Genome-wide identification, expression analysis, and potential roles under low-temperature stress of bHLH gene family in Prunus sibirica.

The basic helix-loop-helix (bHLH) family is one of the most well-known transcription factor families in plants, and it regulates growth, development, and abiotic stress responses. However, systematic analyses of the bHLH gene family in Prunus sibirica have not been reported to date. In this study, 104 PsbHLHs were identified and classified into 23 subfamilies that were unevenly distributed on eight chromosomes. Nineteen pairs of segmental replication genes and ten pairs of tandem replication genes were identified, and all duplicated gene pairs were under purifying selection. PsbHLHs of the same subfamily usually share similar motif compositions and exon-intron structures. PsbHLHs contain multiple stress-responsive elements. PsbHLHs exhibit functional diversity by interacting and coordinating with other members. Twenty PsbHLHs showed varying degrees of expression. Eleven genes up-regulated and nine genes down-regulated in -4°C. The majority of PsbHLHs were highly expressed in the roots and pistils. Transient transfection experiments demonstrated that transgenic plants with overexpressed PsbHLH42 have better cold tolerance. In conclusion, the results of this study have significant implications for future research on the involvement of bHLH genes in the development and stress responses of Prunus sibirica.

Genetic diversity and conservation of Siberian apricot (Prunus sibirica L.) based on microsatellite markers.

Siberian apricot (Prunus sibirica L.) is a woody tree species of ecological, economic, and social importance. To evaluate the genetic diversity, differentiation, and structure of P. sibirica, we analyzed 176 individuals from 10 natural populations using 14 microsatellite markers. These markers generated 194 alleles in total. The mean number of alleles (13.8571) was higher than the mean number of effective alleles (6.4822). The average expected heterozygosity (0.8292) was higher than the average observed heterozygosity (0.3178). Shannon information index and polymorphism information content were separately 2.0610 and 0.8093, demonstrating the rich genetic diversity of P. sibirica. Analysis of molecular variance revealed that 85% of the genetic variation occurred within populations, with only 15% among them. The genetic differentiation coefficient and gene flow were separately 0.151 and 1.401, indicating a high degree of genetic differentiation. Clustering results showed that a genetic distance coefficient of 0.6 divided the 10 natural populations into two subgroups (subgroups A and B). STRUCTURE and principal coordinate analysis divided the 176 individuals into two subgroups (clusters 1 and 2). Mantel tests revealed that genetic distance was correlated with geographical distance and elevation differences. These findings can contribute to the effective conservation and management of P. sibirica resources.

Genome-wide identification and analysis of the WRKY gene family and low-temperature stress response in Prunus sibirica.

BACKGROUND: WRKY transcription factors are a prominent gene family in plants, playing a crucial role in various biological processes including development, metabolism, defense, differentiation, and stress response. Although the WRKY gene family has been extensively studied and analysed in numerous plant species, research on Prunus sibirica's WRKY genes (PsWRKY) remains lacking. RESULTS: This study analysed the basic physicochemical properties, phylogeny, gene structure, cis-acting elements, and Gene ontology (GO) annotation of PsWRKY gene family members using bioinformatics methods based on the whole-genome data of P. sibirica. In total, 55 WRKYs were identified in P. sibirica and were heterogeneously distributed on eight chromosomes. Based on the phylogenetic analysis, these WRKYs were classified into three major groups: Group I, Group II (II-a, II-b, II-c, II-d, II-e), and Group III. Members of different subfamilies have different cis-acting elements, conserved motifs, and intron-exon structures, indicating functional heterogeneity of the WRKY family. Prediction of subcellular localisation indicated that PsWRKYs were mainly located in the nucleus. Twenty pairs of duplicated genes were identified, and segmental duplication events may play an important role in PsWRKY gene family expansion. Analysis of the Ka/Ks ratio showed that the PsWRKY family's homologous genes were primarily purified by selection. Additionally, GO annotation analysis showed that the WRKY gene family was mainly involved in responses to stimuli, immune system processes, and reproductive processes. Furthermore, quantitative real-time PCR (qRT-PCR) analysis showed that 23 PsWRKYs were highly expressed in one or more tissues (pistils and roots) and PsWRKYs showed specific expression patterns under different low-temperature stress conditions. CONCLUSIONS: Our results provide a scientific basis for the further exploration and functional validation of WRKYs in P. sibirica.

Mining for genes related to pistil abortion in Prunus sibirica L.

In Prunus sibirica, the phenomenon of pistil abortion is very common and seriously affects its fruit quality and yield; however, the molecular mechanisms of pistil abortion remains unclear. In this study, we identified differentially expressed genes (DEGs) and pathways associated with pistil abortion using transcriptome sequencing. After comparative analysis, a total of 1,950 DEGs were identified, of which 1,000 were upregulated, and 950 were downregulated. Gene Ontology (GO) functional enrichment analysis of DEGs showed that metabolic process, cellular process, single-organism process, membrane, membrane part, cell, binding, catalytic activity, and transporter activity contained the largest number of DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the plant-pathogen interaction, starch and sucrose metabolism, and plant hormone signal transduction pathways contained the largest number of DEGs. The NAC, bHLH, and B3 transcription factor families contained the largest number of DEGs. qRT-PCR detection confirmed that the gene expression levels were consistent with the transcriptome sequencing results. This study provides a theoretical basis and scientific basis for further research on the molecular mechanisms of P. sibirica pistil abortion.

Genetic diversity of Prunus armeniaca L. var. ansu Maxim. germplasm revealed by simple sequence repeat (SSR) markers.

The genetic diversity and genetic structure of P. armeniaca var. ansu were analyzed based on SSR markers. The aim was to provide scientific basis for conservation, efficient utilization, molecular marker assisted breeding and improved variety selection of P. armeniaca var. ansu germplasm resources. The results showed that the level of genetic diversity within the population was high. Among the 30 SSR markers, the mean number of observed alleles was 11.433, the mean number of effective alleles was 4.433, the mean of Shannon information index was 1.670, and the mean of polymorphic information content was 0.670. Among the eight provenances, Tuanjie Township, Xinyuan County, Xinjiang had the highest genetic diversity. The observed alleles, effective alleles, Shannon information index and Nei's gene diversity index among provenances were higher than those within provenances. Based on Bayesian mathematical modeling and UPGMA cluster analysis, 86 P. armeniaca var. ansu accessions were divided into three subpopulations and four groups, which reflected individual differences in provenances. Subpopulations classified by Bayesian mathematical modeling and groups classified by UPGMA cluster analysis were significantly correlated with geographical provenance (Sig<0.01) and the provenances significantly impacted classification of groups. The provenances played an important role in classification of groups. The genetic distance between Tuanjie Township of Xinyuan County and Alemale Township of Xinyuan County was the smallest, while the genetic relationship between them was the closest and the degree of genetic differentiation was small.

Hydrophobically Associating Polymers Dissolved in Seawater for Enhanced Oil Recovery of Bohai Offshore Oilfields.

As compared to China's overall oil reserves, the reserve share of offshore oilfields is rather significant. However, offshore oilfield circumstances for enhanced oil recovery (EOR) include not just severe temperatures and salinity, but also restricted space on offshore platforms. This harsh oil production environment requires polymers with relatively strong salt resistance, solubility, thickening ability, rapid, superior injection capabilities, and anti-shearing ability. As a result, research into polymers with high viscosity and quick solubility is recognized as critical to meeting the criteria of polymer flooding in offshore oil reservoirs. For the above purposes, a novel hydrophobically associating polymer (HAP) was prepared to be used for polymer flooding of Bohai offshore oilfields. The synthetic procedure was free radical polymerization in aqueous solutions starting at 0 °C, using acrylamide (AM), acrylic acid (AA), 2-acrylamido-2-methylpropane sulfonic acid (AMPS), and poly(ethylene glycol) octadecyl methacrylate (POM) as comonomers. It was discovered that under ideal conditions, the molecular weight of HAP exceeds 2.1 × 107 g⋅mol-1. In a simulated reservoir environment, HAP has substantially greater solubility, thickening property, and salt resistance than conventional polyacrylamide (HPAM), with equivalent molecular weight. Finally, the injectivity and propagation of the two polymers in porous media were investigated. Compared with HPAM, which has a similar molecular weight, HAP solution with the concentration of 0.175% had a much better oil displacement effect in the porous medium, which can enhance oil recovery by 8.8%. These discoveries have the potential to pave the way for chemical EOR in offshore oilfields.

Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica.

BACKGROUND: The phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica. METHOD: The microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data. RESULTS: Anther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that "protein kinase activity", "apoptosis process", "calcium binding", "cell death", "cytochrome c oxidase activity", "aspartate peptidase activity", "cysteine peptidase activity" and other biological pathways such as "starch and sucrose metabolism" and "proteasome" were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened. CONCLUSION: The occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.

Selection of a core collection of Prunus sibirica L. germplasm by a stepwise clustering method using simple sequence repeat markers.

Prunus sibirica is an economically important tree species that occurs in arid and semi-arid regions of northern China. For this species, creation of a core collection is critical for future ecological and evolutionary studies, efficient economic utilization, and development and management of the broader collection of its germplasm resources. In this study, we sampled 158 accessions of P. sibirica from Russia and China using 30 pair of simple sequence repeat molecular markers and 30 different schemes to identify candidate core collections. The 30 schemes were based on combinations of two different sampling strategies, three genetic distances, and five different sample sizes of the complete germplasm resource. We determined the optimal core collection from among the 30 results based on maximization of genetic diversity among groups according to Number of observed alleles (Na), Number of effective alleles (Ne), Shannon's information index (I), Polymorphic information content (PIC), Nei gene diversity (H) and compared to the initial collection of 158 accessions. We found that the optimal core collection resulted from preferred sampling at 25% with Nei & Li genetic distance these ratios of Na, Ne, I, PIC and H to the complete 158 germplasm resources were 73.0%, 113%, 102%, 100% and 103%, respectively, indicating that the core collection comprised a robust representation of genetic diversity in P. sibirica. The proposed core collection will be valuable for future molecular breeding of this species and management of its germplasm resources.

Pu-miR172d regulates stomatal density and water-use efficiency via targeting PuGTL1 in poplar.

miRNAs play essential regulatory roles in many aspects of plant development and in responses to abiotic and biotic stresses. Here, we characterize Pu-miR172d, which acts as a negative regulator of stomatal density by directly repressing the expression of PuGTL1 in Populus ussuriensis. Quantitative real-time PCR and GUS reporter analyses showed that Pu-miR172d was strongly expressed in the guard cells of young leaves. Overexpression of Pu-miR172d significantly decreased stomatal density, resulting in increases in water use efficiency (WUE) and drought tolerance by reducing net photosynthetic rate, stomatal conductance, and transpiration. Molecular analysis showed that PuGTL1 was a major target of Pu-miR172d cleavage. Moreover, PuGTL1-SRDX plants, in which PuGTL1 is suppressed, phenocopied Pu-miR172d-overexpression lines with reduced stomatal density and enhanced WUE. The expression of PuSDD1, a negative regulator of stomatal development, was significantly increased in young leaves of both Pu-miR172d-overexpression and PuGTL1-SRDX plants. RNA-seq analysis of mature leaves indicated that overexpression of Pu-miR172d decreased the expression of many genes related to photosynthesis. Our findings show that the Pu-miR172d/PuGTL1/PuSDD1 module plays an important role in stomatal differentiation, and hence it is a potential target for engineering improved drought tolerance in poplar.

PuHox52-mediated hierarchical multilayered gene regulatory network promotes adventitious root formation in Populus ussuriensis.

Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15 min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.

Comprehensive analysis of trihelix genes and their expression under biotic and abiotic stresses in Populus trichocarpa.

Trihelix genes play important roles in plant growth and development and responses to biotic and abiotic stresses. Here, we identified 56 full-length trihelix genes in Populus trichocarpa and classified them into five groups. Most genes within a given group had similar gene structures and conserved motifs. The trihelix genes were unequally distributed across 19 different linkage groups. Fifteen paralogous pairs were identified, 14 of which have undergone segmental duplication events. Promoter cis-element analysis indicated that most trihelix genes contain stress- or phytohormone-related cis-elements. The expression profiles of the trihelix genes suggest that they are primarily expressed in leaves and roots. Quantitative real-time reverse transcription polymerase chain reaction analysis indicated that members of the trihelix gene family are significantly induced in response to osmotic, abscisic acid, salicylic acid, methyl jasmonate and pathogen infection. PtrGT10 was identified as a target gene of miR172d, which is involved in the osmotic response. Repression of PtrGT10 could increase reactive oxygen species scavenging ability and decrease cell death. This study provides novel insights into the phylogenetic relationships and functions of the P. trichocarpa trihelix genes, which will aid future functional studies investigating the divergent roles of trihelix genes belonging to other species.

Genome-wide identification and expression profiling of the copper transporter gene family in Populus trichocarpa.

Copper transporters (COPT/Ctr) have important roles in the transport of copper (Cu) across the cell membrane in many different species. A comprehensive phylogeny and a molecular structure analysis of the COPT/Ctr family in plants and animals are presented, with an emphasis and bioinformatic analysis of the copper transporter family in Populus trichocarpa (PtCOPT). Structural analyses of PtCOPTs showed that most have 3 transmembrane domains (TMDs), with an exception of PtCOPT4 (2 TMDs). Gene structure, gene chromosomal location, and synteny analyses of PtCOPTs demonstrated that tandem and segmental duplications have likely contributed to the expansion and evolution of the PtCOPTs. Additionally, promoter analyses showed that the function of PtCOPTs is related to Cu and ferrum (Fe) transport. Tissue-specific expression of PtCOPT genes showed that most had relatively high transcript levels in roots and leaves. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of PtCOPT genes were induced not only in limited and excessive Cu, Fe, zinc (Zn) and manganese (Mn) stress, but also in lead (Pb), and cadmium (Cd) stress.

Genome-Wide Analysis of C2H2 Zinc-Finger Family Transcription Factors and Their Responses to Abiotic Stresses in Poplar (Populus trichocarpa).

BACKGROUND: C2H2 zinc-finger (C2H2-ZF) proteins are a large gene family in plants that participate in various aspects of normal plant growth and development, as well as in biotic and abiotic stress responses. To date, no overall analysis incorporating evolutionary history and expression profiling of the C2H2-ZF gene family in model tree species poplar (Populus trichocarpa) has been reported. PRINCIPAL FINDINGS: Here, we identified 109 full-length C2H2-ZF genes in P. trichocarpa, and classified them into four groups, based on phylogenetic analysis. The 109 C2H2-ZF genes were distributed unequally on 19 P. trichocarpa linkage groups (LGs), with 39 segmental duplication events, indicating that segmental duplication has been important in the expansion of the C2H2-ZF gene family. Promoter cis-element analysis indicated that most of the C2H2-ZF genes contain phytohormone or abiotic stress-related cis-elements. The expression patterns of C2H2-ZF genes, based on heatmap analysis, suggested that C2H2-ZF genes are involved in tissue and organ development, especially root and floral development. Expression analysis based on quantitative real-time reverse transcription polymerase chain reaction indicated that C2H2-ZF genes are significantly involved in drought, heat and salt response, possibly via different mechanisms. CONCLUSIONS: This study provides a thorough overview of the P. trichocarpa C2H2-ZF gene family and presents a new perspective on the evolution of this gene family. In particular, some C2H2-ZF genes may be involved in environmental stress tolerance regulation. PtrZFP2, 19 and 95 showed high expression levels in leaves and/or roots under environmental stresses. Additionally, this study provided a solid foundation for studying the biological roles of C2H2-ZF genes in Populus growth and development. These results form the basis for further investigation of the roles of these candidate genes and for future genetic engineering and gene functional studies in Populus.

Genome-Wide Analysis and Heavy Metal-Induced Expression Profiling of the HMA Gene Family in Populus trichocarpa.

The heavy metal ATPase (HMA) family plays an important role in transition metal transport in plants. However, this gene family has not been extensively studied in Populus trichocarpa. We identified 17 HMA genes in P. trichocarpa (PtHMAs), of which PtHMA1-PtHMA4 belonged to the zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) subgroup, and PtHMA5-PtHMA8 were members of the copper (Cu)/silver (Ag) subgroup. Most of the genes were localized to chromosomes I and III. Gene structure, gene chromosomal location, and synteny analyses of PtHMAs indicated that tandem and segmental duplications likely contributed to the expansion and evolution of the PtHMAs. Most of the HMA genes contained abiotic stress-related cis-elements. Tissue-specific expression of PtHMA genes showed that PtHMA1 and PtHMA4 had relatively high expression levels in the leaves, whereas Cu/Ag subgroup (PtHMA5.1- PtHMA8) genes were upregulated in the roots. High concentrations of Cu, Ag, Zn, Cd, Co, Pb, and Mn differentially regulated the expression of PtHMAs in various tissues. The preliminary results of the present study generated basic information on the HMA family of Populus that may serve as foundation for future functional studies.

Nonlinear optical microscopy of the bronchus.

Because of frequent exposure to carcinogens, the bronchus is prone to early pathologic alterations. The assessment of these early changes is of key significance in physiological studies and disease diagnosis of the bronchus. We utilize nonlinear optical microscopy (NLOM) to image mouse bronchial tissue based on intrinsic nonlinear optical contrast. Our results show that NLOM is effective for imaging the bronchial intact microstructural components, providing quantitative information about the biomorphology and biochemistry of tissue. Our findings also display that NLOM can provide a two-photon ratiometric redox fluorometry, based on mitochondrial signals and reduced pyridine nucleotide (NADH and NADPH) and oxidized flavoproteins (Fp) signals, to assess the metabolic state of the epithelial cells and chondrocytes. It was found that NLOM can offer a sensitive tool, based on the second-harmonic signal depth-dependent decay, to obtain quantitative information on the optical property of the stroma associated with normal and diseased tissue states. Our results suggest that with the advent of the clinical portability of typical nonlinear optical endoscopy, the NLOM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of bronchial disease.