Duplication of NRAMP3 gene in poplars generated two homologous transporters with distinct functions

ABSTRACTTransition metals are essential for a wealth of metabolic reactions, but their concentrations need to be tightly controlled across cells and cell compartments, as metal excess or imbalance has deleterious effects. Metal homeostasis is achieved by a combination of metal transport across membranes and metal binding to a variety of molecules. Gene duplication is a key process in evolution, as emergence of advantageous mutations on one of the copies can confer a new function. Here, we report that the poplar genome contains two paralogues encoding NRAMP3 metal transporters localized in tandem. All Populus species analyzed had two copies of NRAMP3, whereas only one could be identified in Salix species indicating that duplication occurred when the two genera separated. Both copies are under purifying selection and encode functional transporters, as shown by expression in the yeast heterologous expression system. However, genetic complementation revealed that only one of the paralogues has retained the original function in release of metals stored in the vacuole previously characterized in A. thaliana. Confocal imaging showed that the other copy has acquired a distinct localization to the Trans Golgi Network (TGN). Expression in poplar indicated that the copy of NRAMP3 localized on the TGN has a novel function in the control of cell-to-cell transport of manganese. This work provides a clear case of neo-functionalization through change in the subcellular localization of a metal transporter as well as evidence for the involvement of the secretory pathway in cell-to-cell transport of manganese.

Genome-Wide Identification and Characterization of the FAR1/FHY3 Family in Populus trichocarpa Torr. & Gray and Expression Analysis in Light Response

Light is an important environmental factor for plant growth, and in higher plants, phytochrome A (phyA) is the predominant far-red photoreceptor, involved in various photoresponses. The FAR1/FHY3 transcription factor family, derived from transposases, is able to regulate plant development in response to multiple photosensitizers phytochrome. In total, 51 PtrFRSs were identified in the poplar genome, and were divided into 4 subfamilies. Among them, 47 PtrFRSs are located on 17 chromosomes. Upstream cis-acting elements of the PtrFRS genes were classified into three categories: growth and metabolism, stress and hormone, and the hormone and stress categories contained most of the cis-acting elements. Analysis of the regulatory networks and expression patterns showed that most PtrFRSs responded to changes in light intensity and were involved in the regulation of phytochromes. In this study, 51 PtrFRSs were identified and comprehensively bioinformatically analyzed, and preliminary functional analysis and prediction of PtrFRSs was carried out.

NHEJ deficiency develops homologous recombination in poplar meaningfully further than the overexpression of HDR factors

Efficient homology-directed DNA repair (HDR) is a vital difficulty confronting researchers to replace the target genome’s desired fragment. In plants, scientists have performed meticulous investigations on herbal, crops, and citrus trees using HDR effector proteins, CtIP and MRE11, to obtain double-stranded breaks (DSBs) more precisely. Although HDR efficiency in plants previously has been reported, no record has been declared about HDR efficiency in poplars.Here, we hypothesized that inhibition of nonhomologous recombination cofactors XRCC4, together with enhancing the HDR pathway activities, enables us to generate the HDR efficiency in poplar trees. In this study, the BleoR gene was used to integrate into the interested site and develop resistant poplars against Zeocin antibiotics. We designed plasmids, including different fusions of HDR proteins and, together with the XRCC4 target. Furthermore, real-time PCR, western blotting, RT-PCR, RT-qPCR, southern blotting, and DNA sequencing were applied to exhibit and evaluate HDR efficiency.While both applying HDR proteins and XRCC4 deficiency simultaneously could improve HDR efficiency, which showed about 50 times more than usual editing by CRISPR-Cas9, the only using HDR proteins without XRCC4 deficiency showed about 16 times more. We developed a new recombinant poplar genome to generate stable lines resistant to the Zeocin antibiotic.

Identification of RING-H2 Gene Candidates Related to Wood Formation in Poplar

RING-H2 genes, the most abundant RING-type genes encoding putative ubiquitin ligase E3, are involved in diverse biological processes. Whether RING-H2 genes are related to wood formation remains to be identified in trees. In this study, we identified 288 RING-H2 genes in Populus trichocarpa, and found that the segmental and tandem duplication events contributed to RING-H2 gene expansion. Microarray dataset (from Affymetrix poplar genome arrays) showed that 64 of the 249 RING-H2 genes were highly or preferentially expressed in stem xylem. According to the AspWood RNAseq dataset, the transcription levels of genes PtrRHH21, 33, 48, 69, 88, 93, 94, 121, 141, 166, 175, 192, 208, 214, 250 and 257 were significantly increased in the xylem ranging from the expanding xylem to the lignifying xylem, suggesting their association with wood formation. Promoter analyses revealed that most of the preferentially xylem-expressed RING-H2 genes possessed SNBE, TERE, M46RE, AC and SMRE cis-elements, which are involved in secondary cell wall biosynthesis and programmed cell death. Based on the promoter GUS-based analysis result, PtrRHH94 was indicated to be associated with wood formation in transgenic P. trichocarpa. Taken together, dozens of Populus RING-H2 gene candidates associated with wood formation have been identified based on multiple gene expression analyses.

Genome-Wide Analysis of Multiple Organellar RNA Editing Factor Family in Poplar Reveals Evolution and Roles in Drought Stress

Poplar (Populus) is one of the most important woody plants worldwide. Drought, a primary abiotic stress, seriously affects poplar growth and development. Multiple organellar RNA editing factor (MORF) genes—pivotal factors in the RNA editosome in Arabidopsis thaliana—are indispensable for the regulation of various physiological processes, including organelle C-to-U RNA editing and plasmid development, as well as in the response to stresses. Although the poplar genome sequence has been released, little is known about MORF genes in poplar, especially those involved in the response to drought stress at the genome-wide level. In this study, we identified nine MORF genes in the Populus genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the P. trichocarpa (Ptr) MORF family members were classified into six groups (Groups I–VI). A microsynteny analysis indicated that two (22.2%) PtrMORF genes were tandemly duplicated and seven genes (77.8%) were segmentally duplicated. Based on the dN/dS ratios, purifying selection likely played a major role in the evolution of this family and contributed to functional divergence among PtrMORF genes. Moreover, analysis of qRT-PCR data revealed that PtrMORFs exhibited tissue- and treatment-specific expression patterns. PtrMORF genes in all group were involved in the stress response. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes in poplar, and, in particular, for improving the drought resistance of poplar by genetics manipulation.

Comparison of gene order of GIGANTEA loci in yellow-poplar, monocots, and eudicots

GIGANTEA plays an important role in the control of circadian rhythms and photoperiodic flowering. The GIGANTEA gene has been studied in various species, but not in basal angiosperms. Moreover, to the best of our knowledge, no study of the genome organization of a basal angiosperm has yet been published. In this study, we sequenced a bacterial artificial chromosome (BAC) harboring GIGANTEA from yellow-poplar ( Liriodendron tulipifera L.) and compared the genomic organization of this gene in yellow-poplar with that in other species from various angiosperm clades. This is the first report on the gene structure and organization of a large contig in any basal angiosperm species. The BAC clone, covering a region of approximately 122 kb from the yellow-poplar genome, was sequenced and assembled by coupling the 454 pyrosequencing technology with ABI capillary sequencing. In addition to GIGANTEA, the gene RPS18.A (encoding ribosomal protein S18.A) was found in this segment of the genome. We found that gene content and order in this region of the yellow-poplar genome were similar to those in the corresponding region in eudicots but not in Oryza sativa and Sorghum bicolor , implying that clustering of the GIGANTEA and RPS18.A genes is ancestral and separation of the genes occurred after the phylogenetic split of monocots from dicots. Phylogenetic analysis of GIGANTEA amino acid sequences placed yellow-poplar closer to eudicots than to monocots. In addition, evidence for transposition and large insertions and duplications was found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.