Characteristics of ZjCIPKs and ZjbHLH74-ZjCIPK5 regulated cold tolerance in jujube.

CIPKs are kind of serine/threonine (Ser/Thr) protein kinases which play important roles in response to biotic and abiotic stresses, and in plant growth and development. However, CIPKs in jujube (Ziziphus jujuba Mill.) had limited information, especially regarding their response to cold stress. In the current study, a total of 18 ZjCIPKs were identified in jujube genome which unevenly distributed on seven chromosomes. Conserved motif and gene structural analysis depicted them with conserved DEGLSA and APE motifs and similar structures. Phylogenetic analysis indicated that CIPKs were classified into five subgroups (I-V). In addition, three pairs of ZjCIPKs exhibited tandem duplication while the segmental duplication of ZjCIPKs was not identified. Study on the cis-acting elements indicted that stress or hormone related cis-acting elements were distributed unevenly on ZjCIPKs promoters and most ZjCIPKs were down- or up-regulated by the cold stress. VIGS induced silencing of ZjCIPK5 decreased the cold tolerance of sour jujube. Subcellular location analysis showed ZjCIPK5 located in nucleus. Moreover, transcription factor ZjbHLH74 which was induced at 6 h under cold stress could interact with the promoter of ZjCIPK5 to regulate jujube cold tolerance. These findings provided insights to a molecular basis of CIPK5 in jujube cold tolerance breeding for future.

A triphosphate tunnel metalloenzyme from pear (PbrTTM1) moonlights as an adenylate cyclase.

Adenylyl cyclase (AC) is the vital enzyme for generating 3',5'-cyclic adenosine monophosphate, an important signaling molecule with profound nutritional and medicinal values. However, merely, a dozen of AC proteins have been reported in plants so far. Here, a protein annotated as triphosphate tunnel metalloenzyme (PbrTTM1) in pear, the important worldwide fruit plant, was firstly identified to possess AC activity with both in vivo and in vitro methods. It exhibited a relatively low AC activity but was capable of complementing AC functional deficiencies in the E. coli SP850 strain. Its protein conformation and potential catalytic mechanism were analyzed by means of biocomputing. The active site of PbrTTM1 is a closed tunnel constructed by nine antiparallel ß-folds surrounded with seven helices. Inside the tunnel, the charged residues were possibly involved in the catalytic process by coordinating with divalent cation and ligand. The hydrolysis activity of PbrTTM1 was tested as well. Compared to the much higher capacity of hydrolyzing, the AC activity of PbrTTM1 tends to be a moonlight function. Through a comparison of protein structures in various plant TTMs, it is reasonable to speculate that many plant TTMs might possess AC activity as a form of moonlighting enzyme function.

Two triphosphate tunnel metalloenzymes from apple exhibit adenylyl cyclase activity.

Adenylyl cyclase (AC) is the key catalytic enzyme for the synthesis of 3',5'-cyclic adenosine monophosphate. Various ACs have been identified in microorganisms and mammals, but studies on plant ACs are still limited. No AC in woody plants has been reported until now. Based on the information on HpAC1, three enzymes were screened out from the woody fruit tree apple, and two of them (MdTTM1 and MdTTM2) were verified and confirmed to display AC activity. Interestingly, in the apple genome, these two genes were annotated as triphosphate tunnel metalloenzymes (TTMs) which were widely found in three superkingdoms of life with multiple substrate specificities and enzymatic activities, especially triphosphate hydrolase. In addition, the predicted structures of these two proteins were parallel, especially of the catalytic tunnel, including conserved domains, motifs, and folded structures. Their tertiary structures exhibited classic TTM properties, like the characteristic EXEXK motif and ß-stranded anti-parallel tunnel capable of coordinating divalent cations. Moreover, MdTTM2 and HpAC1 displayed powerful hydrolase activity to triphosphate and restricted AC activity. All of these findings showed that MdTTMs had hydrolysis and AC activity, which could provide new solid evidence for AC distribution in woody plants as well as insights into the relationship between ACs and TTMs.