Identification and Expression Analysis of the Populus trichocarpa GASA-Gene Family

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays an important regulatory role in the growth and development of plants. In this study, we identified 19 GASA genes using bioinformatics-based methods in Populus trichocarpa, and these PtGASA genes could be divided into three categories based on their phylogenetic relationships. Based on an analysis of the structure and motifs of these genes, it was concluded that PtGASA class II members are more conserved than class I and class III members are, and the results of collinearity analysis showed that members of class II are collinearly related in poplar. Expression analysis of Populus trichocarpa roots, stems, and leaves showed that most of the PtGASA genes are expressed at higher levels in the stems or roots than in the leaves; a similar expression pattern was found in Vitis vinifera, indicating that the GASA-family members mainly play a role in the morphogenesis of poplar. Considering the phenomenon of gene amplification, we found that the higher the similarity of homologous genes was, the more similar the expression patterns. This study represents the first whole-genome identification and expression-profile analysis of the GASA-gene family in poplar, a model species, laying a foundation for functional studies of poplar GASA genes and serving as a reference for related research on other woody plant species.     

Genome-Wide Analysis of the AAAP Gene Family in Populus and Functional Analysis of PsAAAP21 in Root Growth and Amino Acid Transport

The adventitious root (AR) is the basis for successful propagation by plant cuttings and tissue culture and is essential for maintaining the positive traits of a variety. Members of the amino acid/auxin permease (AAAP) gene family play indispensable roles in various plant metabolisms and have few studies on root growth and amino acid transport. In this study, with a systematic bioinformatics analysis of the Populus AAAP family, 83 PtrAAAPs were identified from Populus trichocarpa and grouped into 8 subfamilies. Subsequently, chromosomal distribution, genetic structure, cis-elements analysis, and expression pattern analysis of the AAAP family were performed and the potential gene AAAP21 regulating root development was screened by combining the results of RNA-Seq and QTL mapping. PsAAAP21 was proven as promoting root development by enhancing AR formation. Differentially expressed genes (DEGs) from RNA-seq results of overexpressing lines were enriched to multiple amino acid-related pathways, and the amino acid treatment to transgenic lines indicated that PsAAAP21 regulated amino acid transport, including tyrosine, methionine, and arginine. Analysis of the AAAP gene family provided a theoretical basis for uncovering the functions of AAAP genes. The identification of PsAAAP21 on root promotion and amino acid transport in Populus will help with breeding new woody plant species with strong rooting ability.     

Gibberellin Oxidase Gene Family in L. chinense: Genome-Wide Identification and Gene Expression Analysis

GAox is a key enzyme for the transformation of gibberellins, and belongs to the 2-ketoglutarate dependent dioxygenase gene family (2ODD). However, a systematic analysis of GAox in the angiosperm L. chinense has not yet been reported. Here, we identified all LcGAox gene family members in L. chinense, which were classified into the three subgroups of GA20ox, C19GA2ox, and C20GA2ox. Comparison of the gene structure, conserve motifs, phylogenetic relationships, and syntenic relationships of gibberellin oxidase gene families in different species indicated that the gene functional differences may be due to the partial deletion of their domains during evolution. Furthermore, evidence for purifying selection was detected between orthologous GAox genes in rice, grape, Arabidopsis, and L. chinense. Analysis of the codon usage patterns showed that mutation pressure and natural selection might have induced codon usage bias in angiosperms; however, the LcGAox genes in mosses, lycophytes, and ambarella plants exhibited no obvious codon usage preference. These results suggested that the gibberellin oxidase genes were more primitive. The gene expression pattern was analyzed in different organs subjected to multiple abiotic stresses, including GA, abscisic acid (ABA), and chlormequat (CCC) treatment, by RNA-seq and qRT-PCR, and the stress- and phytohormone-responsive cis-elements were counted. The results showed that the synthesis and decomposition of GA were regulated by different LcGAox genes in the vegetative and reproductive organs of L. chinense, and only LcGA2ox1,4, and 7 responded to the NaCl, polyethylene glycol, 4 °C, GA, ABA, and CCC treatment in the roots, stems, and leaves of seedlings at different time periods, revealing the potential role of LcGAox in stress resistance.     

Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein–protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.     

Genome-Wide Analysis of SQUAMOSA-Promoter-Binding Protein-like Family in Flowering Pleioblastus pygmaeus

SQUAMOSA Promoter-Binding Protein-Like (SPL) family is well-known for playing an important role in plant growth and development, specifically in the reproductive process. Bamboo plants have special reproductive characteristics with a prolonged vegetative phase and uncertain flowering time. However, the underlying functions of SPL genes in reproductive growth are undisclosed in bamboo plants. In the study, a total of 28 SPLs were screened from an ornamental dwarf bamboo species, Pleioblastus pygmaeus. Phylogenetic analysis indicates that 183 SPLs from eight plant species can be classified into nine subfamilies, and the 28 PpSPLs are distributed among eight subfamilies. Homologous analysis shows that as many as 32 pairs of homologous genes were found between P. pygmaeus and rice, and 83 pairs were found between P. pygmaeus and Moso bamboo, whose Ka/Ks values are all <1. MiRNA target prediction reveals that 13 out of the 28 PpSPLs have recognition sites complementary to miRNA156. To screen the SPLs involved in the reproductive growth of bamboo plants, the mRNA abundance of the 28 PpSPLs was profiled in the different tissues of flowering P. pygmaeus and non-flowering plants by RNA-Seq. Moreover, the relative expression level of eight PpSPLs is significantly higher in flowering P. pygmaeus than that in non-flowering plants, which was also validated by RT-qPCR. Combined with phylogenetic analysis and homologous analysis, the eight significant, differentially expressed PpSPLs were identified to be associated with the reproductive process and flower organ development. Among them, there are four potential miRNA156-targeting PpSPLs involved in the flowering process. Of significant interest in the study is the identification of 28 SPLs and the exploration of four key flowering-related SPLs from P. pygmaeus, which provides a theoretic basis for revealing the underlying functions of SPLs in the reproductive growth of bamboo plants.     

Genetic Architecture and Genome-Wide Adaptive Signatures Underlying Stem Lenticel Traits in Populus tomentosa

The stem lenticel is a highly specialized tissue of woody plants that has evolved to balance stem water retention and gas exchange as an adaptation to local environments. In this study, we applied genome-wide association studies and selective sweeping analysis to characterize the genetic architecture and genome-wide adaptive signatures underlying stem lenticel traits among 303 unrelated accessions of P. tomentosa, which has significant phenotypic and genetic variations according to climate region across its natural distribution. In total, we detected 108 significant single-nucleotide polymorphisms, annotated to 88 candidate genes for lenticel, of which 9 causative genes showed significantly different selection signatures among climate regions. Furthermore, PtoNAC083 and PtoMYB46 showed significant association signals and abiotic stress response, so we overexpressed these two genes in Arabidopsis thaliana and found that the number of stem cells in all three overexpression lines was significantly reduced by PtoNAC083 overexpression but slightly increased by PtoMYB46 overexpression, suggesting that both genes are involved in cell division and expansion during lenticel formation. The findings of this study demonstrate the successful application of an integrated strategy for dissecting the genetic basis and landscape genetics of complex adaptive traits, which will facilitate the molecular design of tree ideotypes that may adapt to future climate and environmental changes.