Genome-Wide Identification and Expression Analysis of the bHLH Transcription Factor Family and Its Response to Abiotic Stress in Mongolian Oak (Quercus mongolica).

The basic helix-loop-helix (bHLH) family, one of the largest families of transcription factors in plants, is extensively involved in the growth, development, and stress response of several woody plants. However, no systematic analysis of the bHLH gene family in Quercus mongolica has been reported. We characterize QmbHLH genes and identify the functions of QmbHLH proteins in Q. mongolica. We used bioinformatics approaches, qRT-PCR analysis, and RNA sequencing data to examine chromosomal distributions, gene structures, and conserved patterns, and identified 89 QmbHLH genes, which were divided into 21 subgroups based on the phylogenetic analysis of bHLH genes in Arabidopsis thaliana. Segmental replication played a more prominent role than tandem duplication in the expansion of the QmbHLH gene family. Based on patterns of tissue-specific expression, protein interactions, and cis-element analysis, QmbHLH genes may be extensively involved in the growth and development of Q. mongolica. In leaves, stems, and roots, 12 selected QmbHLH genes exhibited responsiveness to abiotic stresses (salt, cold, weak light, and drought). Our study facilitates follow-up functional investigations of the bHLH gene family in Q. mongolica and provides novel insights into bHLH superfamilies in woody plants.

Identification of Functional Brassinosteroid Receptor Genes in Oaks and Functional Analysis of QmBRI1.

Brassinosteroids (BRs) play important regulatory roles in plant growth and development, with functional BR receptors being crucial for BR recognition or signaling. Although functional BR receptors have been extensively studied in herbaceous plants, they remain largely under-studied in forest tree species. In this study, nine BR receptors were identified in three representative oak species, of which BRI1s and BRL1s were functional BR receptors. Dispersed duplications were a driving force for oak BR receptor expansion, among which the Brassinosteroid-Insensitive-1 (BRI1)-type genes diverged evolutionarily from most rosids. In oak BRI1s, we identified that methionine in the conserved Asn-Gly-Ser-Met (NGSM) motif was replaced by isoleucine and that the amino acid mutation occurred after the divergence of Quercus and Fagus. Compared with QmBRL1, QmBRI1 was relatively highly expressed during BR-induced xylem differentiation and in young leaves, shoots, and the phloem and xylem of young stems of Quercus mongolica. Based on Arabidopsis complementation experiments, we proved the important role of QmBRI1 in oak growth and development, especially in vascular patterning and xylem differentiation. These findings serve as an important supplement to the findings of the structural, functional and evolutionary studies on functional BR receptors in woody plants and provide the first example of natural mutation occurring in the conserved BR-binding region (NGSM motif) of angiosperm BRI1s.

Genome-wide identification of the auxin response factor (ARF) gene family in Magnolia sieboldii and functional analysis of MsARF5.

Auxin plays an essential role in flowering, embryonic development, seed dormancy, and germination. Auxin response factors (ARFs) are plant-specific key transcriptional factors in mediating the gene expression network of auxin signaling. Although ARFs in model plants such as Arabidopsis had been well characterized, their identities and potential roles in non-model plants are less studied. Here, we performed genome-wide identification of ARFs in Magnolia sieboldii K. Koch, a primitive species with high taxonomic importance and medicinal values. We found 25 ARF genes in M. sieboldii, which were widely distributed across multiple chromosomes. Based on sequence similarity, the encoded proteins could be either transcriptional repressors or activators. Gene expression analysis showed a dynamic pattern for many ARFs including MsARF5 during seed germination. In addition, overexpressing of MsARF5 showed that it restores many developmental defects in the Arabidopsis mutant. Moreover, two phenotypically distinct transgenic Arabidopsis lines were obtained, indicating a link between gene expression levels and developmental phenotypes. Taken together, we provided a systematic investigation of the ARF gene family in M. sieboldii and revealed an important role of MsARF5 in mediating auxin signaling.

Selection and validation of reference genes for quantitative real-time PCR of Quercus mongolica Fisch. ex Ledeb under abiotic stresses.

Quercus mongolica Fisch. ex Ledeb is the main species of coniferous and broadleaved mixed forests in northeast and north China, which has high ornamental, economic, and ecological value. The appropriate reference genes must be selected for quantitative real-time PCR to reveal the molecular mechanisms of stress responses and their contribution to breeding of Q. mongolica. In the present study, we chose 11 candidate reference genes (TUA, CYP18, HIS4, RPS13, ACT97, TUB1, UBQ10, UBC5, SAND, PP2A, and SAMDC) and used four programs (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of the above genes in roots, stems, and leaves under five abiotic stress factors (cold, salt, drought, weak light, and heavy metal). The findings revealed that under various experimental environments, the most stable genes were different; CYP18, ACT97, and RPS13 ranked the highest under most experimental environments. Moreover, two genes induced by stress, CMO and P5CS2, were chosen to demonstrate the reliability of the selected reference genes in various tissues under various stress conditions. Our research provides a significant basis for subsequent gene function studies of Q. mongolica.

A chromosome-scale genome assembly of the Mongolian oak (Quercus mongolica).

Mongolian oak (Quercus mongolica Fisch.) is an ecologically and economically important white oak species native to and widespread in the temperate zone of East Asia. Here, we present a chromosome-scale reference genome assembly of Q. mongolica, a representative white oak species, by combining Illumina and PacBio data with Hi-C mapping technologies that is the first reference genome created for an Asian oak. Our results showed that the PacBio draft genome size was 809.84 Mb, with a BUSCO complete gene percentage of 92.71%. Hi-C scaffolding anchored 774.59 Mb contigs (95.65% of draft assembly) onto 12 pseudochromosomes. The contig N50 and scaffold N50 were 2.64 and 66.74 Mb, respectively. Of the 36,553 protein-coding genes predicted in the study, approximately 95% had functional annotations in public databases. A total of 435.34 Mb (53.75% of the genome) of repetitive sequences were predicted in the assembled genome. Genome evolution analysis showed that Q. mongolica is closely related to Q. robur from Europe, and they shared a common ancestor ~11.8 million years ago (Ma). Gene family evolution analysis of Q. mongolica revealed that the nucleotide-binding site (NBS)-encoding gene family related to disease resistance was significantly contracted, whereas the ECERIFERUM 1 (CER1) homologous genes related to cuticular wax biosynthesis was significantly expanded. This pioneering Asian oak genome resource represents an important supplement to the oak genomics community and will improve our understanding of Asian white oak biology and evolution.

Integrated RNA and miRNA sequencing analysis reveals a complex regulatory network of Magnolia sieboldii seed germination.

Magnolia sieboldii K. Koch (M. sieboldii) is a deciduous Chinese tree species of the Magnoliaceae family with high ornamental, medicinal, and economic benefits. The germination of M. sieboldii seeds under natural conditions is extremely difficult, thereby hindering the cultivation and breeding of this important species. The molecular mechanisms underlying M. sieboldii seed germination remain unclear due to the lack of genomic and transcriptomic resources. Here, we integrated both mRNA and miRNA sequencing to identify the genes and pathways related to M. sieboldii germination. A comprehensive full-length transcriptome containing 158,083 high-quality unigenes was obtained by single-molecule real-time (SMRT) sequencing technology. We identified a total of 13,877 genes that were differentially expressed between non-germinated and germinated seeds. These genes were mainly involved in plant hormone signal transduction and diverse metabolic pathways such as those involving lipids, sugars, and amino acids. Our results also identified a complex regulatory network between miRNAs and their target genes. Taken together, we present the first transcriptome of M. sieboldii and provide key genes and pathways associated with seed germination for further characterization. Future studies of the molecular basis of seed germination will facilitate the genetic improvement M. sieboldii.