Analysis of Differentially Expressed Genes in Tissues of Camellia sinensis during Dedifferentiation and Root Redifferentiation

Tissue culture is very important for identifying the gene function of Camellia sinensis (L.) and exploiting novel germplasm through transgenic technology. Regeneration system of tea plant has been explored but not been well established since the molecular mechanism of tea plant regeneration is not clear yet. In this study, transcriptomic analysis was performed in the initial explants of tea plant and their dedifferentiated and redifferentiated tissues. A total of 93,607 unigenes were obtained through de novo assembly, and 7,193 differentially expressed genes (DEGs) were screened out from the 42,417 annotated unigenes. Much more DEGs were observed during phase transition rather than at growth stages of callus. Our KOG and KEGG analysis, and qPCR results confirmed that phase transition of tea plant was closely related to the mechanism that regulate expression of genes encoding the auxin- and cytokinin-responsive proteins, transcription factor MYB15 and ethylene-responsive transcription factor ERF RAP2-12. These findings provide a reliable foundation for elucidating the mechanism of the phase transition and may help to optimize the regeneration system by regulating the gene expression pattern.

www.nature.com/scientificreports www.nature.com/scientificreports/ with the reference sequences in the Nr, Swiss-Prot, KEGG, COG, KOG, GO and Pfam databases. 51,190 unigenes (54.7%) did not show significant similarity with known genes, while 42,417 unigenes (45.3%) were annotated at least in one database, of which 16,120 sequences were above 1000 bp in length ( Table 2).
Differentially expressed genes during phase transition. Analysis showed that 7,193 differentially expressed genes (DEGs) were obtained after comparison of the expression level between these samples according to the threshold of q value < 0.005 & |log 2 (fold change)| > 1. Although the calli were derived from different explants, these tissues were clustered into a branch according to the gene expression behavior; similarly, the redifferentiated roots derived from stem and leaf were also clustered together (Fig. 2). More than 3,000 DEGs were observed during the phase change from stem explant to stem-derived primary callus (3,014 DEGs) and from leaf explant to leaf-derived primary callus (3,495 DEGs), respectively (Fig. 3). Among them, much more genes were down-regulated (2,133 down vs. 881 up in stem-derived tissues and 2,574 down vs. 921 up in leaf-derived tissues) during these phase changes. Similarly, a large number of DEGs were also found during the phase transition from stem-derived callus to its regenerated root (2,610) and from leaf-derived callus to its regenerated root (2,744), however, more genes were up-regulated (723 down vs. 1,887 up in stem-derived tissues and 937 down vs.  Table 2. Summary of the unigene annotation in different databases. www.nature.com/scientificreports www.nature.com/scientificreports/ 1,807 up in leaf-derived tissues) during root redifferentiation. Meanwhile, relatively fewer DEGs were found during the growth stage from stem-derived primary callus to stem-derived callus (100 down vs. 101 up) and from leaf-derived primary callus to leaf-derived callus (227 down vs. 200 up). It was clear that much more genes were required to change their expression patterns during the phase transition such as dedifferentiation and redifferentiation, while fewer genes changed their expressions at various growth periods. Similar change trends of up-and down-regulated DEGs were obtained from different initial explants (stem and leaf) during dedifferentiation and redifferentiation, indicating that the various phases or statuses of tissue culture could be determined by a set of DEGs and epigenetic variation of these genes might profoundly impact on the fate of the regenerated cells. Interestingly, the number of the down-and up-regulated genes changed reversely during dedifferentiation and redifferentiation, indicating differentiated cells required to activate much more genes for exerting special function of the cells.
KOG classification of the DEGs. KOG analysis showed that around 80% DEGs were mainly categorized into the classes of the "general function prediction only", "posttranslational modification, protein turnover, chaperones", "signal transduction mechanisms", "secondary metabolites biosynthesis, transport and catabolism", "carbohydrate transport and metabolism", "transcription", "inorganic ion transport and metabolism", "energy production and conversion", "amino acid transport and metabolism", and "translation, ribosomal structure and biogenesis", although number of the DEGs among the samples with various tissue culture status was different in these classes (Fig. 4), indicating that the expression change of the genes associated with these classes might be very important for phase transition during tissue culture since levels in the secondary metabolites, proteins, carbohydrates, inorganic ions, energetic metabolism and amino acids are likely quite different between differentiated tissues (such as leaf, stem and root) and dedifferentiated ones.
KEGG pathway enrichment of the DEGs. When the primary callus was formed from the stem explant, the up-regulated DEGs significantly enriched in the KEGG item of "glutathione metabolism", "zeatin biosynthesis", "DNA replication", "nitrogen metabolism", "nicotinate and nicotinamide metabolism", and "plant hormone signal transduction"; while the down-regulated DEGs did in the item of "photosynthesis", "ribosome", "photosynthesis-antenna proteins", "flavonoid biosynthesis", and "cyanoamino acid metabolism" (Fig. 5). When the root was regenerated from the stem-derived callus, the enriched items of the up-regulated DEGs were "phenylpropanoid biosynthesis", "flavonoid biosynthesis", "phenylalanine metabolism", "photosynthesis-antenna proteins", "ribosome", "photosynthesis", as well as "cutin, suberine and wax biosynthesis"; the significantly enriched item of the down-regulated DEGs was "plant hormone signal transduction". During the phase transition from leaf explant to primary callus, the pathway of "plant hormone signal transduction", "DNA replication", "zeatin biosynthesis", and "glutathione metabolism" was significantly up-regulated, while pathway of "photosynthesis", "photosynthesis-antenna proteins", "flavonoid biosynthesis", "porphyrin and chlorophyll metabolism", "starch and sucrose metabolism", "galactose metabolism", and "carbon fixation in photosynthetic organisms" was significantly down-regulated, besides the remarkable change of the "phenylpropanoid biosynthesis". Compared with the leaf-derived callus, the pathway of "phenylpropanoid biosynthesis", "flavonoid biosynthesis", "phenylalanine metabolism", "photosynthesis -antenna proteins", "photosynthesis", "cutin, suberine and wax biosynthesis", as well as "stilbenoid, diarylheptanoid and gingerol biosynthesis" was up-regulated in the regenerated root, while the pathway of "zeatin biosynthesis" was down-regulated significantly, besides remarkable change of the pathway www.nature.com/scientificreports www.nature.com/scientificreports/ of "ribosome". It was apparent that up-regulation of "plant hormone signal transduction", "zeatin biosynthesis", "DNA replication" and "glutathione metabolism" pathways, as well as down-regulation of the photosynthesis-and secondary metabolism-related pathways were necessary for dedifferentiation of the explants although change of pathways might also be influenced by the various initial explants [28][29][30][31][32] . Meanwhile, root redifferentitation requires the slowdown of cell division through deactivating the signal transduction pathway, and activating the pathway related to biosynthesis of the cutin, suberine, wax, and phenylpropanoid 33 . Plastid might also be rebuilt in the rededifferentiated cell through up-regulating the photosynthesis-related pathways.

Expression verification of auxins and CKs regulation-related genes.
As the pathway related with plant hormone signal transduction was significantly fluctuated during dedifferentiation and redifferentiation, nine DEGs, being considered to be closely related to regulation of the auxins and CKs during phase transition, were screened out (Table 3) . The expression pattern of these genes during phase transition was verified by qPCR (Fig. 6). The result showed that expression profiles of these 9 genes in the dedifferentiated and redifferentiated tissues derived from initial stem and leaf explants were quite similar to those obtained from the transcriptomic analysis, with a correlation coefficient being 0.8745 (p < 0.01) (Fig. 7). Among these 9 genes, ARR5, GH3.1, IAA18, IAA29, CYCD3-1, CDKB2-2 and MYB15 were up-regulated in primary callus and callus then www.nature.com/scientificreports www.nature.com/scientificreports/ down-regulated in the regenerated root, while the expressions of ARF18 and ERF RAP2-12 were down-regulated in primary callus and callus but up-regulated in the regenerated roots. These validations indicated that transcriptome analysis faithfully revealed the gene modulation mechanism during the phase changes.

Discussion
Exogenous plant growth regulators are usually applied to induce specific cell types and/or organs during dedifferentiation and redifferentiation of tissue culture. In this study, 2,4-dichlorophenoxyacetic acid (2,4-D, belonging to auxins) and 6-benzylaminopurine (BAP, belonging to CKs) were used for inducing callus from stems and leaves of tea plant. The transcriptome and qPCR results revealed that many DEGs, especially the hormone-related genes, were significantly triggered by the growth regulators treatment, and finally initiated the dedifferentiation process.  www.nature.com/scientificreports www.nature.com/scientificreports/ As shown in Fig. 8, the cell division and callus formation might be induced and facilitated through combination of the two regulating pathways. One was auxin-related pathway, in which the expression of auxin-responsive protein encoding gene IAA18 and IAA29 were up-regulated by addition of exogenous 2,4-D, the increased gene products of the IAA18 and IAA29 would then accelerate the release of the ARFs through combination with different TIR1/AFB (transport inhibitor response 1/auxin-related F-box protein) and ARF proteins and activating the ubiquitinoylation, then the excited ARFs would up-regulate the expression of the downstream gene CDK B2-2 [34][35][36] . The previous reports concerning early gene response to auxin treatment also showed that Aux/IAA are short-lived nuclear proteins and mostly have four conserved domains, where the domain III and IV are responsible for dimerization with other Aux/IAA or heterodimerization with auxin responsive factors (ARFs), and the domain II contains the degron motif and can interact directly with the TIR1/AFB and auxin, leading to the ubiquitination and degradation via 26 S proteasome at higher auxin levels and consequently releasing ARFs to regulate the expression of downstream genes 37,38 . Another was CK-related pathway, in which the expression of response regulator gene B-ARRs (B-type Arabidopsis response regulators) and A-ARR5 were all phosphorylated through AHKs (Arabidopsis histidine kinases) → AHPs (Arabidopsis histidine-containing phosphotransfer proteins) phosphorelay signaling way 39 , and cyclin gene CYC D3-1 was up-regulated as the downstream gene of the phosphorylated B-ARRs, due to the addition of BAP. Our result confirmed that dedifferentiation process is also closely related to the CKs modulation pathway which has been reported in Arabidopsis. When the cell is contacted with CKs, transmembrane receptors, AHKs will transmit signals to the nucleus via the phosphorelay pathway, leading to phosphorylation and activation of transcription factors B-ARRs 40 , B-type ARRs then sequentially induce the expression of some response genes, including A-ARRs 41 and cyclin (CYC) 42 . In addition, extremely up-regulated transcription factor MYB15 was also observed, indicating this gene might also be involved in modulation of the dedifferentiation because MYB15 could modulate the expression of auxin-inducible genes by interacting with ARFs 43 and affect the plant growth and development 34 . Our study also revealed that a GH3 family protein encoding gene GH3.1 was significantly up-regulated in callus, which was consistent with other previous finding that GH3 can maintain auxin homeostasis by conjugating excessive indole-3-acetic acid to amino acids in the presence of a higher level auxin 44 . The up-regulated expression of the MYB15, IAA18 and IAA29 could promote auxin signaling pathways through interactions with related ARFs; up-regulating CDK B2-2 and CYC D3-1, involved in auxin and CKs signaling pathways, might be necessary for callus induction of tea plant. The up-regulation of GH3.1 and A-ARR5 might be a response of tissues against high hormonal levels. After the genes related to signaling transduction changed their expression, many downstream pathways including DNA replication, zeatin biosynthesis, glutathione metabolism and photosynthesis were also be modulated. Zeatin biosynthesis and DNA replication were modified for initiating cell division to form callus [28][29][30] , while photosynthesis was changed due to dedifferentiation of chloroplast. Initiation and proliferation of the callus require the up-regulation of glutathione metabolism to deal with the antioxidant defense, nutrient requisition, and regulation of cellular events 31,32 . www.nature.com/scientificreports www.nature.com/scientificreports/ When the root was regenerated from the callus in the 1/2 MS medium supplemented with 1.5 mg/L naphthalene acetic acid (NAA), the expression level of IAA18 and IAA29 were quite lower than that in callus stage, but similar to that in initial explants; while the genes encoding the auxin-responsive factor ARF18 and ethylene-responsive transcription factor ERF RAP2-12 were remarkably up-regulated compared with the callus stage. According to the published reports, auxin application could promote expression of the ACS4 gene encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase to induce ethylene biosynthesis 45 , leading to enhanced ethylene level and up-regulating expression of ethylene-responsive transcription factor. Furthermore, ethylene could influence auxin level and further facilitate auxin signaling pathway 46 through modulating the expression of the weak ethylene insensitive 2 and 7 (WEI2 and WEI7) which encoded the rate-limiting enzyme anthranilate synthase in the tryptophan biosynthesis 47 . Thus, up-regulating expression of ARF18 and ERF RAP2-12, and down-regulating expression of IAA18 and IAA29 might be essential for root induction, as well as changes in gene expression of some downstream pathways, such as cutin, suberine, wax, and phenylpropanoid metabolism which is required to form the specialized cell wall through activating the pathway related to biosynthesis of the cutin, suberine, wax, and phenylpropanoid 33 .

Methods and Materials
Preparation of tea plant samples. Seedlings of Camellia sinensis cultivar 'Jinxuan' derived through in-vitro technique were micropropagated and maintained on the Murashige and Skoog (MS) medium with addition of 2 mg/L 6-benzylaminopurine (BAP), 0.1 mg/L naphthalene acetic acid (NAA), 30 g/L sucrose and 9 g/L agar through single-node culture. A callus inducing medium (CIM) was prepared and autoclaved at 121 °C for 20 min after adding 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.4 mg/L BAP, 30 g/L sucrose and 9 g/L agar into MS medium, and a root inducing medium (RIM) was also prepared and autoclaved after adding 1.5 mg/L NAA, 30 g/L sucrose and 9 g/L agar into 1/2 MS medium. pH value of the all the media was adjusted to pH5.8.
The stems and leaves were disassociated from the seedlings and cut into small pieces (~0.5 cm for stem and ~0.5 cm 2 for leaf) and used as explants. The explants were inoculated onto the CIM and cultivated in tissue culture room at 24 ± 1 °C under the dark condition. The callus began to appear from the cuts of the stem explant (S-explant) after 10 days, and then surrounded the explant after 16 days. Sampling was conducted on the 10 th day and 16 th day, designated as S-primary callus and S-callus, respectively. The stem-derived callus was then inoculated onto the RIM, the root was induced after 15 days. Sampling was carried out and designated as S-root. Similarly, the leaf-derived primary callus and callus (L-primary callus and L-callus) were also obtained after cultivating the leaf explant (L-explant) on the CIM for 12 and 20 days, and redifferentiated root (L-root) was sampled after inducing the L-callus on the RIM for 20 days. The obtained samples were immediately frozen in liquid nitrogen and stored at −80 °C for further use. All the tests were conducted in biological triplicates.
Observation of the callus and regenerated root. In order to confirm the phase transition of the tissue culture, samples were embedded in paraffin and stained with aniline blue solution 48,49 . Observation was performed on a Nikon Eclipse E100 (Nikon Co., Ltd, Japan) with a Nikon DS-U3 image capture. RNA extraction. RNA extraction was carried out with RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instruction. The purity of RNA was checked by NanoPhotometer ® spectrophotometer (Implen, CA, USA). The concentration of RNA was measured on Qubit ® 2.0 Flurometer (Life Technologies, CA, USA) by using Qubit ® RNA Assay Kit. The integrity of RNA was assessed on Bioanalyzer 2100 system (Agilent Technologies, CA, USA) by using RNA Nano6000 Assay Kit. Transcriptome analysis. The mRNAs were enriched by using Oligo (dT) magnetic beads from the 3 μg total RNA for each sample. Sequencing libraries were generated using NEBNext ® Ultra ™ RNA Library Prep Kit for Illumina ® (NEB, USA) according to the manufacturer's recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, NEB, USA) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. Clean data were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Transcriptome assembly was accomplished from the clean data using Trinity software. Gene function of all non-redundant transcripts was annotated based on the database of the NCBI nonredundant protein sequences (Nr), Protein family (Pfam), Eukaryotic Orthologous Group/Clusters of Orthologous Groups of proteins (KOG/COG), a manually annotated and reviewed protein sequences (Swiss-Prot), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) by using BLASTALL package with the significant threshold of E-value ≤ 10 −5 . FPKM was used as expression strength of the gene, and differential expression analysis of two samples was performed using the DEGseq R package (ver. 2.1.0). p value was adjusted using q value and q value < 0.005 & |log2 (fold change)| > 1 was set as the threshold for significantly differential expression. Samples of two biological replicates at each culture stage were used for transcriptome analysis.
Quantitative real-time PCR analysis. To validate the gene expression results of transcriptome, qRT-PCR was employed to determine the expression of 9 genes related to auxins and cytokinins regulation (Table 3) on Applied Biosystems ™ StepOnePlus ™ Real-Time PCR System (ABI, Carlsbad, CA, USA) by using SYBR Premix Ex TaqTM II (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the previous paper 50 . PCR primers were designed by primer-blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with default parameters except the product size (100-250 bp), and the primers were ordered from Sangon Biotech (Shanghai) Co., Ltd. The PCR cycling conditions were: 40 cycles of 95 °C for 30 s, 55 °C for 5 s and 60 °C for 30 s. The melting-curves were analyzed during the reactions to ensure reaction specificity. The relative expression levels of the different