Multi-omics analysis reveals spatiotemporal regulation and function of heteromorphic leaves in Populus.

Despite the high economic and ecological importance of forests, our knowledge of the adaptive evolution of leaf traits remains very limited. Euphrates poplar (Populus euphratica), which has high tolerance to arid environment, has evolved four heteromorphic leaf forms, including narrow (linear and lanceolate) and broad (ovate and broad-ovate) leaves on different crowns. Here, we revealed the significant functional divergence of four P. euphratica heteromorphic leaves at physiological and cytological levels. Through global analysis of transcriptome and DNA methylation across tree and leaf developmental stages, we revealed that gene expression and DNA epigenetics differentially regulated key processes involving development and functional adaptation of heteromorphic leaves, such as hormone signaling pathways, cell division, and photosynthesis. Combined analysis of gene expression, methylation, ATAC-seq, and Hi-C-seq revealed longer interaction of 3D genome, hypomethylation, and open chromatin state upregulates IAA-related genes (such as PIN-FORMED1 and ANGUSTIFOLIA3) and promotes the occurrence of broad leaves while narrow leaves were associated with highly concentrated heterochromatin, hypermethylation, and upregulated abscisic acid pathway genes (such as Pyrabactin Resistance1-like10). Therefore, development of P. euphratica heteromorphic leaves along with functional divergence was regulated by differentially expressed genes, DNA methylation, chromatin accessibility, and 3D genome remodeling to adapt to the arid desert. This study advances our understanding of differential regulation on development and functional divergence of heteromorphic leaves in P. euphratica at the multi-omics level and provides a valuable resource for investigating the adaptive evolution of heteromorphic leaves in Populus.

Fulvic acid ameliorates drought stress-induced damage in tea plants by regulating the ascorbate metabolism and flavonoids biosynthesis.

BACKGROUND: Fulvic acid (FA) is a kind of plant growth regulator, which can promote plant growth, play an important role in fighting against drought, improve plant stress resistance, increase production and improve quality. However, the function of FA in tea plants during drought stress remain largely unknown. RESULTS: Here, we examined the effects of 0.1 g/L FA on genes and metabolites in tea plants at different periods of drought stress using transcriptomics and metabolomics profiles. Totally, 30,702 genes and 892 metabolites were identified. Compared with controlled groups, 604 and 3331 differentially expressed metabolite genes (DEGs) were found in FA-treated tea plants at 4 days and 8 days under drought stress, respectively; 54 and 125 differentially expressed metabolites (DEMs) were also found at two time points, respectively. Bioinformatics analysis showed that DEGs and DEMs participated in diverse biological processes such as ascorbate metabolism (GME, AO, ALDH and L-ascorbate), glutathione metabolism (GST, G6PDH, glutathione reduced form and CYS-GYL), and flavonoids biosynthesis (C4H, CHS, F3'5'H, F3H, kaempferol, quercetin and myricetin). Moreover, the results of co-expression analysis showed that the interactions of identified DEGs and DEMs diversely involved in ascorbate metabolism, glutathione metabolism, and flavonoids biosynthesis, indicating that FA may be involved in the regulation of these processes during drought stress. CONCLUSION: The results indicated that FA enhanced the drought tolerance of tea plants by (i) enhancement of the ascorbate metabolism, (ii) improvement of the glutathione metabolism, as well as (iii) promotion of the flavonoids biosynthesis that significantly improved the antioxidant defense of tea plants during drought stress. This study not only confirmed the main strategies of FA to protect tea plants from drought stress, but also deepened the understanding of the complex molecular mechanism of FA to deal with tea plants to better avoid drought damage.

Comprehensive proteome analyses of lysine acetylation in tea leaves by sensing nitrogen nutrition.

BACKGROUND: Nε-Acetylation of lysine residues, a frequently occurring post-translational modification, plays important functions in regulating physiology and metabolism. However, the information of global overview of protein acetylome under nitrogen-starvation/resupply in tea (Camellia sinensis) leaves was limited. And the full function of lysine acetylated proteins of tea plants in nitrogen absorption and assimilation remains unclear. RESULTS: Here, we performed the global review of lysine acetylome in tea leaves under nitrogen (N)-starvation/resupply, using peptide prefractionation, immunoaffinity enrichment, and coupling with high sensitive LC-MS/MS combined with affinity purification analysis. Altogether, 2229 lysine acetylation sites on 1286 proteins were identified, of which 16 conserved motifs in E*KacK, Kac*K, Kac*R, Kac*HK, Kac*N, Kac*S, Kac*T, Kac*D, were extracted from 2180 acetylated peptides. Approximately, 36.76% of the acetylated lysines were located in the regions of ordered secondary structures. The most of the identified lysine acetylation proteins were located in the chloroplast (39%) and cytoplasm (29%). The largest group of acetylated proteins consisted of many enzymes, such as ATP synthase, ribosomal proteins and malate dehydrogenase [NADP], which were related to metabolism (38%) in the biological process. These acetylated proteins were mainly enriched in three primary protein complexes of photosynthesis: photosystem I, photosystem II and the cytochrome b6/f complex. And some acetylated proteins related to glycolysis and secondary metabolite biosynthesis were increased/decreased under N-resupply. Moreover, the PPI (protein-protein interaction) analysis revealed that the diverse interactions of identified acetylated proteins mainly involved in photosynthesis and ribosome. CONCLUSION: The results suggested that lysine acetylated proteins might play regulating roles in metabolic process in tea leaves. The critical regulatory roles mainly involved in diverse aspects of metabolic processes, especially in photosynthesis, glycolysis and secondary metabolism. A lot of proteins related to the photosynthesis and glycolysis were found to be acetylated, including LHCA1, LHCA3, LHCB6, psaE, psaD, psaN, GAPDH, PEPC, ENL and petC. And some proteins related to flavonoids were also found to be acetylated, including PAL, DFR, naringenin 3-dioxygenase and CHI. The provided data may serve as important resources for exploring the physiological, biochemical, and genetic role of lysine acetylation in tea plants. Data are available via ProteomeXchange with identifier PXD008931.

Genome-wide analysis of R2R3-MYB transcription factors reveals their differential responses to drought stress and ABA treatment in desert poplar (Populus euphratica).

The R2R3-MYB transcription factors are widely involved in the regulation of plant growth, biotic and abiotic stress responses. Meanwhile, seed germination, which is stimulated by internal and external environments, is a critical stage in the plant life cycle. However, the identification, characterization, and expression profiling of the Populus euphratica R2R3-MYB family in drought response during seed germination have been unknown. Our study attempted to identify and characterize the R2R3-MYB genes in P. euphratica (PeR2R3-MYBs) and explore how R2R3-MYBs trigger the drought and abscisic acid (ABA) response mechanism in its seedlings. Based on the analysis of comparative genomics, 174 PeR2R3-MYBs were identified and expanded driven by whole genome duplication or segment duplication events. The analysis of Ka/Ks ratios showed that, in contrast to most PeR2R3-MYBs, the other PeR2R3-MYBs were subjected to positive selection in P. euphratica. Further, the expression data of PeR2R3-MYBs under drought stress and ABA treatment, together with available functional data for Arabidopsis thaliana MYB genes, supported the hypothesis that PeR2R3-MYBs involved in response to drought are dependent or independent on ABA signaling pathway during seed germination, especially PeR2R3-MYBs with MYB binding sites (MBS) cis-element and/or tandem duplication. This study is the first report on the genome-wide analysis of PeR2R3-MYBs, as well as the other two Salicaceae species. The duplication events and differential expressions of PeR2R3-MYBs play important roles in enhancing the adaptation to drought desert environment. Our results provide a reference for prospective functional studies of R2R3-MYBs of poplars and lay the foundation for new breeding strategies to improve the drought tolerance of P. euphratica.

Phylogeography Reveals Geographic and Environmental Factors Driving Genetic Differentiation of Populus sect. Turanga in Northwest China.

Populus sect. Turanga (hereafter referred to as "Populus"), including Populus euphratica and Populus pruinosa, are the predominant tree species in desert riparian forests in northwestern China. These trees play key roles in maintaining ecosystem balance, curbing desertification, and protecting biodiversity. However, the distribution area of Populus forests has been severely diminished and degraded in recent years due to increased habitat destruction and human activity. Understanding the genetic diversity among Populus individuals and populations is essential for designing conservation strategies, but comprehensive studies of their genetic diversity in northwest China are lacking. Here, we assessed the population structures and genetic diversity of 1,620 samples from 85 natural populations of Populus (59 P. euphratica and 26 P. pruinosa populations) covering all of northwestern China using 120 single nucleotide polymorphism (SNP) markers. Analysis of population structure revealed significant differentiation between these two sister species and indicated that strong geographical distribution patterns, a geographical barrier, and environmental heterogeneity shaped the extant genetic patterns of Populus. Both P. euphratica and P. pruinosa populations in southern Xinjiang had higher genetic diversity than populations in other clades, perhaps contributing to local geographic structure and strong gene flow. Analysis of molecular variance (AMOVA) identified 15% variance among and 85% variance within subpopulations. Mantel tests suggested that the genetic variation among P. euphratica and P. pruinosa populations could be explained by both geographical and environmental distance. The genetic diversity of P. euphratica showed a significant negative correlation with latitude and longitude and a positive correlation with various environmental factors, such as precipitation of warmest quarter and driest month, temperature seasonality, and annual mean temperature. These findings provide insights into how the genetic differentiation of endangered Populus species was driven by geographical and environmental factors, which should be helpful for designing strategies to protect these genetic resources in the future.

Fulvic acid enhances drought resistance in tea plants by regulating the starch and sucrose metabolism and certain secondary metabolism.

The aim of this work was to gain insight into the molecular mechanisms underlying the effect of fulvic acid on drought-exposed tea plants. We performed proteomic analysis of fulvic acid-treated tea leaves from the target plants using tandem mass tag quantitative labeling technology and compared the results with those of a previous transcriptomic analysis. We identified 48 and 611 differentially abundant proteins in the leaves of tea plants treated with fulvic acid compared with the control under mild and severe drought, respectively. Comparative analysis showed that, under severe drought, 55 genes had similar expression patterns at the transcriptome and proteome levels, such as PAL, GBE, GBSS and bAS. Bioinformatic analysis revealed that those genes were mainly related to the starch and sucrose metabolism, phenylpropanoid biosynthesis and triterpenoid biosynthesis. SIGNIFICANCE: This study broadens the understanding of the molecular mechanisms underlying the improved drought resistance seen in tea plants in the presence of fulvic acid and provides a basis for further research on the genomics of drought tolerance in these plants. In addition, these findings could be used to develop new guidance strategies for improved drought management systems in tea plantation.

Exogenous abscisic acid induces the lipid and flavonoid metabolism of tea plants under drought stress.

Abscisic acid (ABA) is an important phytohormone responsible for activating drought resistance, but the regulation mechanism of exogenous ABA on tea plants under drought stress was rarely reported. Here, we analyzed the effects of exogenous ABA on genes and metabolites of tea leaves under drought stress using transcriptomic and metabolomic analysis. The results showed that the exogenous ABA significantly induced the metabolic pathways of tea leaves under drought stress, including energy metabolism, amino acid metabolism, lipid metabolism and flavonoids biosynthesis. In which, the exogenous ABA could clearly affect the expression of genes involved in lipid metabolism and flavonoid biosynthesis. Meanwhile, it also increased the contents of flavone, anthocyanins, flavonol, isoflavone of tea leaves under drought stress, including, kaempferitrin, sakuranetin, kaempferol, and decreased the contents of glycerophospholipids, glycerolipids and fatty acids of tea leaves under drought stress. The results suggested that the exogenous ABA could alleviate the damages of tea leaves under drought stress through inducing the expression of the genes and altering the contents of metabolites in response to drought stress. This study will be helpful to understand the mechanism of resilience to abiotic stress in tea plant and provide novel insights into enhancing drought tolerance in the future.

Complete chloroplast genome sequence of Populus pruinosa Schrenk from PacBio Sequel II Platform.

Populus pruinosa Schrenk plays an important role on ecological services in desert areas. The complete chloroplast genome was reported in this study using the PacBio Sequel II Platform. The chloroplast genome with a total size of 157,856 bp consists of two inverted repeats (IR, 27,673 bp) separated by a large single-copy region (LSC, 85,867 bp) and a small single-copy region (SSC, 16,645 bp). Further annotation revealed the chloroplast genome contains 111 genes, including 78 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. A total of 151 simple sequence repeats (SSRs) were identified in the chloroplast genome. This information will be useful for study on the evolution and genetic diversity of P. pruinosa in the future.

Global Ubiquitome Profiling Revealed the Roles of Ubiquitinated Proteins in Metabolic Pathways of Tea Leaves in Responding to Drought Stress.

Drought stress often affects the expression of genes and proteins in tea plants. However, the global profiling of ubiquitinated (Kub) proteins in tea plants remains unearthed. Here, we performed the ubiquitome in tea leaves under drought stress using antibody-based affinity enrichment coupled with LC-MS/MS analysis. In total, 1,409 lysine Kub sites in 781 proteins were identified, of which 14 sites in 12 proteins were up-regulated and 123 sites in 91 proteins down-regulated under drought stress. The identified Kub proteins were mainly located in the cytosol (31%), chloroplast (27%) and nuclear (19%). Moreover, 5 conserved motifs in EKub, EXXXKub, KubD, KubE and KubA were extracted. Several Kub sites in ubiquitin-mediated proteolysis-related proteins, including RGLG2, UBC36, UEV1D, RPN10 and PSMC2, might affect protein degradation and DNA repair. Plenty of Kub proteins related to catechins biosynthesis, including PAL, CHS, CHI and F3H, were positively correlated with each other due to their co-expression and co-localization. Furthermore, some Kub proteins involved in carbohydrate and amino acid metabolism, including FBPase, FBA and GAD1, might promote sucrose, fructose and GABA accumulation in tea leaves under drought stress. Our study preliminarily revealed the global profiling of Kub proteins in metabolic pathways and provided an important resource for further study on the functions of Kub proteins in tea plants.