| Cytokinin(CK) is an important hormone derived from adenine and involves in various aspects in plant development including escaping apical dominance, promoting shoot growth, inhibiting root growth, delaying leaf senescence and chlorophyll decomposition, stimulating leaf expansion, and responding biotic and abiotic stresses. Cytokinin oxidase/dehydrogenase(CKX; EC.1.5.99.12), a flavoenzyme irreversibly degrading CK into adenine/adenosine moiety, is critical for maintaining cytokinin homeostasis in plant kingdom. CKX-overexpressing plants showed increased tolerance to drought and salinity stress. In addition, inhibition of CKX expression caused the accumulation of CK in plant inflorescence meristems, which led to higher seed production.Mulberry, a perennial eco-economic tree, possesses the characterization of wide-distribution and strong adaptive capacities to environment. Mulberry fruit is called as “the third-generation fruit” because of its rich secondary metabolites such as anthocyanin, alkaloids and polyphenol. It has delicious flavor with sweet and acid and is extremely favored by people. However the mechanisms under mulberry important traits in stress resistance, rich in secondary metabolites as well as the different quantities of collective fruits among the resources or varieties are still unclear, which becomes a restrictive factor for mulberry science and industry. As we known that CKXs are important target genes involved in crop yield and stress tolerance in rice, wheat, tobacco and Arabidopsis. Since the biology function of mulberry CKX remains unknown, we then cloned and identified CKX genes in the mulberry genome. Their expression patterns in mulberry flowers, other tissues and under various stresses were investigated in order to explore the functions of mulberry CKX. Obtained data could lay the foundation for improving the production of mulberry and resistance to stresses.Such results will expand our knowledge of CKX functions in woody plants.Six mulberry CKX genes were identified based on the mulberry genome database and named as Mn CKX1~Mn CKX6. Their sequence characterizations were analyzed. The spatial-temporal expression patterns of Mn CKX were detected by real-time PCR(q RT-PCR). To study the functions of Mn CKX, Mn CKX-overexpressing transgenic tobacco plants were obtained. The following is our main results.Identification, cloning and bioinformatics analysis of Mn CKX and their spatial-temporal expression patternsSix Mn CKX genes were disrupted by introns with numbers varying from three to five. All of them had typical CKX domains, the FAD binding domain, and the cytokinin bind domain. Mn CKX5 and Mn CKX6 were tandemly duplicated pair according to the M. notabilis genome annotation results. The signal peptides were predicted in Mn CKX1, Mn CKX5 and Mn CKX6 using signal P software, which indicated that these three Mn CKX genes encoded secretory proteins. The numbers of glycation sites were significant different among Mn CKXs using Net Glycate sotware. The least numbers of glycation sites in Mn CKX3 were eight, while the most numbers in Mn CKX2 were 17. A phylogenetic tree was constructed using the CKX proteins sequences from Arabidopsis thaliana and M. notabilis. The results indicated that Mn CKX5 and Mn CKX6 were clustered into a group with the At CKX3, suggesting they probably had similar biological function. In addition, many types of cis-elements including abiotic and biotic stress responding elements were found in the upstream 1.5kb sequence of Mn CKX according to the Plantcare website, suggesting that Mn CKX genes maybe involved in the resistance of abiotic and biotic stresses.The expressions of Mn CKX in male flower, female flower, root, leaf, bark and shoot of M. notabilis were investigated. According to the q RT-PCR results, Mn CKX1 and Mn CKX6 had the highest levels of expression in root and shoot, respectively. In the female flower of M. notabilis, Mn CKX4 was exclusively expressed. Compared with other Mn CKX, Mn CKX2 and Mn CKX3 exhibited the lowest expression in the above six tissues. Transcriptome sequencing results indicated that Mn CKX2 probably was a male biased gene. To test this hypothesis, several different flowers of M. alba were used to detect the expression of Mn CKX2. Our results showed that the highest expression ofMnCKX2 was detected in the male flower.We further examined the effect of 6-BA on the expressions of Mn CKX. Under continuous 6-BA stimulation, the expression of Mn CKX5 was high and those of Mn CKX1 and Mn CKX2 were low in the M. notabilis seedlings. To test the effect of short period 6-BA stimulation on Mn CKX expressions, we treated the M. notabilis seedlings using 6-BA for 12 hours and 36 hours. We found that the expressions of Mn CKX1, Mn CKX2, Mn CKX3 and Mn CKX4 were lightly up-regulated on 12 hours and then declined on 36 hours. However, the expressions of Mn CKX5 and Mn CKX6 were accumulated consistently. And the expression of Mn CKX6 was even over 50-fold compared with control.M. alba cv Pendula, with the minimum collective fruits per mulberry fruit, and Morus laevigata, with the maximum collective fruits per mulberry fruit were selected to study the expressions of Mn CKX in an effort to examine whether Mn CKX are involved in the development of reproductive organs. The results showed differences in the levels of expression of Mn CKX3 and Mn CKX4 in the two mulberry species. The expressions of Mn CKX3 and Mn CKX4 were higher in M. alba cv Pendula than those in M. laevigata. The results indicated that Mn CKX3 and Mn CKX4 would be involved in the development of mulberry reproductive organs.The expression patterns of Mn CKX genes in M. notabilis under abiotic stressWe analyzed the expression patterns of three candidate genes including Mn POD, Mn RD22 and Mn CAT1 under drought and salt stresses. The expression of Mn POD was quantitative difference under the drought and salinity stresses, indicating that Mn POD was a relative good mulberry stress-marker gene.Based on the above results, we examined the expressions of Mn CKX genes under the drought and salinity stresses. The q RT-PCR results showed that expressions of Mn CKX1 and Mn CKX6 were continuously up-regulated under both stresses. The expressions of other Mn CKX genes were stable with light variation.Functional identification of Mn CKX1 and Mn CKX6 in transgenic tobacco Because of the high expression of Mn CKX1 and Mn CKX6 in root, shoot and under stress condition, we constructed two plant overexpression vectors harboring Mn CKX1 and Mn CKX6 and transformed them into tobacco using leaf discs transformation to investigate their function. GUS histochemical stains results showed that the staining were different among leaves in different transgenic tobaccos. The results of the genome PCR and q RT-PCR showed that the transgenic tobaccos were successfully obtained. The transgenic positive tobaccos had the stunted shoots traits, indicating that the content of CK was probably decreased in transgenic tobaccos over-expressed of Mn CKX1 and Mn CKX6. The transgenic tobaccos had typical cytokinin-deficient phenotype impacted on the development of shoots and underground parts. Leaf discs experiment indicated that chlorophyll content of transgenic tobacco CKX1-1 was higher than that of wild-type tobacco, which was consistent with the expression of Mn CKX1 in transgenic tobacco CKX1-1. The expression of Mn CKX6 in transgenic CKX6-3 was high, but chlorophyll content of CKX6-3 didn’t reach a significant level comparing with wild-type tobacco. The possible reason was that the activity of CKX enzyme ecoded by Mn CKX6 was lower than that ecoded by Mn CKX1.In the present study, six Mn CKX genes were identified using bioinformatics. Their spatial-temporal expression patterns were investigated and their functions were studied by transgenic technology. Data on the expressions of Mn CKX genes coupled with functional analysis provides valuable information for further studies of Mn CKX genes in the breeding and crop improvement. |
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