Identification and genomic distribution of CCT family in pear
In total, 42 PbCCTs were identified in the pear genome (Additional file 2). CCT family members were classified into COL, PRR, ZIM, TCR1 and ASML2 subfamilies, then systematically named according to their family name and sequence similarity. Our analysis revealed that COL subfamily consisted of the highest number of CCT genes in pear, with 33.3% (14 PbCOLs) of the total PbCCTs (Additional file 2). Both PRRs and ASMLs constituted the second largest subfamily with 19.0% (8 PbPRRs and 8 PbASML2s) of the PbCCTs. The ZIM and TCR1 subfamilies were smallest, with 14.3% (6 PbZIMs and 6 PbTCR1s) of the PbCCTs. The molecular weight of these PbCCT proteins ranged from 22.8 kD to 93.6 kD, and their values of isoelectric point were between 4.27 and 9.45.
PbCCTs were unevenly distributed over 15 of the 17 pear chromosomes, with no PbCCT gene found on chromosome 2 and 4 (Fig. 1). Among these, chromosome 1, 3, 8 and 12 contained the fewest PbCCTs with only one member (2.4%) on each chromosome, while chromosome 17 possessed the highest number of PbCCTs with five (11.9%) of the 42 members. However, it should be noted, eight PbCCTs were remained on as yet unmapped scaffolds. Among all identified PbCCTs, a total of seven segmental duplication events were identified in the pear genome (Fig. 1 and Additional file 3), indicating that segmental duplication events were the major contributors to the expansion of the pear CCT family. With one exception (PbTCR5-PbTCR6), the Ka/Ks ratios of the other duplicated pairs were less than 0.26, implying that the pear CCT gene family had mainly undergone strong purifying selection (Additional file 3).
To further explore the synteny relationships of CCT family genes between pear and the other two representative species, Arabidopsis (dicot) and rice (monocot), we performed interspecies comparative synteny analysis in a pairwise manner (Fig. 2). A total of 109 and 35 collinear CCT gene pairs were identified in the pear/Arabidopsis and pear/rice pairs, respectively (Additional file 4). The synteny analysis also showed that multiple pear CCT genes that correspond to single other plant species gene, for examples, PbCOL1/PbCOL6/PbCOL8-At5g24930 and PbPRR1/PbPRR2/PbPRR3/PbPRR8-At2g46790 (Additional file 4). These results indicated that CCT genes may have a common evolutionary ancestor among these species. Some CCT collinear gene pairs of pear/Arabidopsis were anchored to highly conserved collinear blocks, in which the number of syntenic gene pairs was up to 41, whereas none of counterparts of pear/rice pairs contained more than 28 genes (Additional file 4). The high level of syntenic conservation between the pear and Arabidopsis indicated that CCT genes in pear might share similar structures and functions with orthologous genes in Arabidopsis.
Phylogenetic analyses of CCT genes
To explore the phylogenetic relationship of the CCT family, an unrooted neighbor-joining phylogenetic tree was established based on the alignment of the full-length CCT protein sequences from the pear and Arabidopsis (Fig. 3). In most clades, internal nodes were supported by confidence values of at least 80%, indicative of good consistency in the topology, which further corroborates the reliability of the tree. In order to test the reliability of the tree topology, protein domain architecture was used to provide additional support for the proposed phylogeny. The majority of members belonging to the same phylogenetic group exhibited common motif compositions (Fig. 4D). For example, TIFY and C2C2-GATA zinc-finger domains are specifically shared by ZIM subfamily. Presence of the Pseudo-receiver domain is also clade dependent in the PRR subfamily. The conserved intron/exon structural characteristics also supported the fine structure of the phylogenetic trees. For example, all the coding sequences of the PbPRRs were interrupted by 5 or 7 introns, while the TCR1 subfamily contained no more than two introns.
According to the classification criteria of CCT family in Arabidopsis, PbCCTs were classified into seven major clades, named Clade I-VII (Fig. 3). 14 PbCOLs were categorized into three clades, with well-supported bootstrap values: five PbCOLs in Clade I, four PbCOLs in Clade II, and five PbCOLs in Clade III (Fig. 3 and Fig. 4A). Clade I comprised of five PbCOLs (PbCOL1/4/5/8/9), featuring a conserved CCT domain with two upstream zinc-finger B-box domains (Fig. 4D). PbCOL members (PbCOL2/3/6/7) in Clade II exhibited one B-box domain and a CCT domain. The gene structures of Clade I and II were highly conserved, containing two exons and one intron (Fig. 4C). The PbCOL homologs (PbCOL10-PbCOL14) were clustered with the Clade III of Arabidopsis COL subfamily that possesses a normal B-box domain, a second divergent B-box domain and a CCT domain. We found that this phylogenic classification of PbCOLs in Clade I and Clade II was the same as the classification of Arabidopsis COL subfamily based on the difference of the B-box domain (Fig. 3 and Fig. 4D). However, PbCOL10, PbCOL11 and PbCOL12 in Clade III contained only one or no B-box domain. These patterns suggest that the corresponding genes may have lost the B-box type zinc finger domain.
The PRR subfamily was further divided into three main subgroups based on their phylogenetic relationship, named as Clade IVa, Clade IVb and Clade IVc (Fig. 3). Four members (PbPRR1/4/5/6) in Clade IVa and two members (PbPRR2/3) in Clade IVb were highly conserved, containing eight exons and seven introns (Fig. 4C). Clade IVc included two pear PRR genes (PbPRR7/8) that clustered with AtPRR1 (AtTOC1) gene from the same branch (Fig. 3).
The TCR subfamily could be divided into two subgroups, i.e. Clade Va and Clade Vb (Fig. 3). For the Clade Va, PbTCR1 and PbTCR4 were clustered with two AtTCR genes (At5g57180 and At4g25990). Clade Vb contained four pear members (PbTCR2/3/5/6), which were clustered with two AtTCR genes (AtTCR1 and At5g14370).
All pear ZIM genes were also divided into two subgroups (Fig. 3). In detail, Clade VIa was comprised of three pear PbZIM members (PbZML4/5/6), which clustered with Arabidopsis ZIM subfamily genes (AtZIM, AtZML1 and AtZML2), while three pear ZIM homologs (PbZML1/2/3) were identified as a distinct subgroup (Clade VIb) that had no counterpart in Arabidopsis.
Pear ASML2 members were classified into four subgroups and were characterized by only conserved CCT domain (Fig. 3 and Fig. 4D). Clade VIIa and Clade VIIb were divided from the same branch, and contained PbASML3/4 and PbASML7/8, respectively. PbASML1/2 were clustered to Clade VIIc and PbASML5/6 belonged to Clade VIId.
Expression profiles of CCT genes in different tissues and under varying light signals environments
To investigate the tissue expression profiles of the PbCCTs in pear, we analyzed their transcript levels based on a publically available RNA-seq data of different tissues, including leaf, ovary, petal, shoot, stigma and fruit (Fig. 4B). In general, the candidate PbCCTs showed variation in tissue expression patterns. Many PbPRRs and PbCOLs exhibited high transcript abundance level in all six tissues, whereas most PbASMLs were expressed at relatively lower levels in multiple tissues. On the other hand, several PbCCTs exhibited tissue-specific expression. For example, PbCOL6 and PbTCR3 were mainly expressed in leaf, whereas PbCOL9 showed the highest transcript abundance in the petal. Some duplicated gene pairs also showed divergent transcript levels. For instance, PbZML3 showed very low expression in six different tissues; whereas its duplicated gene, PbZML2, was highly expressed in all tested tissues. These results suggest that duplicated genes may evolve to have diverse functions.
We investigate the environmental light spectrum changes among different pear tree canopy positions. Compared with the exterior part of the canopy, the levels of R and B light decreased significantly in the interior part of the canopy, indicating that intensity changes of light quality are important signatures in fruit orchards (Additional file 5). Partial genes from CCT family have showed to regulate growth and development by responding to light signals; therefore, the response of the PbCCTs induction to light quality treatments was further characterized by qRT-PCR. Overall, most of selected PbCCTs showed highly diverse expression patterns under the enhancement of R/B light radiation (Fig. 5 and Fig. 6). These results suggested that they are sensitive to light quality signals, thus, intriguing different responses according to the external light conditions. Under the R light treatments, the expression of three genes (PbCOL6, PbTCR1, PbTCR2) reached a peak at the early stage (R1000), and then down-regulated during subsequent increased exposure to R light (Fig. 5). Additionally, PbCOL11 and PbTCR3 presented an increasing trend with the increasing R light process. It was found that one homeologous pair (PbPPR2 and PbPPR3) displayed strong rhythmic expression patterns, suggesting that these genes could respond to R light changes during their regulation on pear growth and development. Noteworthy, compared with the control (R500), the abundance of PbPRR2 and PbTCR3 transcripts dramatically increased more than 13.5-fold at R2500 stage and 107.6-fold at R3000 stage, respectively. Meanwhile, we also analyzed the expression pattern of the PbCCTs in the B light treatment (Fig. 6). Among them, the transcript levels of four genes (PbPPR2, PbPRR3, PbTCR1 and PbTCR3) were induced by enhanced B light, some of which decreased markedly at the highest abundance (B3500). Notably, the relative expression level of PbPPR2 approximately increased 208.9-fold at B3000 stage relative to control and that of PbTCR3 approximately increased 172.0-fold at B3500 stage. In addition, two PbCCTs (PbCOL5 and PbTCR4) showed the decreased expression levels in response to progressively increasing B light signals.
PbPRR2 is a candidate for negatively regulating photosynthetic performance
Combining the previous transcriptomic study, present bioinformatics analysis and expression analysis [41], PbPRR2 (LOC103943360), a close homologue of Arabidopsis circadian clock gene AtPRR5, was chosen as a strong candidate for functional verification. Because AtPRR5 are implicated photomorphogenesis in R light that is considered as the most efficient wavelength for driving photosynthesis [52–55], these facts prompted us to examine the photosynthetic performance of plants overexpressing PbPRR2 under a broad range of R light intensity. PbPRR2 has a 2013-bp open reading frame and encodes a protein of 670 amino acids (GenBank accession number: MZ826141). The amino acid sequences encoded by PbPRR2 and AtPRR5 (AT5G24470) - the ortholog of PbPRR2 from Arabidopsis - are 40.12% identical (Additional file 6). PbPRR2 protein featured a PR domain at the N terminus and a CCT motif at the C terminus.
To further test the role of PbPRR2 in the regulation of photosynthetic properties under the changing R light signals, PbPRR2 was transiently over-expressed in N. benthamiana leaves and compared with the control leaves. Fluctuating profiles of net photosynthetic rate, stomatal conductance and internal CO2 were observed in both PbPRR2 infiltrated and control leaves with the increasing R light intensity (Fig. 7). However, significantly reduced levels of these photosynthetic parameters were observed in leaves inoculated with the pHEX2-PbPRR2 construct compared to the pHEX2-GUS control. These observations indicate that PbPRR2 may suppress the red light-dependent enhancement of photosynthetic performance.