Leaf growth parameters
The leaf growth traits were significantly different from control after UV-B treatment (Table 2). The leaf area was increased by 1.98 times, the leaves number, leaf width, leaf length and whole-plant fresh weight (FW) increased after UV-B radiation, and were 132.0 ± 6.6, 9.24 ± 0.46 cm, 17.32 ± 0.87 cm, and 247 ± 12.36 g, respectively. The results from table 1 showed that leaf thickness and biomass were significantly higher than that of the control after UV treatment.
Table 2 The effects of UV-B treatment on the growth parameters of EUO leaves
Leaf traits
|
Control
|
UV-B
|
Number of leaves
|
139.00±6.95a
|
132.00±6.6a
|
Leaf width (cm)
|
6.62±0.33a
|
9.24±0.46a
|
Leaf length (cm)
|
12.59±0.63b
|
17.32±0.87a
|
Leaf area (cm2)
|
61±3.05b
|
121±6.05a
|
Leaf thickness (mm)
|
0.23±0.012b
|
0.31±0.016a
|
Leaf fresh weight (g)
|
163.77±8.19b
|
247.12±12.36a
|
Leaf dry weight (g)
|
57.39±2.87b
|
92.84±4.64a
|
Values are means ± SD from 30 developing leaves.
Different lower-case letters show significant differences according to Duncan’s multiple test (P < 0.05). Leaf anatomy analysis
Anatomical observation of leaf cross-sections indicated that the epidermal cells were larger than the control (Fig. 1). Compared with control, the palisade tissue became long and loose, and the spaces between cells increased, the cells of the spongy tissue deform and become dispersed (Fig. 2). The palisade tissue cells of the leaf after UV-B treatment were 2.5 times larger than that of the control. These results indicate that the anatomy of the cells in the leaves changed significantly after UV-B treatment.
De novo assembly and sequence annotation
The clean reads were assembled into 62,287 transcripts and 42,333 unigenes using Trinity, among a total length of 70,449,627 bp and 41,056,860 bp, a mean length of 1,131 bp and 969 bp, and an N50 of 1,762 bp and 1,582 bp, respectively (Fig 2a). The quality of the whole sequencing was high and thus adequate for follow-up analyses. The 200-1000 bp transcripts accounted for 62% of the total transcripts; 22% (13,533) of the transcripts ranged from 1,100 to 1,900 bp; and 10,045 (16%) of the transcripts were longer than 2,000 bp. Most (29,473) of the unigenes ranged from 201 to 1,000 bp, accounting for 70% of the total unigenes; 18% (7,700) of the unigenes ranged from 1,100 to 1,900 bp; and 5,159 (12%) of the unigenes were longer than 2,000 bp (Fig. 2a). The differentially expressed genes (DEGs) were screened, and an MA map was generated to display the level of expression of all genes between groups (FPKM) and the distribution of DEGs (Fig. 2b). Differential gene cluster analysis showed that there were significant differences in gene expression patterns between the control and the UV-B treatment (Fig. 2c).
3.4. Sequence annotation and classification
After aligning the unigene sequences to protein databases, 42,333 unigenes were annotated. The percentage and numbers of unigenes annotated within the SwissProt, TrEMBL, nonredundant (NR), Pfam, Clusters of orthologous groups for eukaryotic complete genomes (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) databases were 14,988 (35.40%), 24,272 (57.30%), 24,500 (57.90%), 19,708 (46.60%), 19,523 (46.10%), 19,282 (45.5%) and 6,790 (16%), respectively (Table 2).
Table 2 The number of unigenes annotated to different databases
Unigene
|
SwissProt
|
TrEMBL
|
NR
|
Pfam
|
KOG
|
GO
|
KO
|
42333
|
14988
|
24272
|
24500
|
19708
|
19523
|
19282
|
6790
|
100%
|
35.40%
|
57.30%
|
57.90%
|
46.60%
|
46.10%
|
45.50%
|
16.00%
|
Functional classification of differentially expressed genes
The Fig 3a showed that the up-regulated genes involved in the translation, ribosomal structure and biogenesis[J] and Transcription[K] among the 19 categories in the information storage and processing; in the cellular processes and signaling, the up-regulated genes involved in posttranslational modification, protein turnover, chaperones[V]; Defense mechanism and Signal transduction mechanisms[T]; in the Metabolism, the up-regulated genes involved in Energy production and conversion[C]; Carbohydrate transport and metabolism[G] and Inorganic ion transport and metabolism[P]. Meantime, in the Information storage and processing, the down-regulated genes involved in Translation, ribosomal structure and biogenesis[J] and Replication, recombination and repair [L]; in the Cellular processes and signaling, the down-regulated genes involved in Posttranslational modification, protein turnover, chaperones[O]; in the Metabolism, the down-regulated genes involved in Carbohydrate transport and metabolism[G]; Energy production and conversion[C]; Amino acid transport and metabolism[E]. Compared with the up-regulated gene, the pathway of the down-regulated gene was significantly less than that of the up-regulated gene, only enriched to 4 metabolic pathways, ribosome, Flavonoid biosynthesis, Carbon fixation in photosynthetic organisms and one carbon pool by folate (Fig 3b). The up-regulated genes are enriched in Photosynthesis in the KEGG database, Phenylpropanoid biosynthesis, Plant hormone signal transduction, Starch and sucrose metabolism, Nitrogen metabolism, ABC transporters, Oxidative phosphorylation, Nicotinate and nicotinamide metabolism and Cyanoamino acid metabolism. 6,985 unigenes were annotated in the KEGG database and were assigned to 14 pathways. We studied explicitly the complex biological factors of genes and obtained pathway annotations for unigenes. (Fig 3c). In summary, the upregulated genes were mainly involved in Cell wall/membrane biogenesis and Plant hormone signal transduction after UV treatment, which might promote plant metabolism and accelerates plant growth.
DEGs Related to plant auxin and Phenylpropanoid biosynthesis
According to the results of KOG and KEGG, DEGs related to the Photosynthesis pathway, Plant auxin and Phenylpropanoid biosynthesis pathways (associated with cell wall biogenesis) were analyzed. In the field of plant hormone signal transduction, the related genes from the auxin response protein aux/iaa, and Saur family protein were upregulated (Fig 4), which could promote the elongation and growth of plant cells and accelerate plant growth. The CRE1 (EC: 2.7.13.3) high expression is involved in zeatin biosynthesis metabolism, which could also promote cell division and then accelerates plant growth.
In phenylpropanoid biosynthesis pathway, peroxidase [EC:1.11.1.7], beta-glucosidase [EC:3.2.1.21], and caffeic acid 3-O-methyltransferase [EC:2.1.1.68] were upregulated, and involved mostly in the biosynthesis of guaiacyl- lignin and 5-hydroxy-guaiacyl lignin (Fig 5).
Validation and expression analyses of key enzyme genes
To verify the changes of gene expression analyses, 10 candidate unigenes associated with leaf development were randomly selected, including the photosynthesis pathway (unigene TR10048 and unigene TR19418), plant auxin signal pathway (unigene TR11070 and unigene TR14945), phenylpropanoid biosynthesis (unigene TR11546, unigene TR11244, unigene TR11203, and unigene TR10148), and flavonoid biosynthetic process (unigene TR8189 and unigene TR15898). As shown in Fig. 6, these results of genes expression by RT-qPCR were identical to the differential expression from two libraries exhibited. Thus, it is useful to further investigate the genes related to leaf growth in Eucommia ulmoides Oliver (EUO).