- Overview of the SMRT sequencing
To obtain the gene expression profiles under sheltered and unsheltered conditions, SMRT sequencing and Illumina RNA-Seq were carried out for leaves and fruits at three developmental stages (DAF35, 45, 55). A total of 1,048,866 post-filter polymerase reads (21.55G) were scanned. The subreads from the same polymerase read sequence generated a circular consistent sequence (CCS), which yielded 685,339 CCS sequences. Among them, 542,795 full-length non-chimera (FLNC) sequences with 5′-primer, 3′-primer, and poly-A were obtained, including 79.20% of all CCSs being FLNCs (300–22,293 bp). This proportion varied slightly between the two tissues, at 34.96% in the leaf data set and 44.25% in the fruit data set. The mean length of FLNC reads is shown in Table 1, and the length distribution of FLNC is shown in Figure S1. FLNC length of leaf and fruit libraries >2 kb accounted for 63.95% and 65.21% of the corresponding FLNC, respectively. Comparison between the protein-coding gene transcripts and those of FLNC revealed strong concordance, which exhibited better recovery of large transcripts than previous Illumina RNA-Seq data for gene model prediction, particularly in the 2,500–4,000-bp size range [27].
Table 1 Summary of PacBio Sequel real-time sequencing
Library
|
Polymerase N50 lengtha
|
Subreads N50
lengthb
|
Mean number
of passesc
|
Number of CCSd
|
Number of FLNCe
|
Mean length
of FLNC
|
|
|
SL and UL1
|
41,750
|
4,031
|
10.11
|
69,562
|
52,291
|
3,468
|
|
SL and UL2
|
43,250
|
4,009
|
11.26
|
21,950
|
17,739
|
3,549
|
|
SL and UL3
|
36,750
|
2,808
|
10.73
|
278,447
|
231,786
|
2,886
|
|
SL and UL4
|
45,750
|
4,117
|
12.06
|
1,692
|
1,355
|
3,631
|
|
SF and UF
|
39,750
|
2,742
|
11.49
|
313,688
|
239,624
|
2,834
|
|
The four rows of “SL and UL1-4” represent the leaf library, and the row “SF and UF” represent the fruit library. aPolymerase, the original read generated by PacBio Sequel; bSubreads, Post-filter polymerase reads; cSequencing times of insert; dNumber of circular consensus sequences; eNumber of full-length non-chimeric.
- Annotation and functional classification of unigenes
The functions of unigenes were annotated by BLAST comparison and were predicted by comparative analysis with five databases. Among the 45,825 unigenes distributed to each of the databases, 45,747 (97.35% of the total) for Nr, 28,481 (60.61%) for COG, 38,629 (82.20%) for SwissProt, 18,202 (38.73%) for KEGG, and 26,065 (55.46%) for GO were investigated (Figure 1a).
Analysis of the Nr database indicated that the highest homologies of cherry were with Prunus mume or with P. persica, with 22,919 (50.09%) and 18,545 (40.53%) unigenes annotated, respectively. In total, 28,481 annotated unigenes were classified into 37 functional groups of the three GO main categories: nine groups for molecular function (MF), 18 for biological process (BP), and 10 for cellular component (CC) (Figure 1b). The top three GO terms for the classified genes were “protein binding” (4,812), “ATP binding” (3,907), and “protein kinase activity” (2,272) for MF; “protein phosphorylation” (2,273), “oxidation-reduction process” (1,833), and “signal transduction” (1,255) for BP; and “membrane” (1,656), “integral component of membrane” (1,571), and “nucleus” (748) for CC (Supplementary Table S2).
A total of 18,202 unigenes were distributed into 265 KEGG database pathways. The top three KEGG pathways were “Metabolism, Carbohydrate Metabolism” (1,719); “Environmental Information Processing, Folding, Sorting, and Degradation” (1,563); and “Genetic Information Processing, Translation” (1,491) (Figure 1c, Supplementary Table S3).
- Functional classification of DEGs under sheltered covering
After filtering the low-quality reads, 17.25 billion clean reads were acquired by Illumina RNA-Seq (Table S4). According to the Illumina data, pair-wise comparisons (UL vs. SL, UF vs. SF) of gene expression among the three stages were performed. In response to microclimatic change with low-PAR conditions, in total, 38,621 (UL35 vs. SL35, UL45 vs. SL45, and UL55 vs. SL55) and 3,584 (UF35 vs. SF35, UF45 vs. SF45, and UF55 vs. SF55) DEGs were detected from the GO database. Additionally, 38,621 DEGs for UL45 vs. SL45 and 2,871 DEGs for UF45 vs. SF45 were acquired (Figure 2a). Throughout the three developmental stages, a total of 6,911 and 1,755 DEGs were detected from the KEGG database in leaves and fruits; 6,868 and 1,552 DEGs for UL45 vs. SL45 and UF45 vs. SF45 were obtained, respectively (Figure 2b).
Compared with UL and UF, the upregulated genes from GO terms of SL were significantly enriched in “catalytic activity,” “biological process,” “oxidoreductase activity,” and “oxidation-reduction process,” among others (Figure 2c). The upregulated genes of SF were primarily associated with “catalytic activity,” “biological process,” “oxidoreductase activity,” and “oxidation-reduction process,” among others, at DAF45 (Figure 2d). Upregulated genes from KEGG terms of RL in contrast to UL included those associated with “circadian rhythm-plant,” “glyoxylate and dicarboxylate metabolism,” “porphyrin and chlorophyl metabolism,” and “carbon fixation in photosynthetic organisms,” among others (Figure 2e); moreover, the upregulated genes of RF were particularly associated with “biosynthesis of amino acids,” “phenylpropanoid biosynthesis,” “phenylalanine metabolism,” and “flavonoid biosynthesis,” among others, at DAF45 (Figure 2f).
Common expression patterns were employed to further analyze the DEGs between UL vs. SL and UF vs. SF at three stages; overall, 7,244 (leaf) and 1,707 (fruit) DEGs were placed into four clusters (Figure 3). Most of the candidate DEGs was categorized into either leaf Cluster 1 (3796 genes) or fruit Cluster 1 (1271 genes). For the top six enriched pathways in leaf Cluster 1, the DEGs exhibited peak expression at DAF45 of SL. For the top five accumulated pathways in leaf Cluster 2, the DEGs showed peak expression at DAF45 of UL (Figure 3a). Meanwhile, for the top four enriched pathways in fruit Cluster 1, the DEGs showed peak expression at DAF35 of SF. Finally, for the top five enriched pathways in fruit Cluster 2, the DEGs showed peak expression at DAF55 of SF (Figure 3b).
Transcriptional results indicated that the adaptability of cherry to low PAR began at DAF35. The adaptability of leaves to the microclimate was primarily attributed to the regulation of photosynthetic characteristics, assimilation, antioxidant status, as well as circadian rhythm. The genes implicated in the biosynthesis of anthocyanins and sugars in the sheltered fruits were upregulated at DAF35 until DAF55, reflecting the prior accumulation of nutrition in comparison to that under shelter-free conditions.
To confirm their authenticity, 12 DEGs were randomly selected to analyze their expression profiles by qRT-PCR. The results of qRT-PCR analysis showed that the expression profiles of the 12 DEGs were similar to those obtained through high-throughput sequencing (Supplementary Table S2). These results confirmed the reliability of the genome-wide transcriptome profiling analysis.
To verify the authenticity of the RNA-Seq results, 12 DEGs were randomly selected and their expression profiles were analyzed by qRT-PCR. The results of qRT-PCR analysis showed that the expression profiles of these DEGs were similar to those of RNA-Seq (Figure 4), confirming the reliability and accuracy of our RNA-Seq data.
- Expression of genes involved in photosynthetic system in sheltered leaves
To clarify the molecular adaptability of cherry trees to the microclimatic change during fruit development upon exposure to the shelter covering, genes involved in environmental sensitivity were screened out from the filtered DEGs for further investigation (Figure 4). Most of the DEGs encoding antenna proteins, or proteins involved in electron transport, reaction center in photosystem I (PSI) and photosystem II (PSII), as well as components of CO2 fixation, were highly expressed in SL compared with UL at DAF45 (Figure 4a).
More than 70 genes were annotated to three metabolic pathways: photosynthesis-antenna proteins (ko00196), photosynthesis (ko00195), and carbon fixation in photosynthetic organisms (ko00710). Therefore, we focused on the transcriptional levels of those genes closely related to photosynthetic efficiency (Figure 5a). All annotated DEGs of light-harvesting chlorophyll a/b binding protein complex I and II (LHCs), namely, 17 genes encoding the chlorophyl a/b binding protein complex I (Lcha1, Lcha2, Lcha3, Lcha4, Lcha5) and 20 genes encoding chlorophyl a/b binding protein complex II (Lchb1, Lchb2, Lchb3, Lchb4, Lchb5, Lchb6, Lchb7), were upregulated in SL at DAF45 (Figure 6a). Moreover, 13 genes encoding proteins involved in reaction center and electron transport in photosynthesis, including, PSI reaction center subunit X (PsaK), reaction center subunit VI (PsaH), PSI reaction center subunit PsaN (PsaN), PSII oxygen-evolving enhancer protein 2 (PsbP), PSII 10 kDa protein (PsbR), PSII repair protein Psb27-H1 (Psb27), ferredoxin of photosynthetic electron transport (PetF), and H+/Na+-transporting ATPase subunit beta (AtpF), were upregulated under sheltered conditions (Figure 6b), whereas one gene of cytochrome f complex (PetA) and one gene of H+/Na+-transporting ATPase subunit alpha (ATPF1AI) had lower transcription levels. Within the pathway of carbon fixation, D-ribulose 1,5-bisphosphate (RuBP) and CO2 produced 3-phosphate-glycerate (3-PGA) under the action of ribulose-bisphosphate carboxylase large chain (rcbL) and ribulose-bisphosphate carboxylase small chain (rcbS); then, 3-PGA was reduced to glyceraldehyde-3P (3-PGAld) by glyceraldehyde 3-phosphate dehydrogenase (GADPH) and glyceraldehyde-3-phosphate dehydrogenase (GAPA), which completes the energy storage process of photosynthesis and increases the production and accumulation of photosynthate; among them, one gene of rbcL, nine genes of rbcS, one gene of GADPH, and seven genes of GAPA were significantly upregulated under sheltered conditions. Notably, the expression of rbcL and GAPA was upregulated more than 10-fold (Figure 6c).
In combination with the PAR-Pn and CO2-Pn curves, the Pn of sheltered leaves was lower, but there were no significant differences in the first two stages of fruit development (Figure 7). The AQY and ACE of sheltered leaves visibly increased by 13.0% and 23.5%; meanwhile, the LCP and CCP parameters decreased to 13.87 and 75.62 µmol·m−2·s−1 at DAF45 (Table S5). In general, combined with photosynthetic characteristics and transcriptomic results of leaves, the findings illustrated that sheltered leaves had stronger abilities to capture and utilize the weak light, while also maintaining stable efficiency of CO2 utilization. This indicated the possibility of good adaptation to the weak light conditions under the sheltered covering in a short time.
- Expression of genes encoding photosynthetic pigments in sheltered leaves
The adaptability of plants to weak light is inextricably related to the photosynthetic pigmentation synthesis pathway [28]. In this study, 34 genes involved in porphyrin and chlorophyl metabolism (ko00860) and 1 gene related to each of terpenoid backbone biosynthesis (ko00900) and carotenoid biosynthesis (ko00906) in sheltered leaves were upregulated at DAF45 (Figure 5b). Regarding the DEGs annotated to chlorophyl synthesis and carotenoid biosynthesis, one gene encoding geranylgeranyl diphosphate reductase (CHLP), one gene encoding oxygen-independent coproporphyrinogen III oxidase (hemN), two genes encoding glutamyl-tRNA reductase (hemA), 23 genes encoding magnesium chelatase subunit H (ChlH), and two genes encoding magnesium chelatase subunit I (ChlI), among others, were upregulated under sheltered conditions at DAF45 (Figure 6d). Additionally, four genes of 15-cis-phytoene synthase (crtB), two genes of 15-cis-phytoene desaturase (PDS), one gene of prolycopene isomerase (crtISO), and two genes of lycopene beta-cyclase (lcyB) were upregulated under sheltered conditions at DAF45 (Figure 6e). The contents of photosynthetic pigments Chl a, Chl b and Car tended to increase under sheltered conditions. Moreover, the contents were higher than those under Cont; for example, Chl a increased by 14% to 16.7%, Chl b by 13.6% to 24%, and Car consistently increased by 22% under sheltered conditions (Figure 8). The transcription levels of genes related to chlorophyl and carotenoid synthesis in the sheltered leaves also fully confirmed that they would not be adversely affected by the sheltered covering (Figure 5b); on the contrary, sheltered covering would enhance the gene transcription levels, thereby increasing the pigment content.
- Expression of genes involved in antioxidant systems in sheltered leaves
ROS scavenging enzymes such as SOD, catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), as well as nonenzymatic antioxidants (glutathione, carotenoids, etc.), are essential for ROS detoxification [29]. Malondialdehyde (MDA) can be used as an indicator of lipid peroxidation under different stress conditions. Transcription levels of multiple genes relating to antioxidant capacity including peroxisome (ko04146), phenylpropanoid biosynthesis (ko00940), glutathione metabolism (ko00053), and carotenoid biosynthesis (ko00906) fluctuated slightly with the microclimatic conditions (Figure 5c). The expression of most antioxidant-related genes was clearly higher in SL than in UL at DAF45. For example, eight genes involved in peroxisome (encoding SOD, CAT), 32 genes involved in phenylpropanoid biosynthesis [encoding trans-cinnamate 4-monooxygenase, CYP; phenylalanine ammonia-lyase, PAL; 4-coumarate-CoA ligase, 4CL; cinnamyl-alcohol dehydrogenase, CAD; caffeic acid 3-O-methyltransferase, COMT; 5-O-(4-coumaroyl)-D-quinate-3′-monooxygenase, C3’H; caffeoyl-CoA O-methyltransferase, CCoAOMT; POD]; eight genes involved in glutathione metabolism (encoding glutathione synthetase, GSS; glutathione reductase, GSR; glutathione S-transferase, GST); and 16 genes involved in carotenoid biosynthesis (encoding zeaxanthin epoxidase, ZEP; violaxanthin de-epoxidase, VED; beta-carotene 3-hydroxylase, CrtZ; beta-ring hydroxylase, LUT5; lycopene beta-cyclase, lcyB; abscisic acid 8′-hydroxylase, CYP707A) showed markedly higher expression in SL than in UL at DAF45 (Figure 5c).
The antioxidant enzyme activities and MDA content in leaves also changed with the change of microclimate associated with sheltered conditions. At DAF35, there was no remarkable difference in the activities of SOD, POD, and CAT of SL and UL; however, the MDA content was higher under sheltered conditions. Moreover, the activities of antioxidant enzymes were higher than under shelter-free conditions at DAF45, while the MDA content was lower than in UL, with the POD, SOD, and CAT activities increasing by 1.7-, 1.9-, and 1.3-fold, respectively (Figure 9). These results showed that the activities of antioxidant enzymes in leaves increased during RSC, which maintained the ability of plants to scavenge ROS, and prevented the damage of membrane lipid peroxidation to leaves, thus preserving photosynthetic efficiency. The results showed the same trend as the results of gene expression levels.
- Expression of genes associated with anthocyanin synthesis in sheltered fruits
The anthocyanin content of mature cherry fruits under sheltered conditions increased significantly compared with that under unsheltered conditions [18]. The upregulated genes of fruits were mainly involved in phenylpropanoid biosynthesis (ko00940) and flavonoid biosynthesis (ko00941). The transcriptional levels of more than 25 genes in sheltered fruits began to increase from the DAF35 stage until fruit ripening (Figure 5d); these included 12 genes involved in phenylpropanoid biosynthesis, with one gene of PAL, six genes of C4H, and five genes of 4CL. Among these, the expressions of C4H and 4CL were upregulated more than 8.5- and 6.1-fold, respectively. Moreover, for 13 genes involved in flavonoid biosynthesis encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3'-hydroxylase (F3'H), dihydro-flavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), the expressions were remarkably higher in the sheltered fruits at DAF45 (Figure 10). The results showed that the synthesis of anthocyanin could be accelerated and the accumulation cycle could be prolonged in the sheltered fruits.