Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple ‘Golden Delicious’ genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Plant-pathogenic phytoplasmas secrete specific virulence proteins into a host plant to modulate plant function for their own benefit. Identification of phytoplasmal effectors is a key step toward clarifying the pathogenic mechanisms of phytoplasma. In this study, Zaofeng3, also known as secreted jujube witches’ broom phytoplasma protein 3 (SJP3), was a homologous effector of SAP54 and induced a variety of abnormal phenotypes, such as phyllody, malformed floral organs, witches’ broom, and dwarfism in Arabidopsis thaliana. Zaofeng3 can also induce small leaves, dwarfism, and witches’ broom in Ziziphus jujuba. Further experiments showed that the three complete α-helix domains predicted in Zaofeng3 were essential for induction of disease symptoms in jujube. Yeast two-hybrid library screening showed that Zaofeng3 mainly interacts with proteins involved in flower morphogenesis and shoot proliferation. Bimolecular fluorescence complementation assays confirmed that Zaofeng3 interacted with these proteins in the whole cell. Overexpression of zaofeng3 in jujube shoot significantly altered the expression patterns of ZjMADS19, ZjMADS47, ZjMADS48, ZjMADS77, and ZjTCP7, suggesting that overexpressing zaofeng3 might induce floral organ malformation and witches’ broom by altering the expression of the transcriptional factors involved in jujube morphogenesis.

Controlling the tree size of fruit species such as peach can reduce the amount of labor and input needed for orchard management. The phytohormone gibberellin (GA) positively regulates tree size by inducing degradation of the GA signaling repressor DELLA. The N-terminal DELLA domain in this protein is critical for its GA-dependent interaction with the GA receptor GID1 and the resulting degradation of the DELLA protein, which allows for growth-promoting GA signaling. In this study, a DELLA family member, PpeDGYLA, contains a DELLA domain but has amino acid changes in three conserved motifs (DELLA into DGYLA, LEQLE into LERLE, and TVHYNP into AVLYNP). In the absence or presence of GA₃, the PpeDGYLA protein did not interact with PpeGID1c and was stable in 35S-PpeDGYLA peach transgenic callus. The overexpression of PpeDGYLA in both polar and Arabidopsis showed an extremely dwarfed phenotype, and these transgenic plants were insensitive to GA₃ treatment. PpeDGYLA could interact with PpeARF6-1 and -2, supposed growth-promoting factors. It is suggested that the changes in the DELLA domain of PpeDGYLA may, to some extent, account for the severe dwarf phenotype of poplar and Arabidopsis transgenic plants. In addition, our study showed that the DELLA family contained three clades (DELLA-like, DELLA, and DGLLA). PpeDGYLA clustered into the DGLLA clade and was expressed in all of the analyzed tissues. These results lay the foundation for the further study of the repression of tree size by PpeDGYLA.

Jujube production is threatened by jujube witches’ broom (JWB) disease, which is caused by JWB phytoplasma. The jujube TCP transcription factor (TF) ZjTCP16 may be involved in the interaction of jujube plants with JWB phytoplasma. In this study, qRT-PCR proved that the expression pattern of ZjTCP16 was altered by JWB phytoplasma. The gene functions of ZjTCP16 were analyzed by its overexpression in Arabidopsis and jujube, as well as knock-down in. The overexpression of ZjTCP16 in Arabidopsis and jujube resulted in dwarfism and small leaves, while the zjtcp16 CRISPR mutants were higher than the WT. Microscopic observation of paraffin sections of jujube stems showed that ZjTCP16 affected the size of cells. The interactions of ZjTCP16 with ZjAS2 and ZjLOB in both the cytoplasm and nucleus were demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Yeast one-hybrid (Y1H) assays and qRT-PCR further confirmed that ZjTCP16 affected the expression of genes involved in leaf morphogenesis and cell proliferation (ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6) at the mRNA level through the ZjAS2 and ZjLOB pathways. In conclusion, ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube.