids and abnormal lamellar structure [
17,
18]. Wu et al., (1999) mapped the recessive nuclear gene controlling this variegated leaf trait on the chromosome 4 of
Arabidopsis [
19]. IM protein serves as a plastid terminal oxidase regulating electron transport and redox, deficient in which would result in the formation of reactive oxygen, in turn leading to photo-oxidized plastids in the white sectors containing lower level of carotenoids and higher level of carotenoid precursor phytoene [
11,
17,
19,
20]. Another variegation-related mutant
var2 was isolated by Martínez-Zapater (1993), which has yellow-green variegated leaves that were susceptible to light and temperature, and was controlled by a pair of recessive nuclear genes [
21,
22]. The candidate gene
FtsH, belonging to the AAA (ATPase associated with various cellular activities) ATPase superfamily, which were anchored on the transmembrane domain in thylakoids. Mutated
FtsH led to abnormal thylakoid function, and affected the chloroplast development. Based on previous studies and these typical mutants, Yu et al., (2007) tentatively summarized seven types of molecular mechanisms underlying variegated leaf formation: genotype chimerism, transposon insertion, RNA silencing, plastome mutation, nuclear gene variation, mitochondrial genome mutation and plastid–nucleus incompatibility [
23]. Moreover, three classic hypothesis models of variegated leaf formation have been proposed, namely, the photo-oxidation hypothesis, the FtsH complex threshold hypothesis and the chloroplast development and homeostasis hypothesis [
23]. These results lay a solid foundation for uncovering the molecular mechanism of variegation formation throughout the plant kingdom.
The occurrence of leaf variegation in kale renders it with richer color patterns and ornamental characteristics, which would attract more interest, so it is necessary to explore the formation mechanism behind it for a better application. However, so far, there is no direct report on the molecular genetic mechanism of variegation formation in kale. In the present study, we characterized a novel variegated leaf mutant, the ‘JC007-2B’, appearing a peculiar green-white leaf variegation. Detailed morphological observation and physiological assays at different development stages revealed that chloroplast development and photosynthetic pigment contents were abnormal in the white sectors. Genetic analyses suggested that the leaf variegation was controlled by a dominant nuclear gene, BoVl. BSR, SSR and Indel markers were applied to conduct the fine mapping of the candidate gene. Sequence alteration with consensus detected in multiple variegated plants of two base substitutions and a base insertion on exon 2 of Bo3g002080, along with its distinctly differential high expression levels, further confirmed that Bo3g002080 was the candidate gene for variegated leaf phenotype. Bo3g002080 is homologous to Arabidopsis MED4 encoding a hydroxyproline-rich glycoprotein family protein, but the function of MED4 in affecting chloroplast development or determining pigment distribution has not been reported yet. To gain insight into the molecular mechanism underlying leaf variegation under the control of BoVl, we compared the transcriptomes of the green and white sectors in ‘JC007-2B’ and analyzed the DEGs, among which the related pathways of photosynthesis, chloroplast development and energy metabolism were significantly enriched in the white sectors, and relevant genes were almost down-regulated. These results contribute to uncovering the molecular mechanism of variegated leaf formation, and provides new insights for germplasm innovation in ornamental kale breeding.
3. Discussion
Leaf variegation can attach rare appearances and unusual esthetic value to ornamental plants. In recent years, ornamental kale has been widely introduced as a decorative landscape plant around the world because of its attractive leaf color pattern and low-temperature tolerance. In this study, the variegated leaf mutant ‘JC007-2B’ (
Figure 1a) and the
BoVl loci are of great value for breeding
B. oleracea new varieties. The variegated leaves with white centers and green edges in ‘JC007-2B’ were gradually developed along with the daily mean temperature dropping to roughly 12 °C. Notably, as a novel type of ornamental phenotype in kale, the variegated leaf phenotype in ‘JC007-2B’ would not disappear as the plant growth, which is distinctive from the intermediate phenotype in common white-leaf kale (
Figure 1b). Such kinds of mutants in which variegated leaves can be stably inherited and preserved make it possible for artificial selection and the development of new ornamental cultivars. Accordingly, the coloration mechanisms of variegated leaves mutants aroused more interests and have been studied at multiple levels in multiple species [
24,
25,
26,
27].
The color variations in green-white variegated leaves were almost accompanied by prominent characteristics of the alterations in chlorophyll and carotenoid contents as well as the abnormality in chloroplast development of the white sectors. Leaf-variegated mutants are thereby regarded as ideal materials for investigating the chloroplast function. Likewise, we determined total chlorophyll, chlorophyll
a, chlorophyll
b and carotenoid contents at three development stages, respectively. It was found that these pigment contents reached the maximum at green leaf stage (S1) and the lowest at albino stage (S2), as well as significantly lower in the white sectors (S3_C) than the green sectors (S3_S) at variegated leaf stage (
Figure 2 and
Figure 3). By observing the ultrastructure of chloroplasts in the green and white areas of variegated leaf, there were abnormal chloroplasts about to collapse in the S3_C rather than structurally clear chloroplasts with highly stacked thylakoids in the S3_S (
Figure 4). Similarly, aberrant chloroplasts with loose grana lamellae and indistinct thylakoid membrane structure were also observed in other green-white variegated leaf mutants including
Csvl of cucumber (
Cucumis sativus L.),
Hvcmf7 of barley (
Hordeum vulgare) and white sectors in variegated leaves of milky stripe fig (
Ficus microcarpa) [
28,
29,
30]. Furthermore, chloroplast is the main place for plant energy transformation, and most of the photosynthetic pigments exist in the thylakoid membrane absorbing light energy through the photosystem for photosynthesis [
11,
19,
20]. The white centers in S3 showed an increase in Chl
a/Chl
b ratio opposite to the decreasing trend in each single pigment, which suggested the Chl
b contents declined to a greater extent. Chl
a/Chl
b ratio can reflect the plant utilization rate of light energy, and we speculate that the white leaves might be subjected to a certain degree of light stress. The Chl
b is only present in the antenna complex and plays an indispensable role in affecting photosynthetic performance [
31]. Accordingly, the DEGs involved in photosynthesis-antenna proteins and photosynthesis were identified, most of which were up-regulated and expressed in the green sectors (S3_S). Based on the aforementioned findings, we speculated that the abnormal photosynthetic pigment contents and damaged chloroplast structure in the white centers led to the green-white appearance of variegated leaves in ‘JC007-2B’ at the physiological level.
Currently, relevant studies of variegated leaf formation in other species besides
Arabidopsis mainly focused on unravelling potential functional pathways and mutant analysis, whereas the genetic models accounting for the variegated leaf phenotype have not been fully identified. In this study, two mapping parents, variegated leaf ornamental kale ‘JC007-2B’ and green-leaf inbred line ‘BS’ of
Brassica oleracea, were used to map the candidate gene. It was proved that the variegated leaf trait was controlled by a dominant nuclear gene. Genetic mapping of the
BoVl locus was carried out by BSR seq and molecular markers, which was located in a 6.74 Kb interval of C03, flanked by Q3I-8 and Q3I-15. A total of three genes were detected in the interval. Through sequence alignment, two non-synonymous base substitutions and a base insertion were found in the exon 2 of
Bo3g002080 (
Figure 5), which occurs in the 132
nd amino acid, within the Vitamin-D-receptor-interacting Mediator subunit 4 conserved domain (
Figure S5a). The nucleotide mutation at the
BoVl locus shared a consistency among the variegated leaf kale lines, indicating that
Bo3g002080 was the most likely candidate gene for
BoVl (
Figure S4).
Bo3g002080 is homologous to
Arabidopsis MED4 encoding a hydroxyproline-rich glycoprotein family protein, which is a component of the middle module in the mediator, wherein seven proteins that are conserved across evolutionary time and eukaryotes constitute this module [
32,
33,
34,
35]. In yeast, MED4 serves as a hub and interacts with all of the other proteins in the middle module [
32]. Studies showed that the
Arabidopsis MED4 mutations exhibit the embryonic mortality phenotype, and MED4 can interact with three types of RNA polymerases [
36]. Up to now, there are few studies on the function of MED4, and whether MED4 affects chloroplast development remains to be studied. In the future, additional experiments, such as transgenic complementation test, CRISPR/Cas9 and RNAi, are needed to uncover the role of
BoVl (
Bo3g002080) in leaf variegation.
Analysis of the differentially expressed genes and related gene regulatory network in variegated leaf formation will facilitate our understanding of the molecular genetic mechanism of kale color pattern and decipher the potential function of
BoVl. To date, a total of three classic hypotheses were proposed for explaining the intricate mechanism underlying leaf variegation. The first one is that the mutation of chlorophyll and carotenoid biosynthesis or related genes that lead to changes in pigments content, such as
MUTATOR,
CLOROPLASTOS ALTERADOS,
GGPS1 and
CRL mutants [
9,
11,
12,
13,
14,
15]; the second hypotheses is that the mutation of chloroplast developmental genes that lead to anormal chloroplast development therefore indirectly affects pigments synthesis, such as
VARIEGATED 2 (
var2) and
VARIEGATED 3 (
var3) mutants [
9,
10]; the third possibility is that photooxidation, which results from gene deletion or mutation during photosynthetic electron transport, will affect the equilibrium between electron transport and plastid quinone’s redox reaction, leading to defects in chloroplast development, similar to those seen in
im mutants [
11,
17]. Likewise, we also observed abnormal chloroplast development and decreased photosynthetic pigments content in the white sector of variegated leaves of ‘JC007-2B’. On the basis of these classic theories and the physiological assays, we put emphases on the photosynthesis, pigments biosynthesis and chloroplast-related pathways and DEGs in the comparative transcriptome analysis. Relevant DEGs related to carotenoid biosynthesis, chloroplast, thylakoid membrane, and photosynthesis were identified between the green margins (S3_S) and the white centers (S3_C) (
Figure 7 and
Figure 8). Thus, it was speculated that the blockage of carotenoid and chlorophyll biosynthesis resulted in the abnormality of chloroplast development, which ultimately induced the formation of variegated leaves.
Among these DEGs, we only found a gene,
ZDS, encoding a ζ-carotene desaturase in carotenoid biosynthesis. Carotenoids as a class of natural pigments, were involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid, and chloroplast biogenesis. In addition, carotenoids play an important role in photoprotection by protecting plants from these oxidative damages [
37]. The
Arabidopsis SPC1/ZDS gene mutation resulted in damaged carotenoid biosynthesis, decreased chlorophyll content, and abnormal chloroplast structure, with a bleached phenotype of leaves. Similarly to the
Arabidopsis im mutants, they were all subjected to different degrees of photooxidation [
20,
38]. Therefore, we speculated that the blockage of carotenoid biosynthesis and the decrease in contents might occur in the white region, which in turn could lead to a certain degree of photooxidation damage and abnormal chlorophyll synthesis, thereafter inducing abnormal chloroplast development. In addition, the expression of most DEGs involved in chloroplast thylakoid development and photosynthesis was decreased in S3_C. Gathering up these threads, a putative formation pattern of leaf variegation of ‘JC007-2B’ was proposed (
Figure 10), which could contribute to the understanding of potential genetic mechanisms and the regulatory network of
BoVl in leaf variegation of ornamental kale.
4. Materials and Methods
4.1. Plant Materials
The double-haploid ‘JC007-2B’ kale line was grown in the greenhouses at Shenyang Agricultural University (Shenyang, China). The kale exhibited a variegated leaf phenotype with white centers and green margins. Variegated phenotype during plant development were as follows: S1, leaves are green; S2, white appears at the leaf center, although the margins remain green when the plant undergoes a period of chilling temperature; and S3, the leaf center is white, while the margins are green (
Figure 1). To investigate the genetic mechanism of variegated leaf formation, we crossed ‘JC007-2B’ with a
Brassica oleracea inbred line ‘BS’ to produce F
1. The F
1 populations were selfed to generate F
2 populations, and they were backcrossed with two mapping parents to produce BC
1 lines, respectively. A total of 50 variegated leaf individuals and 20 green leaf individuals of F
2 populations were used for BSR-seq analysis and then another 1331 green leaf individuals from F
2 population were planted to fine map the
BoVl gene. All populations were grown on land at Shenyang Agriculture University, Shenyang, China.
The leaf color pattern was determined by visual inspection when the populations underwent a period of chilling temperature. Chi-square test was used for verifying the segregation rate.
4.2. Measurement of Chlorophyll and Carotenoid Content
Green and white sectors in variegated leaves were selected for the measurement of chlorophyll and carotenoid levels. Dry weight samples (20 mg) were soaked in 10 mL of 96% ethanol solution (
v/
v), at 25 °C, for 24 h. The samples were centrifuged for 30 s every 12 h [
39]. The absorbance was measured three times each at wavelengths of 649, 665, and 470 nm on an ultraviolet spectrophotometer (T6 New Century; Persee, Beijing, China). Total chlorophyll, chlorophyll
a, chlorophyll
b, and carotenoid contents were calculated using the following formulae:
4.3. Transmission Electron Microscopy
Leaves at three stages were cut into 1 mm
2 sections, fixed in glutaraldehyde solution (2.5% glutaraldehyde, 0.1 M Na
2HPO
4, 0.1 M NaH
2PO
4 [pH 7.0]), washed in 0.1 M phosphate buffer, fixed in 1% OsO
4 in the same phosphate buffer, and dehydrated in a graded series of acetone before embedding and polymerization in Epon 812. After ultrathin sectioning (LKB2088 type ultramicrotome; LKB Co., Bromma, Sweden), the samples were stained with uranyl acetate and lead citrate solutions and observed under a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan). Images were acquired at 30,000× magnification [
40].
4.4. BSR-Seq Analysis
Fifty variegated leaf individuals and fifty green leaf individuals of F2 populations were selected to construct two bulks, Var and CK, for BSR-seq analysis. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen), and were quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. The RNA with RIN value above 7 from fifty individual variegated leaves and fifty green leaves was mixed to construct two libraries, respectively. The next-generation sequencing library preparations were constructed according to the manufacturer’s protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) was used to perform the poly(A) mRNA isolation. The mRNA fragmentation and priming was performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First-strand cDNA and second-strand cDNA were synthesized using ProtoScript II Reverse Transcriptase and Second Strand Synthesis Enzyme Mix, respectively. Two libraries with different indices were multiplexed and loaded on an Illumina HiSeq X Ten instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). The sequences were processed and analyzed by GENEWIZ (Nanjing, Jiangsu, China).
Trimmomatic (v0.30) was used to obtain high quality clean data. Then, clean data were aligned to reference genome (EnsemblPlant, B. oleracea, v2.1) via software Hisat2 (v2.0.1). HTSeq (v0.6.1) estimated gene and isoform expression levels from the paired-end clean data. DESeq Bioconductor package, a model based on the negative binomial distribution, helps us to analysis differential expression genes (DEGs). Additionally, the threshold was set as log2 (fold change) > 1 and statistical significance (p < 0.05). Samtools v0.1.18 with command mpileup and Bcftools v0.1.19 were used to do SNV calling. Additionally, ED value was calculated based on an mpileup file, which generated by samtools v0.1.18.
4.5. Genomic DNA Extraction, PCR and Molecular Marker Development for Fine Mapping
Young leaf samples of green leaf individuals of F
2 populations and two parents were collected and frozen in liquid nitrogen. Genome DNAs were extracted from leaves from modified cetyltrimethylammonium bromide (CTAB) method [
40]. The SSR loci were detected via SSR Hunter based on the
Brassica oleracea genome, while the Indel loci were identified according to the results of genome re-sequencing and BSR-seq analysis. Both two types of markers were designed by Primer Premier 5.0. A total of 77 SSR markers were used to confirm the mapping region, then 15 Indel markers were used to narrow the region. Map distances were calculated referring to Kosambi’s (2011) mapping function [
41].
The PCR reaction contains ~80 ng genome DNA, 0.5 μM of each primer, 200 μM dNTPs, 1× reaction buffer and 0.5 U Taq DNA polymerase (Tiangen, Beijing, China). The PCR program was as follows: 95 °C for 5 min, 35 cycles of 95 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR products were separated on a 6% polyacrylamide gel by electrophoresis, and electrophoresis at 200 V for 1.5 h. The gels were stained in 0.1% AgNO3 solution and then were transferred into a developing solution (1.5% sodium hydroxide, 0.4% formaldehyde).
4.6. Gene Annotation and Candidate Gene Identification
Candidate gene prediction was based on the
Brassica oleracea genome database (
ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea, accessed on 1 October 2022). The functions of the genes in the interval were analyzed using the BLASTP tool from TAIR. The gene and promoter sequence of the genes were amplified from two parents genomic DNA with PrimeSTAR
®vMax DNA Polymerase (TAKARA, Dalian, China). All primers used for sequencing are listed in
Supplementary Materials Table S3. The PCR products were purified using the Gel Extraction Kit (CWBIO, Beijing, China), introduced into the PMD 18-T Vector (Takara, Dalian, China), and transformed into TOP10 competent cells (CWBIO). The recombinant plasmids were sequenced by Genewiz (Tianjin, China) and sequences were aligned using DNAMAN 6 (
https://www.lynnon.com/, accessed on 1 October 2022).
4.7. Phylogenetic Analysis and Alignment of BoVl and Its Homologous Proteins
The amino acid sequences of
BoMED4 and its homologous protein in different species were downloaded from the NCBI database. Protein sequence alignments were performed with Clustal W, and a neighbor-joining tree was constructed with 1000 replications. The proteins sequences used in the neighbor-joining tree were from the following species: the amino acid sequence, isoelectric point (PI) and molecular weight (MW) of the gene protein were estimated by Edit-seq; three candidate genes were sequenced by DNAman; the conserved domains of candidate genes were analyzed using Hmmer (
http://plants.ensembl.org/hmmer/index.html/, accessed on 1 October 2022). We used: smart (
http://smart.embl-heidelberg.de/, accessed on 1 October 2022) to predict the amino acid sequence of the candidate gene and the conserved domain of the candidate gene; sopma (
http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl, accessed on 1 October 2022) to predict the secondary structure of candidate genes; and Protscale (
https://web.expasy.org/protscale/, accessed on 1 October 2022) to hydrophobicity prediction of candidate genes. The phylogenetic tree of reference genes
Bo3g002080, ‘BS’ and ‘JC007-2B’ in kale and other species was constructed using MEGA11.
4.8. RNA-Seq Analysis
Total RNA was extracted using a Total RNA Purification kit (LC Science, Houston, TX, USA; TRK1001) according to the manufacturer’s protocol. RNA quantity and purity were evaluated using a Bioanalyzer 2100 and RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara, CA, USA), with an RNA integrity number > 7.0. Poly(A) mRNA was isolated from 10 μg RNA using the poly-T oligo method (Invitrogen, Carlsbad, CA, USA). Cleaved RNA fragments were used to generate a cDNA library using an mRNA-Seq Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Samples from S3 (leaf margin and center) with three independent biological replicates were used to construct 6 cDNA libraries designated as S3_S_1, S3_S_2, S3_S_3, S3_C_1, S3_C_2, and S3_C_3. Paired-end sequencing was carried out using the Illumina HiSeq 4000 system (LC Sciences, Hangzhou, China) according to the manufacturer’s protocol. After removing reads of low quality, those that remained were mapped to the
B. oleracea reference genome (
ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/, accessed on 1 October 2022) using the HISAT package, allowing for a maximum of two mismatches and multiple alignments per read (up to 20 by default).
Mapped reads of each sample were assembled using StringTie. The final transcriptome was generated by merging all transcriptomes using Perl scripts. mRNA expression levels were calculated by the fragments per kilobase million (FPKM) method using StringTie (
https://ccb.jhu.edu/software/stringtie/, accessed on 1 October 2022), whereas differentially expressed mRNAs were identified based on log
2 (fold change) > 1 and statistical significance (
p < 0.05) using the R package Ballgown (R Foundation for Statistical Computing, Vienna, Austria). All DEGs were mapped to GO terms and KEGG pathways. Significantly enriched GO terms and KEGG pathways in DEGs were identified by hypergeometric tests with Bonferroni correction;
p ≤ 0.05 was defined as the threshold.
The RNA samples used for RNA-seq were also used for qRT-PCR analysis. The first-strand cDNA was synthesized using a cDNA synthesis kit (Vazyme, Nanjing, China). Nine DEGs involved in chloroplast development and photosynthesis and three candidate genes in the region were selected for evaluation. Gene-specific primers were designed with Primer Premier Software v.5.0 (Premier Biosoft, Palo Alto, CA, USA) (
Table S4), while the actin gene was used as an internal control. An amount of 2 μL cDNA (1:50 dilution), 25 μL of 2× Ultra SYBR Mix (CWBIO, Beijing, China), and 1 µL of each primer (100 nM final concentration) formed the 50-μL reaction, followed by the program of 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A melting curve analysis (55–95 °C) was performed at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s. Experiments were performed using a QuantStudio 6 PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with three independent biological replicates. Relative expression level was calculated with the 2
−ΔΔCt method [
42].
4.9. Statistical Analysis
Part of the statistical analyses in this study were performed using Student’s t-test. Values were considered as significantly different with p < 0.05 (*). For the rest, analysis of variance (followed by Duncan’s test) was used to test differences between samples, with p < 0.05 considered statistically significant.