3.1. Pigment levels and rosmarinic acid contents in four perilla cultivars
The transplanted seedlings grew well in the experimental field and the front and back leave colors of ‘94’ and ‘M357’ kept steady with green and purple color, respectively, in all the growth stage (Fig. 1). The cultivar ‘272’ and ‘103’ underwent quick color changes, and then the leaf colors were steady in all growth stage (Fig. 1). Finally, the back of mature leaf in ‘272’ and ‘103’ turned light and dark purple, respectively. At same time, the front leaf color of ‘272’ was still green, and the front leaf color was green with light purplish vein at the bottom of leaf in ‘103’.
For chlorophyll and carotenoid contents, it was found notable difference among four cultivars. The total chlorophyll and carotenoid contents of 357-PP were highest among the four cultivars (Fig. 2A). For anthocyanin, the largest content was also in 357-PP, followed by 103-GPP, and the low contents were in 94-GG and 272-GLP (Fig. 2B). The anthocyanin content was around 20 fold higher in 357-PP than that of in 272-GLP (Fig. 2B). The total content of flavonoid was also high in 357-PP (Fig. 2C).
3.2. Analysis of photosynthetic parameters in four perilla cultivars
Four photosynthetic parameters were shown in Fig. 3. For the net photosynthetic rate, 357-PP had highest level, followed by 103-GPP, and the lowest level was in 272-GLP. Concerning the Gs, it was closely matched the Pn curve and the highest level was in 357-PP. Similarly, the Tr and Gs curves had the same trends of Pn. According to the chlorophyll contents of four cultivars, perilla cultivar with higher chlorophyll contents has higher photosynthetic rate.
3.3. Identification of leave color in four perilla cultivars
The chromatic parameters of the front and back leaves of each samples were recorded using the CIE L*a*b* color system (Table 1). The values of L*a*b* of original leaf colors of 272-GLP, and 103-GPP were similar with those in 94-GG (Table 1). After leaf color steady, the values of a* b* in the front leaf colors were ranged from − 15.81 to 4.79, and 4.55 to 22.77, respectively, while they were ranged from − 14.12 to 8.76, and 2.19 to 22.16, respectively, in the back leaf colors (Table 1). The a* represents red and green color. The values of the chromatic parameters a* in 357-PP was distinct from those in other three cultivars, because it showed positive a* values (mean 4.79) in the front leaf and lowest (mean 2.19) among four perilla cultivars in back leaves. The L* values (mean 40.28, 48.86 in front and back, respectively) of the front and back leaves were high in green leaves of ‘94’, and low in front leave of ‘M357’ (mean 25.56) and back leave of ‘No.101’ (mean 27.21) and ‘M357’ (28.45). These samples show no significant differences in hue (Table 1). The lowest chroma value (C*) was in 6.61. As described, these measured results were consistent with the leaf pigmentation appearance.
Table 1
The CIE L*a*b*chromatic parameters in front and back leaf of four perilla cultivars.
Cultivars
|
Front leaf
|
Back leaf
|
L*
|
a*
|
b*
|
C*
|
hc
|
L*
|
a*
|
b*
|
C*
|
hc
|
94-GG
|
40.28
|
-15.81
|
22.77
|
27.72
|
-0.96
|
48.86
|
-14.12
|
22.16
|
26.28
|
-1.00
|
272-GLP
|
36.43
|
-13.58
|
20.34
|
24.46
|
-0.98
|
36.42
|
-1.27
|
11.12
|
11.19
|
-1.46
|
103-GPP
|
35.23
|
-11.93
|
18.93
|
22.38
|
-1.01
|
27.21
|
8.43
|
3.16
|
9.00
|
0.36
|
357-PP
|
25.56
|
4.79
|
4.55
|
6.61
|
0.76
|
28.45
|
8.76
|
2.19
|
9.03
|
0.24
|
272-orignal
|
39.86
|
-17.06
|
26.65
|
31.64
|
-1.00
|
45.59
|
-12.97
|
20.57
|
24.32
|
-1.01
|
272-middle
|
37.12
|
-14.02
|
22.22
|
26.27
|
-1.01
|
40.25
|
-6.97
|
15.33
|
16.84
|
-1.14
|
103-orginal
|
40.32
|
-13.24
|
21.07
|
24.88
|
-1.01
|
47.64
|
-11.30
|
19.78
|
22.78
|
-1.05
|
103-middle
|
34.09
|
-11.82
|
16.58
|
20.36
|
-0.95
|
34.90
|
2.35
|
7.07
|
7.45
|
1.25
|
The correlative analysis between pigment contents and the L*a*b* values in front leaf showed that chlorophyll, carotenoid, anthocyanin, and flavonoid were positive related to a* value, and they together determined purple leaf color formation (Table 2). In back leaf, only anthocyanin and flavonoid has positive correlative relationship with a* value (Table 2). It was indicated that back leaf color change was controlled by anthocyanin.
Table 2
The correlative analysis between different pigment contents and L*a*b* values of front and back leaf.
Pigment
|
L* (front)
|
a* (front)
|
b* (front)
|
L* (back)
|
a* (back)
|
b* (back)
|
chlorophyll
|
-0.562
|
0.593
|
− .656*
|
− .735**
|
.800**
|
− .714*
|
carotenoid
|
− .499*
|
.616**
|
− .559*
|
-0.101
|
0.202
|
-0.180
|
anthocyanin
|
− .892**
|
.889**
|
− .889**
|
-0.471
|
0.505
|
-0.520
|
flavonoid
|
− .525*
|
.609**
|
− .548*
|
-0.145
|
0.183
|
-0.215
|
Tri-dimenstional graphics allowed to evidence that separation among the four leaf color types. The front leaf color analysis showed that 357-PP in one room, while the other cultivars in the same room. The original color of 272-GLP and 103-GPP and 94-GG was close in the same room, while the color of their middle stage was close to their steady color (Fig. 4A). For the back leaf color analysis, purple leaf of 103-GPP and 357-PP were in the same room and steady color of 272-GLP and color of middle stage in 103-GPP was in other room (Fig. 4B). As a whole, for 272-GLP and 103-GPP, the front leaf color were relatively steady, and their back leaf color were gradually change during growth and steady at last (Fig. 4).
3.4. Illumina sequencing and de novo transcriptome assembly
In this work, 51.43 Gb nucleotides were produced by Illumina sequencing BGISEQ platform. After the removal of adapter sequences, ambiguous reads and low quality reads, total of 42.86 Gb clean reads were assembled into 128,041 unigenes, with average sequence length of 1,488 bp (Table S2). The N50 length, N90 length, and GC percentage of assembled unigenes were 2,229 bp, 807 bp, and 40.93%, respectively (Table S2). The top three sequence length distribution and percentage of the unigenes were 200–300 bp (15.04%), 300–400 bp (6.74%), and 400–500 bp (4.57%), respectively (Fig. S1A). A total of 13,714 unigenes were longer than 3000 bp (10.71%) (Fig. S1A). These results showed that the quality of sequencing data was good. BUSCO quality evaluation also showed assembled unigenes were complete and reliable, and complete(C) and single-copy(S) of all unigenes arrived at 96 (Fig. S1B). A total of 78,105 protein coding sequences (CDS) with 84,896,910 bp total length were detected using Transdecoder, and the 300–400 bp CDS (11.96%) were highly aligned (Fig. S2, Table S3). The maximum CDS length reached to 15,303 bp, and a total of 2,201 CDS were longer than 3000 bp (2.82%) (Table S3).
3.5. Functional annotation of perilla leaf transcriptomes
The assembled unigenes were annotated to seven public databases, including nonredundant protein sequence database (Nr), nucleotide sequence database (Nt), Swiss-prot (Swissprot), Kyoto encyclopedia of genes and genomes (KEGG), clusters of orthologous groups of proteins (KOG/COG), Pfam, and gene ontology (GO). In total, 98,086 unigenes (76.61%) could be annotated in at least on database, while there were 37,219 unigenes (29.07%) successfully annotated in all seven databases (Fig. 5; Table 3). The venn diagram of Nr, Nt, Swissprot, KEGG, and GO database showed that 44,972 unigenes were annotated in all the five public databases (Fig. 5A). The species showed the best BlastX matches were Sesamum indicum which took up 45.39%, the second- and third-ranked species Handroanthus impetiginosus and Erythranthe guttata was contributed to 22.38%, and 12.87%, respectively (Fig. 5B). For GO assignments, 69,508 unigenes matched to Nr database were further divided into 38 major functional groups. Of all the GO term categories, binding (34,680), membrane part (20,741), and cellular process (14,769) were the highest ranking in the three GO categories molecular function, cellular component, and biological process, respectively (Fig. 5C). A total of 74,907 unigenes were mapped to KEGG pathways, and global and overview maps (15,265, 20.38%), carbohydrate metabolism (6,206, 8.28%), and translation (5,472, 7.31%) were occupied the top three ranking (Fig. 5D). The 73,203 unigenes were classified into 25 KOG classifications. General function prediction only (15,060, 20.57%) had the most representation, followed by signal transduction mechanisms (9,836, 13.44%) and function unknown (6,570, 8.98%) (Fig. 5E).
Table 3
The annotations of perilla leaf unigenes against the seven public databases.
Values
|
Total
|
NR
|
NT
|
SwissProt
|
KEGG
|
KOG
|
Pfam
|
GO
|
Intersection
|
Overall
|
Number
|
128,041
|
93,319
|
78,001
|
70,961
|
74,907
|
73,203
|
71,543
|
69,508
|
37,219
|
98,086
|
Percentage
|
100%
|
72.88%
|
60.92%
|
55.42%
|
58.50%
|
57.17%
|
55.88%
|
54.29%
|
29.07%
|
76.61%
|
Gene structure analysis suggested that 3,577 simple sequence repeats (SSRs) were detected in 26,807 unigenes (Fig. S3A). In addition, 3,577 transcript factors were predicted in this study (Fig. S3B) Principle components analysis (PCA) showed that four cultivars were clustered different positions and purple leaf ‘M357’ was apart from the other three perilla cultivars (Fig. S4A). Gene expression profiles of accumulative bar diagram composite column-diagram showed that many unigenes (FPKM values = 1–10) were distributed in M357-PP (Fig. S4B). These results indicated that transcriptome sequencing could be used to identify the leave color regulated genes in perilla. A total of 78,700 unigenes were expressed in all the four cultivars were seen in Fig. S4C.
3.6. Analysis of differentially expressed genes (DEGs) with different pairwise comparisons
In order to explore the differentially expressed genes (DEGs) among four perilla cultivars with different leaf colors, the gene expression levels between each pairwise comparison were studied, and then DEGs were identified by different pairwise comparisons (Fig. S5; Fig. S6). The highest number of DEGs was appeared in 272-GLP vs 357-PP (35,381) among six pairwise comparisons, with 17,824 and 17,577 unigenes up- and down-regulated, respectively (Fig. S5). In contrast, the lowest number of DEGs was observed in 272-GLP vs 103-GPP (19,376), with 13,369 and 12,088 unigenes up- and down-regulated, respectively (Fig. S5). The venn diagrams of any three pairwise comparison showed that highest number of DEGs (9,670) were common to the 357-PP vs 94-GG, 357-PP vs 272-GLP and 357-PP vs 103-GPP comparisons, and the lowest number of DEGs were common in 94-GG vs 103-GPP, 103-GPP vs 272-GLP and 94-GG vs 272-GLP comparisons (Fig. S6A-D). Furthermore, much more DEGs (1,795) were observed in many purple leaf attended pairwise comparisons including 357-PP vs 94-GG, 357-PP vs 103-GPP, 103-GPP vs 272-GLP and 357-PP vs 272-GLP than those pairwise comparisons with many green leaf attended comparisons, ‘94-GG vs 272-GLP’, ‘94-GG vs 357-PP’, ‘94-GG vs 103-GPP’ and 103-GPP vs 272-GLP (Fig. S6E-F). A total of 611 DEGs were common to the 357-PP vs 94-GG, 357-PP vs 103-GPP, 357-PP vs 272-GLP, 94-GG vs 272-GLP, and 94-GG vs 103-GPP comparisons (Fig. S6G). These results suggested that the genes regulated green and purple color had more significant difference. Notably, many DEGs were appeared in the pairwise comparisons contained purple leaf of 357-PP.
3.7. GO functional and KEGG pathway enrichment of DEGs and their expression patterns analysis
GO functional enrichment analysis of DEGs revealed that of top three main enrichment of the GO among five comparisons viz. 357-PP vs 94-GG, 357-PP vs 103-GPP, 357-PP vs 272-GLP, 94-GG vs 103-GPP, and 103-GPP vs 272-GLP, were ribosome (GO:0005840), and translation (GO:0006412) and structural constituent of ribosome (GO:0003735), respectively (Fig. S7). In 94-GG vs 272-GLP, the top three ranking GO functional enrichment were ADP binding (GO:0043531), photosystem I (GO:0009522), and photosynthesis, light harvesting (GO:0009765), respectively (Fig. S7E). The GO functional enrichment further analysis showed that the categories of photosynthesis (GO:0015979), chloroplast thylakoid membrane (GO:0009535), photosystem I (GO:0009522), and II (GO:0009523), chloroplast (GO:0009507), were all enriched among the above six pairwise comparisons. The categories of photosystem I reaction center (GO:0009538), and L-phenylalanine catabolic process (GO:0006559) were specifically distributed in 357-PP vs 103-GPP. We also found the GO functional enrichment of pairwise comparisons with green leaf cultivar was different with those with purple leaf cultivar. These results were also suggested that the content, synthesis and metabolim of chlorophyll, and photosynthesis ability were different in four perilla cultivars with different leave colors.
A total of 50 DEGs related to photosystem I and II, photosynthesis, light harvesting, chloroplast thylakoid membrane, chlorophyll binding, chloroplast, chloroplast envelope, photosynthesis, photosystem I reaction center, ferredoxin-NADP + reductase activity and PS II associated light-harvesting complex II catabolic process, and photosynthesis were identified and summarized in Table S4.
It is interested that the DEGs enriched in photosynthesis, light harvesting in photosystem I, such as chlorophyll a/b-binding protein genes (CL125.Contig9_All, Unigene136_All, Unigene127_All, Unigene128_All) and photosystem I reaction center, photosystem I reaction center subunit genes (CL10496.Contig2_All, CL5429.Contig4_All, CL238.Contig4_All, CL12334.Contig1_All, and CL13338.Contig3_All) showed up-regulated expression pattern in 357-PP (Table S4). These results suggested that purple leaf of perillia have strong ability of light harvesting and photosynthesis in photosystem I, compared with those of green or green plus purple leaves. However, ferredoxin-NADP + reductase activity related genes showed down-regulated expression pattern in purple leaves. The key gene controlled chlorophyll synthesis, such as magnesium-chelatase subunit ChlD (Unigene12797_All) CPO (CL5607.Contig4_All, CL2268.Contig3_All), PPOX (CL3522.Contig4_All, CL1474.Contig27_All), and MgCh (CL9498.Contig1_All), showed up-regulated in 94-GG and 103-GPP. At the same time, the gene chlorophyll M (CHLM, Unigene17397_All), chlorophyll 27 (CHL27, CL1169.Contig13_All), and protochlorophyllide reductase (POR, CL374.Contig6_All) control chlorophyll synthesis were also up-regulated in 357-PP, while the Chlorophyll synthase (CHLG, CL5824.Contig3_All) were down-regulated in 357-PP. However, the MCS/SGR/NYC1 (CL5222.Contig3_All, CL3099.Contig4_All, CL3287.Contig1_All, CL3709.Contig1_All, CL6196.Contig1_All, and CL3865.Contig1_All), with PPH unigenes (Unigene19598_All and Unigene19603_All), and pheophorbide a oxygenase (PAO) (CL9496.Contig2_All) played role in chlorophyll degradation was down- and up-regulated, respectively, in 357-PP. In chlorophyll recycling process, chlorophyll a and b could not be easily recycling in purple leaf 357-PP, according to lower expression of CAO (CL1364.Contig13_All), CBR (CL3043.Contig4_All), and HCAR (CL6020.Contig12_All) (Fig. S8A). As a whole, it was deduced that more chlorophyll synthesis in green leaf, while the chlorophyll degradation rate was slow at the beginning, and then fast in purple leaf 357-PP.
In chloroplast enrichment, zeaxanthin epoxidase (ZEP, CL950.Contig28_All) regulated carotenoid synthesis also showed up-regulated expression in 357-PP (Fig. S8B, Table S4). It was suggested that carotenoid may be played in leaf color formation.
KEGG pathway enrichment analyses of DEGs with p < 0.001 as the threshold were also performed in six pairwise comparisons. Like GO functional enrichment, 94-GG vs 272-GLP had a little difference with other five pairwise comparisons. In 357-PP vs 94-GG, 357-PP vs 272-GLP, and 357-PP vs 103-GPP comparisons, the top one to five ranking pathways all include ribosome (ko03010), carbon fixation in photosynthetic organisms (ko00710), photosynthesis-antenna proteins (ko00196), and photosynthesis (ko00195), flavonoid biosynthesis (ko00941, ranked sixth in 357-PP vs 94-GG) (Fig. S9A-C). Notably, porphyin and chlorophyll metabolism (ko00860) in 357-PP vs 94-GG was ranked fifth, and it was also enriched in 357-PP vs 272-GLP, and 357-PP vs 103-GPP (Fig. S9A). It was further suggested that chlorophyll metabolism was different among four cultivars with four kinds of leaf colors. Phenylpropaniod biosynthesis (ko00940), stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945) were also common in the above three pairwise comparisons. In 94-GG vs 272-GLP, 94-GG vs 103-GPP, and 103-GPP vs 272-GLP comparisons, carbon fixation in photosynthetic organisms (ko00710), photosynthesis (ko00195), porphyin and chlorophyll metabolism (ko00860), photosynthesis-antenna proteins (ko00196), phenylpropaniod biosynthesis (ko00940), and flavonoid biosynthesis (ko00941) were common in these three comparisons (Fig. S9D-F). Flavone and flavonol biosynthesis (ko00944), and isoflavonoid biosynthesis (ko00943) were specific observed in 357-PP vs 94-GG and 94-GG vs 272-GLP, and 357-PP vs 103-GPP, respectively comparisons.
A total of 17 DEGs related to carotenoid biosynthesis (ko00906) were also identified in five pairwise comparisons (Fig. S8B, Table S4). The DEGs including 9-cis epoxycarotenoid dioxygenase (NCED, Unigene214_All), carotenoid cleavage dioxygenase (CCD7, CL14084.Contig2_All), and zeaxanthin epoxidase (CL950.Contig28_All), showed much more expression in purple leaf, compared with those in green leaf. The up-regulation of beta-carotene isomerase D27 (DWRF27, Unigene32590_All), phytoene synthase 2 (PSY2, CL6263.Contig2_All), cytochrome P450 CYP97A41 (CYP97A41, CL12610.Contig3_All), 15-cis-zeta-carotene isomerase (ZIISO, CL3672.Contig2_All), and cytochrome P450 CYP97A41 (CL12610.Contig3_All) was showed in green leaf (Fig. 8B). The kinds of carotenoids were different in purple leaf 357-PP and in green leaf 94-GG, according to these genes’ expression. It was deduced that the neoxanthin and carlactone, and lutein were highly accumulated in purple leaf 357-PP and in green leaf 94-GG, respectively.
The DEGs enriched by KEGG pathway related to chlorophyll metabolism, flavonoid biosynthesis, and photosynthesis were analyzed and summarized in Table S4. Most of DEGs enriched photosynthesis, porphyrin and chlorophyll metabolism (ko00860), and photosynthesis - antenna proteins (ko00196) were same as those DEGs in GO functional enrichment. Especially, chlorophyll breakdown gene MCS/SGR/NYC1 (CL5222.Contig3_All, CL3099.Contig4_All and CL3709.Contig1_All) highly expressed in 94-GG suggested fast chlorophyll degradation was in green leaf.
A total of 13 and 24 DEGs related to phenylpropanoid biosynthesis (ko00940), and flavonoid biosynthesis (ko00941) were screened out in KEGG enrichment, respectively. They were took part in perilla leaf color formation. The PAL (CL13983.Contig3_All), C4H (Unigene27773_All, Unigene14839_All, CL6772.Contig2_All), C3H (CL11971.Contig9_All) HCT (CL13936.Contig3_All, Unigene10775_All, and Unigene5083_All) and COMT (Unigene17835_All) all showed up-down expression pattern in 94-GG, 272-GLP, 103-GPP and 357-PP (Fig. 8C, Table S4). Much more expression of PAL, C4H, C3H, and HCT would be caused much more caffeic acid synthesis in green and front green and back purple leaves in 94-GG, 272-GLP, and 103-PP, compared with that of only purple leaf 357-PP. Much more caffeic acid synthesis might be promoted rosomarinic acid synthesis. Rosomarinic acid synthesis gene, RAS (CL13662.Contig2_All) also showed high expression in 272-GLP and 94-GG (Fig. 8C, Table S4). Many CCR (CL14054.Contig2_All, CL1293.Contig1_All), 4CL (CL12689.Contig5_All, Unigene15858_All, Unigene9488_All, and CL3950.Contig8_All) unigenes showed up-regulation in purple cultivar ‘M357’. At the same time, the key anthocyanins synthesized genes CHS (Unigene15383_All) and CHI (CL3453.Contig1_All) all highly expressed in 357-PP.
As a whole, the genes related to flavonoid 3'-hydroxylase (F3’H), flavonoid 3',5'-methyltransferase-like (F3’,5’H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase/leucoanthocyanidin dioxygenase (LODX/ANS) showed specific expression in 103-GPP and 357-PP. Highly expressed of these above genes induced much more anthocyanins accumulation. Much more expression of F3’,5’H gene could be promoted delphinidin-3-glycoside accumulation and further cause leaf of 357-PP turning purple (Fig. 8C). Based on F3’H, F3H, and FSII gene expression, it was suggested that much more flavones, flavonals, and isoflavones might synthesized in green leaf, compared with that in the purple leaf (Fig. 8C, Table S4).
3.8. The hierarchical clustering analysis of key DEGs involved in chlorophyll metabolism and flavonoid biosynthesis
According to the results of venn diagrams and DEGs enriched GO function and KEGG pathway (Fig. S4, Fig. S9), all the genes referred to chlorophyll biosynthesis and metabolisms were searched. Meanwhile, the expression patterns of these genes according to FPKM of RNA-seq data in four perilla cultivars were analyzed, and the relationship between their expression patterns and leaf color formation was investigated. The 23 chlorophyll synthesis and degradation related genes (59 unigenes or contigs) were identified in our work (Table 4). For this study, without those genes with a FPKM value of less than 1, a total of 55 unigenes or contigs were different expressed and probably related to leaf color formation. The hierarchical clustering thermogram of 55 DEGs was shown in Fig. 6A. The different four cultivars could be easily separated by expression levels of 42 unigenes, and it was indicated that these genes may be regulated leaf coloration.
Table 4
The DEGs regulated chlorophyll metabolic pathway.
No.
|
Gene name
|
Unigene ID
|
Length
|
94-GG
|
272-GLP
|
103-GPP
|
357-PP
|
1
|
glutamyl-tRNA synthetase (GluRS)
|
CL1929.Contig3_All
|
2,483
|
2.18 ± 0.80
|
7.51 ± 0.59
|
7.12 ± 0.76
|
13.48 ± 0.95
|
2
|
Glutamyl-tRNA reductase (GluTR/HEMA)
|
CL6762.Contig1_All
|
1,437
|
33.94 ± 0.06
|
35.82 ± 3.05
|
68.24 ± 1.29
|
18.92 ± 3.64
|
CL8793.Contig3_All
|
2,131
|
2.07 ± 0.41
|
2.70 ± 0.10
|
1.75 ± 0.13
|
0.89 ± 0.02
|
Unigene15301_All
|
2,195
|
213.96 ± 4.82
|
236.18 ± 4.02
|
225.26 ± 0.43
|
118.00 ± 0.55
|
3
|
Glutamate-1-semialdehyde 2,1-aminomutase(GSAAT)
|
CL6883.Contig3_All
|
3,679
|
31.31 ± 1.29
|
26.91 ± 0.35
|
30.84 ± 0.90
|
17.67 ± 0.13
|
4
|
5-aminolevulinate dehydratase(ALAD)
|
Unigene9122_All
|
1,646
|
17.24 ± 0.01
|
29.80 ± 0.81
|
31.46 ± 1.14
|
61.15 ± 0.25
|
Unigene15911_All
|
768
|
40.58 ± 0.40
|
2.72 ± 0.45
|
1.94 ± 1.24
|
25.64 ± 0.08
|
5
|
Hydroxymethylbilane synthase(HEMC/HMBS)
|
CL9339.Contig2_All
|
1,495
|
14.25 ± 0.47
|
9.15 ± 1.22
|
12.81 ± 0.72
|
13.28 ± 0.11
|
6
|
uroporphyrinogen III synthase (HEMD/UROS)
|
Unigene20647_All
|
1,228
|
36.17 ± 2.30
|
32.12 ± 0.32
|
39.04 ± 0.22
|
28.15 ± 0.32
|
7
|
Uroporphyrinogen III decarboxylase (UROD)
|
CL13981.Contig7_All
|
1,700
|
10.43 ± 0.51
|
6.88 ± 0.55
|
10.54 ± 1.22
|
1.89 ± 0.23
|
CL4132.Contig1_All
|
2,331
|
9.29 ± 0.44
|
6.23 ± 1.01
|
10.73 ± 0.04
|
12.34 ± 0.20
|
8
|
Coproporphyrinogen III oxidase (CPO)
|
CL5607.Contig4_All
|
3,040
|
1.84 ± 0.14
|
1.59 ± 0.04
|
1.54 ± 0.16
|
0.62 ± 0.35
|
Unigene12050_All
|
1,021
|
0.00 ± 0.00
|
0.19 ± 0.26
|
0.13 ± 0.18
|
9.59 ± 1.36
|
CL2268.Contig3_All
|
1,401
|
31.75 ± 0.40
|
31.67 ± 0.40
|
30.71 ± 0.49
|
15.63 ± 0.26
|
9
|
Protoporphyrinogen oxidase (PPOX)
|
CL3522.Contig4_All
|
3,109
|
5.42 ± 0.18
|
4.83 ± 0.34
|
2.65 ± 0.58
|
0.75 ± 0.00
|
CL1474.Contig27_All
|
2,601
|
3.33 ± 0.76
|
1.96 ± 0.01
|
2.38 ± 0.31
|
0.48 ± 0.30
|
CL2458.Contig10_All
|
2,934
|
0.37 ± 0.11
|
0.70 ± 0.44
|
0.00 ± 0.00
|
0.35 ± 0.16
|
10
|
Magnesium chelatase (MgCh)
|
CL11069.Contig1_All
|
1,554
|
41.61 ± 2.96
|
23.65 ± 2.86
|
44.49 ± 2.45
|
47.46 ± 1.89
|
CL9498.Contig1_All
|
4,986
|
259.57 ± 1.87
|
213.60 ± 0.74
|
217.16 ± 1.80
|
149.00 ± 3.68
|
Unigene12797_All
|
2,580
|
45.46 ± 0.54
|
30.62 ± 0.21
|
37.88 ± 0.28
|
20.19 ± 0.53
|
11
|
Magnesium protoporphyrin IX methyltransferase (CHLM)
|
Unigene17397_All
|
3,320
|
21.20 ± 0.14
|
23.08 ± 0.17
|
29.24 ± 0.06
|
26.46 ± 0.00
|
12
|
Magnesium-protoporphyrin IX monomethylester
cyclase (CHL27)
|
CL1169.Contig13_All
|
4,009
|
36.91 ± 3.45
|
29.77 ± 1.91
|
51.17 ± 0.21
|
57.09 ± 0.13
|
13
|
Protochlorophyllide oxidoreductase (POR)
|
Unigene673_All
|
1,396
|
2.79 ± 0.57
|
1.02 ± 0.26
|
1.44 ± 0.04
|
11.76 ± 0.95
|
CL374.Contig6_All
|
2,832
|
0.23 ± 0.18
|
4.57 ± 0.61
|
3.16 ± 0.33
|
6.47 ± 0.30
|
14
|
Divinyl reductase (DVR)
|
CL1122.Contig2_All
|
1,969
|
12.16 ± 1.15
|
16.61 ± 1.79
|
15.16 ± 0.35
|
48.94 ± 1.17
|
CL8802.Contig2_All
|
1,689
|
144.20 ± 0.66
|
79.99 ± 1.90
|
128.22 ± 2.87
|
77.22 ± 3.02
|
15
|
Chlorophyll synthase (ChlG)
|
CL5824.Contig3_All
|
1,631
|
24.74 ± 0.34
|
26.73 ± 2.98
|
31.35 ± 2.52
|
6.26 ± 0.02
|
16
|
Chlorophyllide a oxygenase(CAO)
|
CL2783.Contig1_All
|
2,272
|
8.01 ± 0.28
|
6.01 ± 0.23
|
9.33 ± 0.90
|
37.49 ± 1.07
|
CL1364.Contig13_All
|
3,400
|
30.19 ± 1.94
|
26.51 ± 1.65
|
25.81 ± 0.13
|
25.83 ± 1.52
|
17
|
Chlorophyll b reductase (CBR)
|
CL2905.Contig6_All
|
2,440
|
1.51 ± 0.19
|
0.30 ± 0.01
|
0.32 ± 0.02
|
0.42 ± 0.04
|
CL3043.Contig4_All
|
1,939
|
24.03 ± 0.73
|
4.70 ± 0.25
|
4.98 ± 0.76
|
2.88 ± 0.64
|
18
|
Hydroxymethyl chlorophyll a reductase (HCAR)
|
CL6020.Contig12_All
|
3,516
|
0.83 ± 0.33
|
1.61 ± 0.09
|
0.79 ± 0.11
|
0.86 ± 0.21
|
19
|
Magnesium dechelatase (STAYGREEN/NON-YELLOWING (SGR/NYE) (MCS/SGR/NYE)
|
CL5222.Contig3_All
|
1,368
|
58.38 ± 1.15
|
38.29 ± 0.62
|
0.18 ± 0.00
|
3.39 ± 0.57
|
Unigene46613_All
|
1,023
|
12.35 ± 2.23
|
4.52 ± 0.83
|
37.06 ± 0.89
|
22.91 ± 0.05
|
CL3099.Contig4_All
|
4,101
|
2.91 ± 0.32
|
4.21 ± 0.42
|
1.30 ± 0.27
|
1.78 ± 0.01
|
CL3287.Contig1_All
|
1,673
|
10.74 ± 0.51
|
6.70 ± 1.01
|
8.69 ± 0.05
|
7.49 ± 0.12
|
CL3709.Contig1_All
|
1,893
|
8.31 ± 0.08
|
5.67 ± 0.49
|
0.53 ± 0.08
|
0.90 ± 0.25
|
CL6196.Contig1_All
|
1,261
|
23.65 ± 2.64
|
14.25 ± 0.59
|
0.68 ± 0.05
|
0.39 ± 0.21
|
CL3865.Contig1_All
|
1,146
|
35.48 ± 0.43
|
20.87 ± 0.05
|
22.75 ± 1.70
|
19.16 ± 0.90
|
20
|
Pheophytinase (PPH)
|
Unigene19598_All
|
1,907
|
4.55 ± 0.99
|
0.00 ± 0.00
|
5.14 ± 0.13
|
2.54 ± 0.37
|
Unigene19602_All
|
1,579
|
5.19 ± 0.08
|
5.16 ± 0.43
|
5.75 ± 1.07
|
2.52 ± 0.06
|
Unigene19603_All
|
1,634
|
6.45 ± 0.54
|
5.58 ± 1.04
|
4.93 ± 0.33
|
2.68 ± 0.59
|
Unigene19608_All
|
2,239
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.53 ± 0.08
|
Unigene42901_All
|
920
|
0.00 ± 0.00
|
0.00 ± 0.00
|
3.79 ± 0.21
|
0.86 ± 0.32
|
CL1060.Contig14_All
|
2,805
|
4.85 ± 0.42
|
4.82 ± 0.93
|
5.91 ± 0.15
|
20.42 ± 0.96
|
CL11133.Contig1_All
|
3,167
|
42.26 ± 1.12
|
31.02 ± 1.87
|
29.02 ± 1.03
|
149.06 ± 1.39
|
CL1652.Contig13_All
|
2,375
|
23.74 ± 1.37
|
17.51 ± 0.24
|
19.73 ± 0.58
|
7.87 ± 0.74
|
CL2285.Contig5_All
|
1,200
|
5.47 ± 0.78
|
10.51 ± 1.62
|
5.43 ± 0.81
|
0.00 ± 0.00
|
21
|
Pheophorbide a oxygenase (PAO)
|
CL9496.Contig2_All
|
1,744
|
1.97 ± 0.19
|
4.19 ± 1.24
|
3.89 ± 0.84
|
12.83 ± 0.32
|
CL1997.Contig6_All
|
1,573
|
2.57 ± 0.19
|
4.08 ± 0.33
|
3.50 ± 0.12
|
11.45 ± 0.87
|
CL4104.Contig8_All
|
2,722
|
78.12 ± 2.96
|
99.38 ± 1.64
|
95.07 ± 1.00
|
154.49 ± 6.92
|
22
|
Red chlorophyll catabolite reductase (RCCR)
|
Unigene35253_All
|
1,187
|
0.10 ± 0.13
|
0.00 ± 0.00
|
0.00 ± 0.00
|
7.05 ± 1.10
|
Unigene4771_All
|
2,897
|
0.45 ± 0.12
|
0.45 ± 0.06
|
0.64 ± 0.15
|
3.05 ± 0.25
|
Unigene7171_All
|
1,358
|
24.11 ± 3.25
|
14.64 ± 1.46
|
27.43 ± 1.90
|
2.53 ± 1.99
|
Unigene9514_All
|
1,853
|
26.22 ± 4.23
|
22.25 ± 0.03
|
5.67 ± 0.16
|
1.57 ± 0.20
|
Unigene19354_All
|
1,471
|
3.13 ± 0.71
|
1.35 ± 1.10
|
10.44 ± 3.46
|
12.05 ± 2.53
|
Unigene19360_All
|
1,399
|
7.80 ± 0.15
|
8.46 ± 2.28
|
15.80 ± 1.08
|
23.77 ± 4.54
|
Unigene4575_All
|
698
|
2.82 ± 0.33
|
2.95 ± 0.33
|
0.59 ± 0.13
|
0.22 ± 0.03
|
23
|
TIC55
|
CL7170.Contig1_All
|
1,922
|
168.47 ± 1.60
|
149.52 ± 5.44
|
156.59 ± 1.68
|
128.71 ± 5.63
|
At the same time, the 20 flavonoid synthesis related genes were also analyzed according to different pairwise comparisons with different groups, and a total of 102 unigenes or contigs were identified (Table 5). Without those genes with a FPKM value of less than 1, the remaining 73 unigenes or contigs were different expressed in four perilla cultivars and they were probably related to perilla leaf color formation. Their hierarchical clustering thermogram was shown in Fig. 6B. According to heatmap cluster, the relationship of cultivar 272 and 94, and 103 and 357 was close, respectively (Fig. 6B). It was suggested that these flavonoid synthesis related genes were regulated perilla leaf color.
Table 5
DEGs in flavonoid biosynthesis pathway.
No.
|
Gene name
|
Unigene ID
|
Length (bp)
|
94-GG
|
272-GLP
|
103-GPP
|
357-PP
|
1
|
Phenyl alanineammonia lyase (PAL)
|
CL13983.Contig3_All
|
2,507
|
24.24 ± 3.40
|
59.41 ± 2.64
|
16.58 ± 0.25
|
9.32 ± 0.62
|
|
|
CL13983.Contig5_All
|
3,223
|
18.26 ± 0.44
|
31.96 ± 0.95
|
12.63 ± 1.41
|
7.29 ± 1.20
|
2
|
Cinnamate 4-hydroxylase (C4H)
|
Unigene27773_All
|
2,536
|
2.69 ± 0.83
|
5.37 ± 0.35
|
2.71 ± 0.43
|
1.18 ± 0.46
|
|
|
CL6772.Contig5_All
|
1,727
|
10.73 ± 0.69
|
29.92 ± 1.87
|
3.03 ± 0.71
|
6.08 ± 1.87
|
|
|
CL6772.Contig2_All
|
2,465
|
23.05 ± 1.58
|
33.07 ± 0.90
|
17.05 ± 0.25
|
13.40 ± 0.09
|
|
|
Unigene14838_All
|
1,856
|
2.55 ± 0.27
|
6.14 ± 0.45
|
1.59 ± 0.52
|
1.44 ± 0.50
|
|
|
Unigene14839_All
|
896
|
8.74 ± 1.45
|
26.01 ± 2.11
|
2.82 ± 0.37
|
5.00 ± 0.05
|
3
|
p-coumarate 3 hydroxylase (C3H)
|
CL11971.Contig9_All
|
1,874
|
43.59 ± 1.56
|
80.73 ± 0.45
|
35.85 ± 1.78
|
23.40 ± 0.78
|
4
|
Caffeic O-methyltransferase (COMT)
|
Unigene17835_All
|
1,561
|
69.91 ± 0.98
|
75.77 ± 4.86
|
68.58 ± 0.20
|
28.48 ± 0.77
|
|
|
CL9808.Contig2_All
|
1,016
|
23.51 ± 0.61
|
57.27 ± 0.78
|
12.97 ± 0.86
|
24.58 ± 2.88
|
|
|
Unigene20838_All
|
1,537
|
5.25 ± 0.18
|
12.69 ± 2.02
|
3.11 ± 0.11
|
6.54 ± 0.59
|
5
|
4-coumarate-CoA Ligase (4CL)
|
CL14054.Contig2_All
|
3,600
|
0.27 ± 0.04
|
0.59 ± 0.07
|
0.44 ± 0.18
|
1.61 ± 0.18
|
|
|
CL1453.Contig1_All
|
3,246
|
40.35 ± 0.13
|
14.44 ± 0.83
|
20.75 ± 0.73
|
4.0.0476 ± 0.45
|
|
|
CL8560.Contig7_All
|
1,706
|
1.79 ± 0.42
|
1.30 ± 0.11
|
1.91 ± 0.24
|
6.54 ± 0.99
|
|
|
Unigene28027_All
|
1,797
|
3.11 ± 0.06
|
4.07 ± 0.21
|
3.75 ± 0.26
|
1.74 ± 0.35
|
|
|
CL1293.Contig1_All
|
2,056
|
33.90 ± 1.46
|
24.04 ± 1.40
|
44.19 ± 1.57
|
81.74 ± 2.96
|
|
|
CL12166.Contig3_All
|
3,110
|
0.58 ± 0.22
|
2.99 ± 0.28
|
1.21 ± 0.21
|
0.71 ± 0.01
|
|
|
CL1293.Contig2_All
|
2,238
|
1.17 ± 1.00
|
28.11 ± 3.44
|
1.40 ± 0.00
|
1.30 ± 0.05
|
6
|
Cynnamoyl CoA reductase (CCR)
|
CL12689.Contig5_All
|
2,099
|
4.27 ± 0.24
|
0.83 ± 0.38
|
2.15 ± 0.27
|
20.82 ± 0.02
|
|
|
CL12689.Contig13_All
|
2,192
|
13.61 ± 1.66
|
7.61 ± 0.06
|
0.70 ± 0.21
|
8.61 ± 0.04
|
|
|
CL329.Contig2_All
|
2,386
|
1.22 ± 0.08
|
3.17 ± 0.30
|
1.28 ± 0.13
|
0.68 ± 0.04
|
|
|
CL3926.Contig3_All
|
1,443
|
5.22 ± 0.12
|
5.17 ± 0.58
|
2.07 ± 0.11
|
0.58 ± 0.50
|
|
|
CL9761.Contig1_All
|
9,154
|
0.08 ± 0.01
|
0.83 ± 0.16
|
0.21 ± 0.05
|
4.05 ± 0.01
|
|
|
Unigene15858_All
|
1,624
|
9.69 ± 0.98
|
17.95 ± 1.43
|
13.27 ± 1.33
|
39.40 ± 0.76
|
|
|
Unigene9488_All
|
1,991
|
0.24 ± 0.06
|
0.68 ± 0.06
|
0.82 ± 0.03
|
2.13 ± 0.22
|
|
|
Unigene10450_All
|
1,381
|
4.58 ± 0.47
|
4.65 ± 0.04
|
9.61 ± 0.28
|
11.55 ± 0.20
|
|
|
Unigene3767_All
|
3,532
|
1.33 ± 0.05
|
0.09 ± 0.00
|
1.46 ± 0.28
|
0.09 ± 0.06
|
|
|
Unigene4710_All
|
774
|
5.64 ± 0.42
|
1.47 ± 0.23
|
6.82 ± 0.36
|
1.10 ± 0.07
|
|
|
Unigene8839_All
|
4,673
|
1.68 ± 0.31
|
4.23 ± 0.46
|
0.95 ± 0.07
|
1.08 ± 0.01
|
|
|
CL3950.Contig8_All
|
1,120
|
8.21 ± 1.70
|
8.76 ± 0.07
|
9.60 ± 0.54
|
18.42 ± 1.34
|
7
|
Hydroxycinnamoyl transferase (HCT)
|
CL13936.Contig3_All
|
1,834
|
0.00 ± 0.00
|
3.74 ± 0.37
|
0.00 ± 0.00
|
0.00 ± 0.00
|
|
|
Unigene10775_All
|
2,903
|
38.97 ± 1.50
|
3.01 ± 0.06
|
10.30 ± 0.49
|
9.50 ± 0.87
|
|
|
Unigene1890_All
|
1,630
|
0.34 ± 0.20
|
13.03 ± 1.00
|
0.30 ± 0.12
|
0.13 ± 0.02
|
|
|
Unigene5083_All
|
3,253
|
5.98 ± 0.54
|
14.00 ± 0.65
|
5.19 ± 0.59
|
3.96 ± 0.45
|
|
|
Unigene3321_All
|
1,073
|
20.25 ± 0.76
|
11.84 ± 0.28
|
6.77 ± 0.19
|
1.42 ± 0.21
|
|
|
CL11508.Contig2_All
|
1,660
|
3.27 ± 0.35
|
5.87 ± 0.16
|
4.37 ± 0.19
|
13.95 ± 1.24
|
8
|
Chalcone synthase (CHS)
|
CL1534.Contig1_All
|
1,663
|
125.35 ± 7.76
|
38.89 ± 1.89
|
177.00 ± 12.99
|
48.87 ± 1.34
|
|
|
Unigene15383_All
|
2,135
|
14.27 ± 0.93
|
5.28 ± 0.59
|
24.59 ± 0.75
|
49.96 ± 1.68
|
9
|
Chalconeisomerase (CHI)
|
CL13206.Contig6_All
|
1,113
|
4.88 ± 0.04
|
3.21 ± 0.41
|
4.56 ± 0.83
|
1.38 ± 0.01
|
|
|
CL13206.Contig9_All
|
1,024
|
26.40 ± 0.26
|
20.51 ± 0.21
|
20.07 ± 1.40
|
6.79 ± 0.06
|
|
|
CL13730.Contig3_All
|
1,617
|
1.29 ± 0.00
|
0.65 ± 0.04
|
0.94 ± 0.06
|
0.09 ± 0.04
|
|
|
CL2151.Contig4_All
|
2,416
|
1.04 ± 0.28
|
1.86 ± 0.36
|
1.69 ± 0.22
|
4.54 ± 0.84
|
|
|
CL3453.Contig1_All
|
2,322
|
8.70 ± 0.04
|
8.95 ± 0.92
|
9.72 ± 0.62
|
28.85 ± 1.46
|
|
|
Unigene533_All
|
2,511
|
24.48 ± 1.62
|
19.11 ± 0.68
|
33.65 ± 0.26
|
4.61 ± 0.22
|
10
|
Flavone synthase II (FSII)
|
Unigene20542_All
|
1,856
|
34.45 ± 2.28
|
28.99 ± 4.13
|
26.76 ± 0.33
|
4.28 ± 0.01
|
|
|
Unigene965_All
|
1,842
|
31.63 ± 2.47
|
14.99 ± 3.68
|
12.68 ± 0.86
|
5.58 ± 0.39
|
|
|
CL7056.Contig3_All
|
1,376
|
2.36 ± 0.01
|
1.46 ± 0.28
|
5.52 ± 0.17
|
2.60 ± 0.16
|
|
|
Unigene20541_All
|
1,870
|
10.55 ± 1.16
|
7.96 ± 0.25
|
46.22 ± 0.11
|
11.92 ± 0.83
|
|
|
Unigene13096_All
|
587
|
0.12 ± 0.17
|
2.14 ± 0.53
|
1.22 ± 0.52
|
4.21 ± 1.01
|
|
|
Unigene31141_All
|
1,066
|
0.35 ± 0.08
|
2.69 ± 0.50
|
0.93 ± 0.16
|
2.98 ± 0.01
|
|
|
CL13434.Contig1_All
|
1,988
|
1.54 ± 0.01
|
0.06 ± 0.03
|
3.45 ± 0.17
|
1.83 ± 0.53
|
|
|
CL5086.Contig2_All
|
1,246
|
1.27 ± 0.12
|
1.45 ± 0.39
|
0.00 ± 0.00
|
0.58 ± 0.23
|
11
|
Flavonol synthase (FLS)
|
CL5086.Contig3_All
|
1,175
|
4.04 ± 0.40
|
3.80 ± 0.23
|
6.88 ± 0.96
|
17.21 ± 0.97
|
|
|
CL7927.Contig15_All
|
813
|
0.13 ± 0.18
|
2.06 ± 0.58
|
0.00 ± 0.00
|
0.15 ± 0.21
|
|
|
CL5086.Contig2_All
|
1,246
|
1.27 ± 0.12
|
1.45 ± 0.39
|
0.00 ± 0.00
|
0.58 ± 0.23
|
|
|
CL3571.Contig5_All
|
927
|
42.25 ± 2.34
|
64.65 ± 3.32
|
105.77 ± 6.07
|
73.73 ± 0.03
|
|
|
CL9805.Contig2_All
|
1,205
|
3.45 ± 0.83
|
1.61 ± 0.08
|
2.68 ± 0.23
|
5.46 ± 0.20
|
|
|
CL5652.Contig1_All
|
1,286
|
5.79 ± 0.70
|
1.62 ± 0.11
|
2.20 ± 1.16
|
0.22 ± 0.30
|
|
|
CL5652.Contig1_All
|
1,286
|
5.79 ± 0.70
|
1.62 ± 0.11
|
2.20 ± 1.16
|
0.22 ± 0.30
|
|
|
Unigene6990_All
|
2,276
|
3.54 ± 0.10
|
2.86 ± 0.18
|
3.47 ± 0.38
|
0.52 ± 0.06
|
|
|
CL3571.Contig5_All
|
927
|
42.25 ± 2.34
|
64.65 ± 3.32
|
105.77 ± 6.07
|
73.73 ± 0.03
|
|
|
CL9805.Contig2_All
|
1,205
|
3.45 ± 0.83
|
1.61 ± 0.08
|
2.68 ± 0.23
|
5.46 ± 0.20
|
12
|
Flavanone 3-hydroxylase (F3H)
|
CL1253.Contig4_All
|
4,042
|
0.00 ± 0.00
|
0.22 ± 0.14
|
5.40 ± 0.52
|
0.39 ± 0.04
|
|
|
CL1253.Contig2_All
|
2,980
|
0.00 ± 0.00
|
1.74 ± 0.01
|
22.82 ± 0.03
|
0.66 ± 0.21
|
|
|
CL7927.Contig6_All
|
4,691
|
1.39 ± 0.18
|
3.44 ± 0.39
|
1.48 ± 0.16
|
0.90 ± 0.10
|
|
|
Unigene14836_All
|
2,203
|
12.97 ± 0.84
|
3.19 ± 0.02
|
0.00 ± 0.00
|
1.31 ± 0.06
|
|
|
Unigene14837_All
|
1,911
|
0.02 ± 0.02
|
0.32 ± 0.45
|
27.03 ± 1.20
|
2.11 ± 0.18
|
|
|
Unigene20877_All
|
1,292
|
0.00 ± 0.00
|
0.56 ± 0.06
|
33.01 ± 0.15
|
34.57 ± 0.34
|
13
|
Flavonol 3′-hydrogenase (F3’H)
|
CL13530.Contig1_All
|
1,885
|
0.00 ± 0.00
|
2.04 ± 0.05
|
17.37 ± 1.26
|
11.60 ± 0.10
|
|
|
CL13530.Contig3_All
|
1,962
|
0.00 ± 0.00
|
0.33 ± 0.05
|
18.49 ± 1.46
|
14.96 ± 1.57
|
|
|
CL13530.Contig4_All
|
2,417
|
0.00 ± 0.00
|
0.10 ± 0.07
|
2.57 ± 0.31
|
1.84 ± 0.32
|
|
|
CL13530.Contig5_All
|
1,976
|
0.02 ± 0.02
|
1.97 ± 0.09
|
26.77 ± 1.34
|
20.68 ± 0.82
|
|
|
CL5548.Contig2_All
|
997
|
0.00 ± 0.00
|
1.49 ± 0.29
|
58.61 ± 2.93
|
33.01 ± 0.88
|
|
|
Unigene10538_All
|
2,644
|
0.89 ± 0.03
|
1.15 ± 0.05
|
4.81 ± 0.03
|
3.11 ± 0.02
|
|
|
Unigene11136_All
|
2,041
|
12.96 ± 1.06
|
7.30 ± 0.82
|
0.78 ± 0.02
|
0.23 ± 0.32
|
|
|
CL2039.Contig3_All
|
8,445
|
10.12 ± 0.69
|
2.56 ± 0.06
|
6.87 ± 0.71
|
0.38 ± 0.04
|
|
|
Unigene8486_All
|
2,369
|
1.94 ± 0.02
|
1.48 ± 0.27
|
0.82 ± 0.05
|
0.67 ± 0.08
|
14
|
Flavonol 3′5′-hydrogenase (F3’,5’H)
|
CL8993.Contig1_All
|
1,594
|
0.34 ± 0.01
|
0.23 ± 0.11
|
0.35 ± 0.35
|
1.72 ± 0.18
|
|
dihydroflavonol dihydroflavonol 4-reductase (DFR)
|
CL12815.Contig11_All
|
2,636
|
0.53 ± 0.20
|
5.67 ± 0.09
|
44.56 ± 1.01
|
24.08 ± 0.28
|
|
|
CL10733.Contig1_All
|
1,513
|
26.85 ± 1.18
|
14.17 ± 0.10
|
20.71 ± 0.11
|
9.48 ± 0.91
|
|
|
CL10733.Contig3_All
|
1,890
|
1.68 ± 0.06
|
2.07 ± 0.11
|
4.30 ± 1.18
|
1.22 ± 0.06
|
15
|
Leucoanthocyanidin dioxygenase (LDOX/ANS)
|
CL1006.Contig1_All
|
1,556
|
0.00 ± 0.00
|
6.55 ± 0.03
|
77.35 ± 0.26
|
29.13 ± 0.81
|
|
|
CL1006.Contig2_All
|
1,626
|
0.04 ± 0.05
|
6.48 ± 0.23
|
178.33 ± 1.94
|
141.63 ± 1.03
|
|
|
CL1006.Contig3_All
|
1,590
|
0.02 ± 0.03
|
11.58 ± 0.81
|
29.12 ± 1.61
|
56.57 ± 2.23
|
|
|
CL7584.Contig1_All
|
1,309
|
0.00 ± 0.00
|
0.28 ± 0.18
|
15.21 ± 1.72
|
15.20 ± 2.06
|
|
|
CL7584.Contig2_All
|
1,272
|
0.00 ± 0.00
|
0.31 ± 0.01
|
25.83 ± 1.66
|
16.12 ± 1.43
|
|
|
CL3950.Contig7_All
|
1,431
|
1.30 ± 0.93
|
7.19 ± 1.03
|
4.49 ± 0.93
|
11.13 ± 0.74
|
|
|
CL3950.Contig8_All
|
1,120
|
8.21 ± 1.70
|
8.76 ± 0.07
|
9.60 ± 0.54
|
18.42 ± 1.34
|
16
|
UDP-glucose:anthocianidin:flavonoidglucosyltransferase(UFGT)
|
CL13864.Contig2_All
|
1,621
|
7.09 ± 0.53
|
1.21 ± 0.08
|
5.25 ± 0.04
|
9.01 ± 0.94
|
|
|
CL13864.Contig1_All
|
1,657
|
8.29 ± 0.13
|
8.29 ± 0.04
|
7.06 ± 0.21
|
13.18 ± 0.62
|
|
|
Unigene5399_All
|
1,709
|
16.00 ± 2.71
|
19.39 ± 0.86
|
17.94 ± 0.71
|
10.65 ± 0.35
|
|
|
CL7065.Contig2_All
|
1,667
|
2.85 ± 0.67
|
0.09 ± 0.12
|
6.09 ± 2.03
|
2.45 ± 0.06
|
|
|
CL7065.Contig3_All
|
1,658
|
13.79 ± 0.01
|
0.90 ± 0.27
|
30.36 ± 0.40
|
7.82 ± 0.40
|
17
|
Anthocyanidinsynthase(ANS)
|
CL3950.Contig1_All
|
1,210
|
43.78 ± 1.75
|
45.33 ± 1.79
|
25.61 ± 1.22
|
34.73 ± 1.68
|
|
|
CL3950.Contig7_All
|
1,431
|
1.30 ± 0.93
|
7.19 ± 1.03
|
4.49 ± 0.93
|
11.13 ± 0.74
|
|
|
CL3950.Contig2_All
|
1,231
|
1.32 ± 0.13
|
1.25 ± 0.13
|
0.42 ± 0.51
|
3.63 ± 1.01
|
|
|
CL3950.Contig5_All
|
1,520
|
3.13 ± 0.12
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
|
|
CL3950.Contig6_All
|
1,402
|
0.07 ± 0.09
|
1.89 ± 0.16
|
1.28 ± 0.28
|
2.41 ± 0.25
|
|
|
Unigene652_All
|
2,044
|
1.77 ± 0.12
|
2.98 ± 0.13
|
2.11 ± 0.13
|
0.62 ± 0.10
|
18
|
Rosmarinic acid synthase(RAS)
|
CL13662.Contig2_All
|
1,665
|
57.38 ± 3.85
|
86.89 ± 0.19
|
22.03 ± 2.23
|
14.04 ± 1.03
|
19
|
UDP-glucoronosyl and UDP-glucosyl transferase (UGT88D7)
|
Unigene12571_All
|
1,765
|
163.21 ± 0.70
|
143.22 ± 3.97
|
114.76 ± 3.61
|
36.67 ± 0.91
|
20
|
Cytochrome P450 CYP98A78 (CYP98A78)
|
CL306.Contig5_All
|
4,827
|
10.84 ± 0.40
|
19.51 ± 0.78
|
8.17 ± 0.05
|
5.56 ± 0.47
|
3.9. The important transcription factor genes involved in perilla leaf color regulation
Transcription factors (TFs) play an important role in plant development and secondary metabolism. In this paper, a total of 28 TF genes were identified and they have different expression patterns in four perilla cultivars. It includes one AP10/EREBP gene, one bZIP gene, one MADS box gene SVP, one TCP gene, four MYB gene, one zf-HD gene, two NAC21/22 genes, three WRKY genes, four WD40 genes, and seven bHLH genes. The AP10/EREBP, bHLH35-like, bHLH29, bHLH137-like, bZIP, SVP, MYB-like, MYB113, NAC21/22, WD40 repeat containing protein 23, U4/U6 small nuclear ribonucleoprotein PRP4-like protein, and autophagy-related protein 18b were up-regulated and the remained 14 TFs were down-regulated (Fig. 7, Table 6). The relationship of four cultivars constructed by heatmap was similar with that constructed by flavonoid. It was suggested that these TFs might control flavonoid accumulation, and effect leaf color formation. The bHLH35-like (CL1476.Contig1_All), and WD repeat-containing protein 43 (CL4426.Contig1_All) and zinc-finger homeodomain protein 3 (CL13045.Contig26_All) were specifically expressed in 272-GLP and 103-GPP, respectively. Previous study showed that MYB113 regulated perilla leaf color, and this TF was also identified in our work. Meanwhile, MYB61 (CL3951.Contig1_All) and MYB-like (CL12881.Contig13_All) showed same expression pattern as MYB113 viz. up-regulated in deep color leaves. TF genes including AP10/EREBP (CL1914.Contig26_All), bZIP (CL403.Contig9_All), SVP (CL11571.Contig4_All), NAC21/22 (Unigene3776_All and Unigene4273_All), and bHLH137-like (Unigene631) also highly expressed in purple leaf 357-PP (Fig. 7B). It was suggested that these TFs may regulated perilla leaf color formation.
Table 6
Different expressed transcript factors in four perilla cultivars.
No.
|
Gene name
|
Unigene ID
|
Length (bp)
|
94-GG
|
272-GLP
|
103-GPP
|
357-PP
|
1
|
AP10/EREBP
|
CL1914.Contig26_All
|
3,675
|
0.64 ± 0.05
|
0.80 ± 0.08
|
2.57 ± 0.00
|
6.51 ± 0.25
|
2
|
bHLH35-like
|
CL1476.Contig1_All
|
1,641
|
0.69 ± 0.05
|
12.43 ± 0.59
|
0.83 ± 0.06
|
4.34 ± 0.11
|
|
|
CL1476.Contig2_All
|
1,259
|
0.73 ± 0.02
|
13.15 ± 0.82
|
0.87 ± 0.33
|
4.45 ± 0.94
|
|
|
CL1476.Contig3_All
|
3,896
|
0.12 ± 0.01
|
3.35 ± 0.10
|
0.11 ± 0.07
|
0.76 ± 0.12
|
|
|
CL1476.Contig4_All
|
1,210
|
2.58 ± 0.28
|
25.97 ± 0.18
|
3.96 ± 0.59
|
11.75 ± 0.31
|
3
|
bHLH29
|
CL3060.Contig3_All
|
1,969
|
4.58 ± 0.35
|
5.55 ± 0.34
|
0.65 ± 0.08
|
20.36 ± 0.59
|
4
|
bHLH63
|
CL3993.Contig2_All
|
2,585
|
11.45 ± 0.29
|
1.94 ± 0.19
|
10.46 ± 0.86
|
0.55 ± 0.12
|
5
|
bHLH96-like
|
CL6326.Contig2_All
|
1,169
|
7.32 ± 0.95
|
1.53 ± 0.39
|
8.38 ± 1.96
|
0.31 ± 0.00
|
6
|
bHLH162-like
|
Unigene39374_All
|
565
|
6.33 ± 0.08
|
9.19 ± 2.35
|
3.67 ± 0.86
|
0.00 ± 0.00
|
7
|
bHLH137-like
|
Unigene631_All
|
2,674
|
2.42 ± 0.11
|
1.22 ± 0.23
|
2.42 ± 0.23
|
9.93 ± 1.18
|
|
|
Unigene632_All
|
2,532
|
0.64 ± 0.21
|
0.00 ± 0.00
|
0.57 ± 0.28
|
1.85 ± 0.87
|
8
|
bZIP
|
CL403.Contig9_All
|
2,216
|
2.50 ± 0.60
|
0.00 ± 0.00
|
3.33 ± 0.06
|
7.98 ± 0.55
|
9
|
MADS-SVP
|
CL11571.Contig4_All
|
1,481
|
0.30 ± 0.33
|
0.06 ± 0.01
|
0.55 ± 0.02
|
2.87 ± 0.28
|
10
|
MYB-like
|
CL12881.Contig13_All
|
3,418
|
0.12 ± 0.00
|
0.09 ± 0.01
|
2.92 ± 0.01
|
18.36 ± 0.64
|
11
|
MYB3R-5
|
Unigene3364_All
|
9,287
|
3.00 ± 0.10
|
2.36 ± 0.37
|
5.92 ± 0.04
|
0.22 ± 0.02
|
|
|
Unigene8633_All
|
3,902
|
5.73 ± 0.70
|
0.79 ± 0.24
|
6.34 ± 0.96
|
2.44 ± 1.39
|
12
|
MYB48-like
|
CL4642.Contig3_All
|
1,604
|
1.29 ± 0.13
|
1.57 ± 0.90
|
3.60 ± 0.02
|
0.23 ± 0.07
|
13
|
MYB49
|
Unigene3294_All
|
3,894
|
2.27 ± 0.23
|
7.28 ± 0.69
|
0.81 ± 0.08
|
0.06 ± 0.01
|
14
|
MYB61
|
CL3951.Contig1_All
|
1,557
|
9.94 ± 1.86
|
12.47 ± 0.52
|
8.14 ± 0.04
|
35.59 ± 1.27
|
|
|
CL3951.Contig4_All
|
1,580
|
6.72 ± 0.51
|
7.01 ± 0.42
|
6.77 ± 1.07
|
30.85 ± 0.60
|
15
|
MYB72
|
CL9015.Contig2_All
|
934
|
10.63 ± 0.12
|
40.00 ± 0.23
|
3.44 ± 0.04
|
2.72 ± 0.30
|
16
|
MYB113
|
CL263.Contig3_All
|
870
|
0.00 ± 0.00
|
0.09 ± 0.12
|
4.65 ± 0.13
|
7.05 ± 0.64
|
|
|
CL263.Contig4_All
|
1,818
|
0.13 ± 0.01
|
0.69 ± 0.11
|
1.00 ± 0.04
|
3.20 ± 0.67
|
|
|
CL2927.Contig1_All
|
978
|
0.00 ± 0.00
|
0.57 ± 0.04
|
1.68 ± 0.65
|
3.63 ± 0.40
|
17
|
NAC21/22
|
Unigene3776_All
|
1,599
|
0.13 ± 0.04
|
0.04 ± 0.06
|
1.53 ± 0.20
|
10.45 ± 0.81
|
|
|
Unigene4273_All
|
1,677
|
0.29 ± 0.24
|
0.25 ± 0.09
|
1.31 ± 0.01
|
22.39 ± 2.94
|
18
|
TCP15
|
Unigene37688_All
|
1,362
|
5.71 ± 0.34
|
2.10 ± 0.31
|
5.30 ± 0.35
|
0.42 ± 0.28
|
19
|
WRKY1
|
CL6289.Contig1_All
|
1,375
|
0.00 ± 0.00
|
0.00 ± 0.00
|
2.77 ± 0.13
|
29.50 ± 4.60
|
20
|
WRKY33
|
CL12462.Contig1_All
|
1,861
|
0.15 ± 0.13
|
0.02 ± 0.03
|
0.40 ± 0.04
|
3.31 ± 0.65
|
21
|
WRYK69
|
CL10325.Contig8_All
|
1,977
|
9.09 ± 0.11
|
2.80 ± 1.59
|
9.16 ± 0.92
|
22.36 ± 1.46
|
22
|
PRP4-like protein (contains WD40 repeats)
|
CL293.Contig68_All
|
1,882
|
0.05 ± 0.02
|
0.51 ± 0.05
|
0.75 ± 0.09
|
4.36 ± 1.26
|
23
|
autophagy-related protein 18b (contains WD40 repeats)
|
Unigene11308_All
|
3,223
|
0.45 ± 0.09
|
1.69 ± 0.06
|
1.76 ± 0.03
|
1.56 ± 0.41
|
24
|
WD40 repeat-containing protein 23
|
CL6654.Contig5_All
|
1,415
|
0.62 ± 0.47
|
3.17 ± 0.08
|
2.85 ± 0.95
|
2.33 ± 0.58
|
25
|
WD40 repeat-containing protein
|
CL3532.Contig6_All
|
3,410
|
0.53 ± 0.38
|
2.26 ± 0.05
|
0.02 ± 0.02
|
0.00 ± 0.00
|
|
WD40 repeat-containing protein
|
CL6654.Contig3_All
|
1,543
|
1.13 ± 0.93
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.11 ± 0.16
|
|
WD 40 repeat protein
|
CL8837.Contig1_All
|
697
|
11.53 ± 0.87
|
1.64 ± 0.67
|
2.05 ± 0.53
|
2.56 ± 0.74
|
26
|
WD repeat-containing protein 43
|
CL4426.Contig1_All
|
1,267
|
0.77 ± 0.21
|
0.00 ± 0.00
|
2.94 ± 0.43
|
0.00 ± 0.00
|
27
|
WD repeat domain-containing protein 83
|
Unigene23099_All
|
2,401
|
3.20 ± 0.39
|
0.12 ± 0.07
|
1.29 ± 0.30
|
1.27 ± 0.11
|
28
|
zinc-finger homeodomain protein 3
|
CL13045.Contig26_All
|
2,855
|
1.27 ± 0.20
|
0.56 ± 0.25
|
4.04 ± 0.36
|
0.00 ± 0.00
|
3.10. Validation of candidate DEGs by qRT-PCR
In order to further verify the reliability of the RNA-Seq data, 14 genes from the DEGs related to the flavonoid synthesis and cholophyll metabolism were randomly selected, and validated them using qRT-PCR. The qRT-PCR results showed that most of these selected genes had similar expression patterns with those identified in the RNA sequencing data (Fig. 8), which indicated that the transcriptome sequencing data had high repeatability and accuracy.