Metabolomics and Transcriptomics Integration of Early Response of Populus tomentosa to Reduced Nitrogen Availability

Nitrogen (N) is one of the most crucial elements for plant growth and development. However, little is known about the metabolic regulation of trees under conditions of N deficiency. In this investigation, gas chromatography-mass spectrometry (GC-MS) was used to determine global changes in metabolites and regulatory pathways in Populus tomentosa. Thirty metabolites were found to be changed significantly under conditions of low-N stress. N deficiency resulted in increased levels of carbohydrates and decreases in amino acids and some alcohols, as well as some secondary metabolites. Furthermore, an RNA-sequencing (RNA-Seq) analysis was performed to characterize the transcriptomic profiles, and 1,662 differentially expressed genes were identified in P. tomentosa. Intriguingly, four pathways related to carbohydrate metabolism were enriched. Genes involved in the gibberellic acid and indole-3-acetic acid pathways were found to be responsive to low-N stress, and the contents of hormones were then validated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Coordinated metabolomics and transcriptomics analysis revealed a pattern of co-expression of five pairs of metabolites and unigenes. Overall, our investigation showed that metabolism directly related to N deficiency was depressed, while some components of energy metabolism were increased. These observations provided insights into the metabolic and molecular mechanisms underlying the interactions of N and carbon in poplar.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa (P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

Association Study and Mendelian Randomization Analysis Reveal Effects of the Genetic Interaction Between PtoMIR403b and PtoGT31B-1 on Wood Formation in Populus tomentosa

MicroRNAs (miRNAs), important posttranscriptional regulators of gene expression, play a crucial role in plant growth and development. A single miRNA can regulate numerous target genes, making the determination of its function and interaction with targets challenging. We identified PtomiR403b target to PtoGT31B-1, which encodes a galactosyltransferase responsible for the biosynthesis of cell wall polysaccharides. We performed an association study and epistasis and Mendelian randomization (MR) analyses to explore how the genetic interaction between PtoMIR403b and its target PtoGT31B-1 underlies wood formation. Single nucleotide polymorphism (SNP)-based association studies identified 25 significant associations (P < 0.01, Q < 0.05), and PtoMIR403b and PtoGT31B-1 were associated with five traits, suggesting a role for PtomiR403b and PtoGT31B-1 in wood formation. Epistasis analysis identified 93 significant pairwise epistatic associations with 10 wood formation traits, and 37.89% of the SNP-SNP pairs indicated interactions between PtoMIR403b and PtoGT31B-1. We performed an MR analysis to demonstrate the causality of the relationships between SNPs in PtoMIR403b and wood property traits and that PtoMIR403b modulates wood formation by regulating expression of PtoGT31B-1. Therefore, our findings will facilitate dissection of the functions and interactions with miRNA-targets.

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.

High-Frequency Homologous Recombination Occurred Preferentially in Populus

Homologous recombination (HR), the most significant event in meiosis, has important implications for genetic diversity and evolution in organisms. Heteroduplex DNA (hDNA), the product of HR, can be captured by artificially induced chromosome doubling during the development of the embryo sac to inhibit postmeiotic segregation, subsequently, and hDNAs are directly detected using codominant simple sequence repeat (SSR) markers. In the present study, two hybrid triploid populations derived from doubling the chromosomes of the embryo sac induced by high temperature in Populus tomentosa served as starting materials. Eighty-seven, 62, and 79 SSR markers on chromosomes 01, 04, and 19, respectively, that were heterozygous in the maternal parent and different from the paternal parent were screened to detect and characterize the hDNA in P. tomentosa. The results showed that the hDNA frequency patterns on chromosomes changed slightly when the number of SSR primers increased. The highest hDNA frequency occurred at the adjacent terminal on chromosomes, which was slightly higher than those at the terminals in the two genotypic individuals, and the hDNA frequency gradually decreased as the locus-centromere distance decreased. With the increase in the number of SSR markers employed for detection, the number of recombination events (REs) detected significantly increased. In regions with high methylation or long terminal repeat (LTR) retrotransposon enrichment, the frequency of hDNA was low, and high frequencies were observed in regions with low sequence complexity and high gene density. High-frequency recombination occurring at high gene density regions strongly affected the association between molecular markers and quantitative trait loci (QTLs), which was an important factor contributing to the difficulty encountered by MAS in achieving the expected breeding results.

Characteristics and chemical reactivity of biogenic volatile organic compounds from dominant forest species in the Jing-Jin-Ji area, China

Background Biogenic volatile organic compounds (BVOCs) play an essential role in tropospheric atmospheric chemical reactions. There are few studies conducted on BVOCs emission of dominant forest species in the Jing-Jin-Ji area of China. Based on the field survey, forest resources data and the measured standard emission factors, the Guenther model developed in 1993 (G93) was applied in this paper to estimate the emission of BVOCs from several dominant forest species (Platycladus orientalis, Quercus variabilis, Betula platyphylla, Populus tomentosa, Pinus tabuliformis, Robinia pseudoacacia, Ulmus pumila, Salix babylonica and Larix gmelinii) in the Jing-Jin-Ji area in 2017. Then the spatiotemporal emission characteristics and atmospheric chemical reactivity of these species were extensively evaluated. Results The results showed that the total annual BVOCs emission was estimated to be 70.8 Gg C·year− 1, consisting 40.5 % (28.7 Gg C·year− 1) of isoprene, 36.0 % (25.5 Gg C·year− 1) of monoterpenes and 23.4 % (16.6 Gg C·year− 1) of other VOCs. The emissions from Platycladus orientalis, Quercus variabilis, Populus tomentosa and Pinus tabulaeformis contributed 56.1 %, 41.2 %, 36.0 % and 31.1 %, respectively. The total BVOCs emission from the Jing-Jin-Ji area accounted for 61.9 % and 1.8 % in summer and winter, respectively. Up to 28.8 % of emission was detected from Chengde followed by Beijing with 24.9 %, that mainly distributed in the Taihang Mountains and the Yanshan Mountains. Additionally, the Robinia pseudoacacia, Populus tomentosa, Quercus variabilis, and Pinus tabulaeformis contributed mainly to BVOCs reaction activity. Conclusions The BVOCs emission peaked in summer (June, July, and August) and bottomed out in winter (December, January, and February). Chengde contributed the most, followed by Beijing. Platycladus orientalis, Quercus variabilis, Populus tomentosa, Pinus tabulaeformis and Robinia pseudoacacia represent the primary contributors to BVOCs emission and atmospheric reactivity, hence the planting of these species should be reduced.