Basic leucine zipper (bZIP), a conserved transcription factor widely found in eukaryotes, has important regulatory roles in plant growth. To understand the information related to the bZIP gene family in walnut, 88 JrbZIP genes were identified at the genome-wide level and classified into 13 subfamilies (A, B, C, D, E, F, G, H, I, J, K, M, and S) using a bioinformatic approach. The number of exons in JrbZIPs ranged from 1 to 12, the number of amino acids in JrbZIP proteins ranged from 145 to 783, and the isoelectric point ranged from 4.85 to 10.05. The majority of JrbZIP genes were localized in the nucleus. The promoter prediction results indicated that the walnut bZIP gene contains a large number of light-responsive and jasmonate-responsive action elements. The 88 JrbZIP genes were involved in DNA binding and nucleus and RNA biosynthetic processes of three ontological categories, molecular functions, cellular components and biological processes. The codon preference analysis showed that the bZIP gene family has a stronger bias for AGA, AGG, UUG, GCU, GUU, and UCU than other codons. Moreover, the transcriptomic data showed that JrbZIP genes might play an important role in floral bud differentiation. The results of a protein interaction network map and kegg enrichment analysis indicated that bZIP genes were mainly involved in phytohormone signaling, anthocyanin synthesis and flowering regulation. qRT-PCR demonstrated the role of the bZIP gene family in floral bud differentiation. Co-expression network maps were constructed for 29 walnut bZIP genes and 6 flowering genes, and JrCO (a homolog of AtCO) was significantly correlated (p < 0.05) with 13 JrbZIP genes in the level of floral bud differentiation expression, including JrbZIP31 (homolog of AtFD), and JrLFY was significantly and positively correlated with JrbZIP10,11,51,59,67 (p < 0.05), and the above results suggest that bZIP family genes may act together with flowering genes to regulate flower bud differentiation in walnut. This study was the first genome-wide report of the walnut bZIP gene family, which could improve our understanding of walnut bZIP proteins and provide a solid foundation for future cloning and functional analyses of this gene family.

The 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme for the process of ABA synthesis that plays key roles in a variety of biological processes. In the current investigation, genome-wide identification and comprehensive analysis of the NCED gene family in ‘Kuerle Xiangli’ (Pyrus sinkiangensis Yu) were conducted using the pear genomic sequence. In total, nineteen members of PbNCED genes were identified from the whole genome of pear, which are not evenly distributed over the scaffolds, and most of which were focussed in the chloroplasts. Sequence analysis of promoters showed many cis-regulatory elements, which presumably responded to phytohormones such as abscisic acid, auxin, etc. Synteny block indicated that the PbNCED genes have experienced strong purifying selection. Multiple sequence alignment demonstrated that these members are highly similar and conserved. In addition, we found that PbNCED genes were differentially expressed in various tissues, and three PbNCED genes (PbNCED1, PbNCED2, and PbNCED13) were differentially expressed in response to exogenous Gibberellin (GA₃) and Paclobutrazol (PP₃₃₃). PbNCED1 and PbNCED13 positively promote ABA synthesis in sepals after GA₃ and PP₃₃₃ treatment, whereas PbNCED2 positively regulated ABA synthesis in ovaries after GA₃ treatment, and PbNCED13 positively regulated ABA synthesis in the ovaries after PP₃₃₃ treatment. This study was the first genome-wide report of the pear NCED gene family, which could improve our understanding of pear NCED proteins and provide a solid foundation for future cloning and functional analyses of this gene family. Meanwhile, our results also give a better understanding of the important genes and regulation pathways related to calyx abscission in ‘Kuerle Xiangli’.

The GRF gene family plays an important role in plant growth and development as regulators involved in plant hormone signaling and metabolism. However, the Juglandaceae GRF gene family remains to be studied. Here, we identified 15, 15, 19, and 20 GRF genes in J. regia, C. illinoinensis, J. sigillata, and J. mandshurica, respectively. The phylogeny shows that the Juglandaceae family GRF is divided into two subfamilies, the ε-group and the non-ε-group, and that selection pressure analysis did not detect amino acid loci subject to positive selection pressure. In addition, we found that the duplications of the Juglandaceae family GRF genes were all segmental duplication events, and a total of 79 orthologous gene pairs and one paralogous homologous gene pair were identified in four Juglandaceae families. The Ka/KS ratios between these homologous gene pairs were further analyzed, and the Ka/KS values were all less than 1, indicating that purifying selection plays an important role in the evolution of the Juglandaceae family GRF genes. The codon bias of genes in the GRF family of Juglandaceae species is weak, and is affected by both natural selection pressure and base mutation, and translation selection plays a dominant role in the mutation pressure in codon usage. Finally, expression analysis showed that GRF genes play important roles in pecan embryo development and walnut male and female flower bud development, but with different expression patterns. In conclusion, this study will serve as a rich genetic resource for exploring the molecular mechanisms of flower bud differentiation and embryo development in Juglandaceae. In addition, this is the first study to report the GRF gene family in the Juglandaceae family; therefore, our study will provide guidance for future comparative and functional genomic studies of the GRF gene family in the Juglandaceae specie.

MIKC-type MADS-box transcription factors constitute one of the largest gene families in plants, playing pivotal roles in regulating plant growth and development, hormone signaling transduction, and responses to biotic and abiotic stresses. However, there have been no reports on the systematic identification and characterization of MIKC-type MADS-box proteins in walnuts. In this study, we identified 52 JrMADS genes in the walnut genome and transcriptome, and categorized them into 14 subfamilies through structural domain and phylogenetic tree analysis. It was found that these genes were unevenly distributed across 16 chromosomes. Within the MIKC-type MADS-box gene family, we identified three pairs of tandem-duplicated genes and 40 pairs of segmental duplicated genes, indicating that segmental duplication was the primary mechanism of gene amplification in walnut. Ka/Ks analysis showed that the family genes have undergone purifying selection during evolutionary processes. The promoter was predicted to contain cis-acting elements related to growth, development, plant hormones, and stress response. Expression profile analysis showed that JrMADS genes have different expression patterns in various tissues and developmental stages of male and female flower buds. Notably, an ancient clade of TM8 (JrMADS43) genes was found, which is absent in Arabidopsis but present in other flowering plants. Another gene, TM6 gene (JrMADS4), belongs to the AP3 subfamily and is a clade that has diverged from tomatoes. Through qPCR analysis, we verified the differential expression of JrMADS genes at different developmental stages (MB-1/2/3 and FB-1/2/3), with JrMADS5, JrMADS8, JrMADS14, JrMADS24, JrMADS40, JrMADS46, JrMADS47, JrGA3ox1, and JrGA3ox3 showing significantly higher expression in male than in female flower buds. In summary, our results provide valuable information for further biological functions research on MIKC-type MADS-box genes in walnut, such as flower organ development, and lays a solid foundation for future studies.