C2H2-Type Zinc Finger Proteins (DkZF1/2) Synergistically Control Persimmon Fruit Deastringency.

Hypoxic environments are generally undesirable for most plants, but for astringent persimmon, high CO2 treatment (CO2 > 90%), also termed artificial high-CO2 atmosphere (AHCA), causes acetaldehyde accumulation and precipitation of soluble tannins and could remove astringency. The multiple transcriptional regulatory linkages involved in persimmon fruit deastringency have been advanced significantly by characterizing the ethylene response factors (ERFs), WRKY and MYB; however, the involvement of zinc finger proteins for deastringency has not been investigated. In this study, five genes encoding C2H2-type zinc finger proteins were isolated and designed as DkZF1-5. Phylogenetic and sequence analyses suggested the five DkZFs could be clustered into two different subgroups. qPCR analysis indicated that transcript abundances of DkZF1/4 were significantly upregulated during AHCA treatment (1% O2 and 95% CO2) at day 1, DkZF2/5 at both day 1 and 2, while DkZF3 at day 2. Dual-luciferase assay indicated DkZF1 and DkZF2 as the activators of deastringency-related structural genes (DkPDC2 and DkADH1) and transcription factors (DkERF9/10). Moreover, combinative effects between various transcription factors were investigated, indicating that DkZF1 and DkZF2 synergistically showed significantly stronger activations on the DkPDC2 promoter. Further, both bimolecular fluorescence complementation (BiFC) and yeast two hybrid (Y2H) assays confirmed that DkZF2 had protein-protein interactions with DkZF1. Thus, these findings illustrate the regulatory mechanisms of zinc finger proteins for persimmon fruit deastringency under AHCA.

Phylogenetic, Molecular, and Functional Characterization of PpyCBF Proteins in Asian Pears (Pyrus pyrifolia).

C-repeat binding factor/dehydration-responsive element (CBF/DRE) transcription factors (TFs) participate in a variety of adaptive mechanisms, and are involved in molecular signaling and abiotic stress tolerance in plants. In pear (Pyrus pyrifolia) and other rosaceous crops, the independent evolution of CBF subfamily members requires investigation to understand the possible divergent functions of these proteins. In this study, phylogenetic analysis divided six PpyCBFs from the Asian pear genome into three clades/subtypes, and collinearity and phylogenetic analyses suggested that PpyCBF3 was the mother CBF. All PpyCBFs were found to be highly expressed in response to low temperature, salt, drought, and abscisic acid (ABA) as well as bud endodormancy, similar to PpyCORs (PpyCOR47, PpyCOR15A, PpyRD29A, and PpyKIN). Transcript levels of clade II PpyCBFs during low temperature and ABA treatments were higher than those of clades I and III. Ectopic expression of PpyCBF2 and PpyCBF3 in Arabidopsis enhanced its tolerance against abiotic stresses, especially to low temperature in the first case and salt and drought stresses in the latter, and resulted in lower reactive oxygen species (ROS) and antioxidant gene activities compared with the wild type. The increased expression of endogenous ABA-dependent and -independent genes during normal conditions in PpyCBF2- and PpyCBF3-overexpressing Arabidopsis lines suggested that PpyCBFs were involved in both ABA-dependent and -independent pathways. All PpyCBFs, especially the mother CBF, had high transactivation activities with 6XCCGAC binding elements. Luciferase and Y1H assays revealed the existence of phylogenetically and promoter-dependent conserved CBF-COR cascades in the pear. The presence of a previously identified CCGA binding site, combined with the results of mutagenesis of the CGACA binding site of the PpyCOR15A promoter, indicated that CGA was a core binding element of PpyCBFs. In conclusion, PpyCBF TFs might operate redundantly via both ABA-dependent and -independent pathways, and are strongly linked to abiotic stress signaling and responses in the Asian pear.

Genome wide identification and predicted functional analyses of NAC transcription factors in Asian pears.

BACKGROUND: NAC proteins contribute to diverse plant developmental processes as well as tolerances to biotic and abiotic stresses. The pear genome had been decoded and provided the basis for the genome-wide analysis to find the evolution, duplication, gene structures and predicted functions of PpNAC transcription factors. RESULTS: A total of 185 PpNAC genes were found in pear, of which 148 were mapped on chromosomes while 37 were on unanchored scaffolds. Phylogeny split the NAC genes into 6 clades (Group1- Group6) with their sub clades (~ subgroup A to subgroup H) and each group displayed common motifs with no/minor change. The numbers of exons in each group varied from 1 to 12 with an average of 3 while 44 pairs from all groups showed their duplication events. qPCR and RNA-Seq data analyses in different pear cultivars/species revealed some predicted functions of PpNAC genes i.e. PpNACs 37, 61, 70 (2A), 53, 151(2D), 10, 92, 130 and 154 (3D) were potentially involved in bud endodormancy, PpNACs 61, 70 (2A), 172, 176 and 23 (4E) were associated with fruit pigmentations in blue light, PpNACs 127 (1E), 46 (1G) and 56 (5A) might be related to early, middle and late fruit developments respectively. Besides, all genes from subgroups 2D and 3D were found to be related with abiotic stress (cold, salt and drought) tolerances by targeting the stress responsive genes in pear. CONCLUSIONS: The present genome-wide analysis provided valuable information for understanding the classification, motif and gene structure, evolution and predicted functions of NAC gene family in pear as well as in higher plants. NAC TFs play diverse and multifunctional roles in biotic and abiotic stresses, growth and development and fruit ripening and pigmentation through multiple pathways in pear.

DkNAC7, a novel high-CO2/hypoxia-induced NAC transcription factor, regulates persimmon fruit de-astringency.

Artificial high-CO2 atmosphere (AHCA, 95% CO2 and 1% O2) has been widely applied as a postharvest de-astringency treatment for persimmon fruit. AHCA increases expression of transcription factors, including ethylene response factors (DkERF), that target de-astringency genes. Here, the promoter of DkERF9, a previously characterized AHCA-inducible and de-astringency regulator, was utilized to screen a cDNA library by yeast one hybrid assay. A novel NAC transcription factor, named DkNAC7, was identified. Dual-luciferase assay indicated that DkNAC7 could not only trans-activate the promoter of DkERF9, but also activated the previously identified deastringency-related gene DkPDC2. Real-time PCR analysis showed that DkNAC7 was up-regulated by AHCA treatment, in concert with the removal of astringency from persimmon fruit and subcellular localization showed DkNAC7 was located in the nucleus. Thus, these results indicate that DkNAC7 is a putative transcriptional activator involved in regulating persimmon fruit deastringency by trans-activition on both DkERF9 and DkPDC2, which encodes pyruvate decarboxylase.