Rrelated with chlorophyll content in carnation flower petals [51]. Additionally, HCAR expression is strongly up-regulated during the stage in which etiolated A. thaliana seedlings turn green [52]. These results suggest that HCAR is essential for chlorophyll turnover during the greening stage. Three HCAR unigenes were identified in our database, and only one (unigene 168719) was significantly up-regulated during the G stage. High expression levels of this unigene in the G stage might contribute to the higher chlorophyll a concentrations during this stage compared with the other two stages (Fig. 5a). Pheophytinase (PPH) has a key function in chlorophyll degradation. The expression of PPH is induced in darkness, which accelerates chlorophyll degradation. In PPH mutant plants, chlorophyll degradation is inhibited, and the plants exhibit a sustained green phenotype during senescence [53]. In our study, the PRIMA-1 expression of PPH (unigene 5416) was lower in the YG stage than in the W and G stages (Fig. 5b), suggesting the lowest chlorophyll a degradation rate occurred during the YG stage. However, the chlorophyll a concentration in the YG stage was lower than that during the G stage. This may have been because the expression level for unigene 4795, which encodes chlorophyll synthase (CHLG), was significantly higher in the G stage PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 than in the YG stage (Fig. 5c). The higher chlorophyll a concentration in the G stage than in the YG stage might be because chlorophyll a was synthesized faster than it was degraded. Chlorophyllase (CLH) catalyzes the conversion of chlorophyll a to chlorophyllide a. In citrus plants, CLH expression levels are negatively correlated with chlorophyll contents [54]. We identified six candidate CLH unigenes in our database (Fig. 5b), four of which were significantly differentially expressed between the YG and G stages. The expression levels of unigene 56286 and unigene 90639 were up-regulated during the YG and W stages, respectively, while two unigenes were up-regulated in the G stage (unigene 11687 and unigene 131928) (Fig. 6c). Unigene 56286 and unigene 90639 might contribute to chlorophyll a degradation to lower the chlorophyll concentration more during the YG and W stages than in the G stage. The expression levels of PaO, which encodes pheophorbide a oxygenase, are closely correlated with the rate of chlorophyll breakdown [55]. Three PaO unigenes were detectedLi PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 et al. BMC Plant Biology (2016) 16:Page 10 ofFig. 5 Chlorophyll concentrations and the related unigenes. a The chlorophyll a and b concentrations were determined at different stages. The asterisk indicates a significant difference between the YG or W stages and the G stage (P < 0.05; Student's t-test). b Chlorophyll biosynthesis pathway. The bracketed numbers in red following each gene name indicate the number of corresponding unigenes identified in our database. c All differentially expressed genes involved in chlorophyll biosynthesis were hierarchically clustered and mapped using the fragments per kilobase of exon per million mapped reads values. Colors indicate the normalized signal intensity as defined in the barin our database, but only one (unigene 89999) was significantly up-regulated from the YG stage to the G stage. However, the chlorophyll content in the G stage was relatively high (Fig. 5a). Identical results were reported for carnations [51], potentially because the post-transcriptional regulation of PaO inhibits the activity of the encoded enzyme [56].