Ligand and Strain Synergistic Effect in NiFeP0.32 LDH for Triggering Efficient Oxygen Evolution Reaction.

Developing efficient water-splitting electrocatalysts to accelerate the slow oxygen evolution reaction (OER) kinetics is urgently desired for hydrogen production. Herein, ultralow phosphorus (P)-doped NiFe LDH (NiFePx LDH) with mild compressive strain is synthesized as an efficient OER electrocatalyst. Remarkably, NiFePx LDH with the phosphorus mass ratio of 0.32 wt.% and compressive strain ratio of 2.53% (denoted as NiFeP0.32 LDH) exhibits extraordinary OER activity with an overpotential as low as 210 mV, which is superior to that of commercial IrO2 and other reported P-based OER electrocatalysts. Both experimental performance and density function theory (DFT) calculation demonstrate that the doping of P atoms can generate covalent Fe─P coordination bonds and lattice distortion, thus resulting in the consequent depletion of electrons around the Fe active center and the downward shift of the d-band center, which can lead to a weaker adsorption ability of *O intermediate to improve the catalytic performance of NiFeP0.32 LDH for OER. This work provides novel insights into the distinctive coordinated configuration of P in NiFePx LDH, which can result in superior catalytic performance for OER.

The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ-WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ-WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37-MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC-NBS-LRR resistance gene MdRPM1. DAP-seq, Y1H, EMSA, and RT-qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37-MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ-WRKY complex in plant pathogen defence responses.

Metabolomics and Transcriptomics Analyses Reveals the Molecular Regulatory Mechanisms of Walnut (Juglans regia L.) Embryos in Response to Shade Treatment.

The walnut is an important nut that has numerous uses worldwide. However, due to dwarf and close plantation methods as well as continuous cloudy or rainy days that occur during periods of walnut oil accumulation, the walnut fruit exhibits varying degrees of stress under low-light conditions. However, the effects of shade on metabolites and genes in walnut embryos remain unclear in the literature. The purpose of this study is to investigate the lipid biosynthesis process that occurs in walnut embryos under shade treatment via the use of metabolomics and transcriptomics analyses. The results indicate that the oil content decreases significantly under shaded conditions, while the protein content increases significantly. The expression levels of fatty acid desaturase 2 (FAD2) and stearoyl-ACP-desaturase (SAD) involved in the lipid biosynthesis mechanism were significantly reduced in the shaded group, which resulted in reductions in oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids. The reduced oil content was consistent with the downregulation of genes associated with the lipid biosynthesis mechanism. In the amino acid biosynthesis process, the upregulated cysteine synthase (cscK) and anthranilate synthase beta subunit 2 (trpG) genes promoted the accumulation of L-aspartic acid and L-citrulline. The increase in protein content was consistent with the upregulation of genes related to amino acid biosynthesis. Thus, our study provides new insights into the regulatory mechanisms of shade underlying overall walnut fruit quality.

Systematical Characterization of the AT-Hook Gene Family in Juglans regia L. and the Functional Analysis of the JrAHL2 in Flower Induction and Hypocotyl Elongation.

AT-hook motif nuclear localization (AHL) proteins play essential roles in various plant biological processes. Yet, a comprehensive understanding of AHL transcription factors in walnut (Juglans regia L.) is missing. In this study, 37 AHL gene family members were first identified in the walnut genome. Based on the evolutionary analysis, JrAHL genes were grouped into two clades, and their expansion may occur due to segmental duplication. The stress-responsive nature and driving of developmental activities of JrAHL genes were revealed by cis-acting elements and transcriptomic data, respectively. Tissue-specific expression analysis showed that JrAHLs had a profound transcription in flower and shoot tip, JrAHL2 in particular. Subcellular localization showed that JrAHL2 is anchored to the nucleus. Overexpression of JrAHL2 in Arabidopsis adversely affected hypocotyl elongation and delayed flowering. Our study, for the first time, presented a detailed analysis of JrAHL genes in walnut and provided theoretical knowledge for future genetic breeding programs.

Genome-Wide Identification, Expression, and Interaction Analysis of BEL-Like Homeodomain Gene Family in Peach.

BEL1-like homeodomain (BLH) family genes as homeodomain transcription factors are found ubiquitously in plants to play important regulatory roles in reproductive development, morphological development, and stress response. Although BLH proteins have been reported in some species, there is little information about BLH genes in peach. In this study, we identified 11 peach PpBLH genes based on the conserved domain. Phylogenetic analysis suggested that the PpBLH proteins could be divided into five groups, which might be involved in different aspects of morphogenesis. Genomics structure analysis revealed that there were four exons in the PpBLH gene, and the length of the third exon was 61 bp. Chromosomal location analysis showed that the PpBLH genes were not distributed uniformly on six chromosomes. Promoter analysis showed that the promoter sequences of six PpBLH genes contained multiple cis-acting elements for hormones and stress. Six PpBLH genes were cloned by RT-PCR, and PpBLH1, PpBLH4, and PpBLH7 showed different expression patterns in the tested fruits under common temperature and high temperature. Y2H results indicated that PpBLH7 andPpBLH10 interacted with the PpOFP6 protein, and PpBLH1 interacted with the PpOFP1, PpOFP2, PpOFP4, and PpOFP13 proteins. These results provide new insight for further study of PpBLH genes, and construction of regulatory networks of PpBLH proteins in the growth, development, and stress response of peach.

Genome-wide analysis of the apple CaCA superfamily reveals that MdCAX proteins are involved in the abiotic stress response as calcium transporters.

BACKGROUND: Calcium (Ca2+) plays an important role in plant growth and development, and the maintenance of calcium homeostasis is necessary for the survival of all plant species. Ca2+/H+ exchangers (CAXs) are a subgroup of the CaCA (Ca2+/cation antiporter) superfamily. In general, CAX proteins mediate cytosolic Ca2+ entry into vacuoles to prevent excessive accumulation of Ca2+ in the cytosol. The CaCA superfamily has been identified and characterised in many plant species; however, characterisation of the CaCA superfamily and functional study of apple CAX proteins have yet to be conducted in apple (Malus × domestica Borkh.). RESULTS: Here, we identified 21 CaCA family proteins in apple for the first time. Phylogenetic and gene structure analysis, as well as prediction of conserved motifs, suggested that these proteins could be classified into four groups: CAX, CCX, NCL, and MHX. Expression analysis showed that the 10 MdCAX genes we cloned strongly responded to calcium and abiotic stress treatments. Collinearity analysis and characterisation of calcium transport capacity resulted in the identification of a pair of segmental duplication genes: MdCAX3L-1 and MdCAX3L-2; MdCAX3L-2 showed strong calcium transport capacity, whereas MdCAX3L-1 showed no calcium transport capacity. Yeast two-hybrid (Y2H) assays showed that these two proteins could interact with each other. The high sequence similarity (94.6%) makes them a good model for studying the crucial residues and structural basis of the calcium transport of CAX proteins. Prediction of the protein interaction network revealed several proteins that may interact with CAX proteins and play important roles in plant stress responses, such as SOS2, CXIP1, MHX, NRAMP3, and MTP8. CONCLUSIONS: Our analysis indicated that MdCAX proteins have strong calcium transport capacity and are involved in the abiotic stress response in apple. These findings provide new insight and rich resources for future studies of MdCAX proteins in apple.

Genome-wide identification, expression, and interaction analysis for ovate family proteins in peach.

Ovate family proteins (OFPs), which are involved in aspects of plant development and growth, is a class of plant-specific transcription factors. Although OFPs have been reported in some species, little is known about their evolution, structure, fruit developmental expression, and interactions among OFP members in peach (Prunus persica). In this study, 15 peach OFP (PpOFP) genes were identified. Phylogenetic analysis showed that 716 OFPs from 32 species were divided into 15 subgroups; 10 subgroups (Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, and Ij) were composed of dicotyledonous plants only and five (IIa, IIb, IIc, IId, and IIe) were composed of monocotyledonous plants only. Structure analysis showed that the OFP genes in monocotyledonous and dicotyledonous plants had no introns. Chromosomal localization analysis showed that 15 PpOFP genes were unevenly mapped on seven chromosomes. Furthermore, eight of the 15 PpOFP genes were cloned successfully by the RT-PCR method. To some extent, eight PpOFPs were expressed in all the detected peach tissues. In addition, analysis by Y2H and BiFC technologies indicated that both PpOFP4 and PpOFP5 formed homodimers with themselves, and PpOFP5 interacted with PpOFP7 or PpOFP8 to form heterodimers. These results serve as the theoretical basis for the analysis of the biological function and regulation of peach OFP transcription factors in the growth, development and other conditions, as well as evolution studies of OFP transcription factors in higher plants.

Genome-wide analyses of genes encoding FK506-binding proteins reveal their involvement in abiotic stress responses in apple.

BACKGROUND: The FK506-binding proteins (FKBPs) play diverse roles in numerous critical processes for plant growth, development, and abiotic stress responses. However, the FKBP gene family in the important fruit crop apple (Malus × domestica Borkh.) has not been studied as thoroughly as in other species. Our research objective was to investigate the mechanisms by which apple FKBPs enable apple plants to tolerate the effects of abiotic stresses. RESULTS: Using bioinformatics-based methods, RT-PCR, and qRT-PCR technologies, we identified 38 FKBP genes and cloned 16 of them in the apple genome. The phylogenetic analysis revealed three major groups within that family. The results from sequence alignments, 3-D structures, phylogenetics, and analyses of conserved domains indicated that apple FKBPs are highly and structurally conserved. Furthermore, genomics structure analysis showed that those genes are also highly and structurally conserved in several other species. Comprehensive qRT-PCR analysis found various expression patterns for MdFKBPs in different tissues and in plant responses to water-deficit and salt stresses. Based on the results from interaction network and co-expression analyses, we determined that the pairing in the MdFKBP62a/MdFKBP65a/b-mediated network is involved in water-deficit and salt-stress signaling, both of which are uniformly up-regulated through interactions with heat shock proteins in apple. CONCLUSIONS: These results provide new insight for further study of FKBP genes and their functions in abiotic stress response and multiple metabolic and physiological processes in apple.

Mapping QTLs for water-use efficiency reveals the potential candidate genes involved in regulating the trait in apple under drought stress.

BACKGROUND: Improvement of water-use efficiency (WUE) can effectively reduce production losses caused by drought stress. A better understanding of the genetic determination of WUE in crops under drought stress has great potential value for developing cultivars adapted to arid regions. To identify the genetic loci associated with WUE and reveal genes responsible for the trait in apple, we aim to map the quantitative trait loci (QTLs) for carbon isotope composition, the proxy for WUE, applying two contrasting irrigating regimes over the two-year experiment and search for the candidate genes encompassed in the mapped QTLs. RESULTS: We constructed a high-density genetic linkage map with 10,172 markers of apple, using single nucleotide polymorphism (SNP) markers obtained through restriction site-associated DNA sequencing (RADseq) and a final segregating population of 350 seedlings from the cross of Honeycrisp and Qinguan. In total, 33 QTLs were identified for carbon isotope composition in apple under both well-watered and drought-stressed conditions. Three QTLs were stable over 2 years under drought stress on linkage groups LG8, LG15 and LG16, as validated by Kompetitive Allele-Specific PCR (KASP) assays. In those validated QTLs, 258 genes were screened according to their Gene Ontology functional annotations. Among them, 28 genes were identified, which exhibited significant responses to drought stress in 'Honeycrisp' and/or 'Qinguan'. These genes are involved in signaling, photosynthesis, response to stresses, carbohydrate metabolism, protein metabolism and modification, hormone metabolism and transport, transport, respiration, transcriptional regulation, and development regulation. They, especially those for photoprotection and relevant signal transduction, are potential candidate genes connected with WUE regulation in drought-stressed apple. CONCLUSIONS: We detected three stable QTLs for carbon isotope composition in apple under drought stress over 2 years, and validated them by KASP assay. Twenty-eight candidate genes encompassed in these QTLs were identified. These stable genetic loci and series of genes provided here serve as a foundation for further studies on marker-assisted selection of high WUE and regulatory mechanism of WUE in apple exposed to drought conditions, respectively.

Genome-Wide Identification and Analysis of Apple NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER Family (NPF) Genes Reveals MdNPF6.5 Confers High Capacity for Nitrogen Uptake under Low-Nitrogen Conditions.

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) proteins play important roles in moving substrates such as nitrate, peptides, amino acids, dicarboxylates, malate, glucosinolates, indole acetic acid (IAA), abscisic acid (ABA), and jasmonic acid. Although a unified nomenclature of NPF members in plants has been reported, this gene family has not been studied as thoroughly in apple (Malus × domestica Borkh.) as it has in other species. Our objective was to provide general information about apple MdNPFs and analyze the transcriptional responses of some members to different levels of nitrate supplies. We identified 73 of these genes from the apple genome and used phylogenetic analysis to organize them into eight major groups. These apple NPFs are structurally conserved, based on alignment of amino acid sequences and analyses of phylogenetics and conserved domains. Examination of their genomic structures indicated that these genes are highly conserved among other species. We monitored 14 cloned MdNPFs that showed varied expression patterns under different nitrate concentrations and in different tissues. Among them, NPF6.5 was significantly induced by both low and high levels of nitrate. When compared with the wild type, 35S:MdNPF6.5 transgenic apple calli were more tolerant to low-N stress, which demonstrated that this gene confers greater capacity for nitrogen uptake under those conditions. We also analyzed the expression patterns of those 73 genes in various tissues. Our findings benefit future research on this family of genes.

Genome-Wide Analysis and Cloning of the Apple Stress-Associated Protein Gene Family Reveals MdSAP15, Which Confers Tolerance to Drought and Osmotic Stresses in Transgenic Arabidopsis.

Stress-associated proteins (SAPs) are novel A20/AN1 zinc finger domain-containing proteins that are now favorable targets to improve abiotic stress tolerance in plants. However, the SAP gene family and their biological functions have not been identified in the important fruit crop apple (Malus × domestica Borkh.). We conducted a genome-wide analysis and cloning of this gene family in apple and determined that the overexpression of MdSAP15 enhances drought tolerance in Arabidopsis plants. We identified 30 SAP genes in the apple genome. Phylogenetic analysis revealed two major groups within that family. Results from sequence alignments and analyses of 3D structures, phylogenetics, genomics structure, and conserved domains indicated that apple SAPs are highly and structurally conserved. Comprehensive qRT-PCR analysis found various expression patterns for MdSAPs in different tissues and in response to a water deficit. A transgenic analysis showed that the overexpression of MdSAP15 in transgenic Arabidopsis plants markedly enhanced their tolerance to osmotic and drought stresses. Our results demonstrate that the SAP genes are highly conserved in plant species, and that MdSAP15 can be used as a target gene in genetic engineering approaches to improve drought tolerance.

The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants.

In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees.

[Genome-wide identification and expression analysis of the WRKY gene family in peach].

The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles.

MdVQ17 negatively regulates apple resistance to Glomerella leaf spot by promoting MdWRKY17-mediated salicylic acid degradation and pectin lyase activity.

Glomerella leaf spot (GLS) is a fungal disease caused by Colletotrichum fructicola, which severely restricts the yield and quality of apples. Valine-glutamine (VQ) proteins are transcriptional regulators involved in the regulation of plant growth and stress responses. However, little is known about the role of VQ proteins in the biotic stress response in apple. Here, a VQ gene, MdVQ17, that was highly induced by C. fructicola infection was identified. Overexpression of MdVQ17 in apple increased susceptibility to C. fructicola and significantly reduced the salicylic acid content and ß-1,3-glucanase and chitinase activities. Based on yeast two-hybrid screening, MdWRKY17, which promotes susceptibility to C. fructicola, was identified as an MdVQ17-interacting protein. Co-expression of MdVQ17 can promote the binding and transcriptional activation activity of MdWRKY17 on the promoter of Downy Mildew Resistant 6 (MdDMR6), thereby promoting MdWRKY17-mediated salicylic acid degradation. Based on DNA affinity purification sequencing, the pectin lyase-encoding gene MdPL-like was identified as a direct target of MdWRKY17. MdWRKY17 can directly bind to the promoter of MdPL-like and activate its transcription, and the binding and activation of MdWRKY17 on the MdPL-like promoter were significantly enhanced by MdVQ17 co-expression. Functional identification showed that MdPL-like promoted pectin lyase activity and susceptibility to C. fructicola. In sum, these results demonstrate that the MdVQ17-MdWRKY17 module mediates the response to C. fructicola infection by regulating salicylic acid accumulation and pectin lyase activity. Our findings provide novel insights into the mechanisms by which the VQ-WRKY complex modulates plant pathogen defense responses.

Waxberry-like hydrophilic Co-doped ZnFe2O4 as bifunctional electrocatalysts for water splitting.

Water splitting is a promising technique for clean hydrogen production. To improve the sluggish hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), the development of efficient bifunctional electrocatalysts for both HER and OER is urgent to approach the scale-up applications of water splitting. Nowadays transition metal oxides (TMOs) are considered as the promising electrocatalysts due to their low cost, structural flexibility and stability, however, their electrocatalytic activities are eager to be improved. Here, we synthesized waxberry-like hydrophilic Co-doped ZnFe2O4 electrocatalysts as bifunctional electrocatalysts for water splitting. Due to the enhanced active sites by electronic structure tuning and modified super-hydrophilic characteristics, the spinel ZFO-Co0.5 electrocatalyst exhibits excellent catalytic activities for both OER and HER. It exhibits a remarkable low OER overpotential of 220 mV at a current density of 10 mA cm-2 and a Tafel slope of 28.2 mV dec-1. Meanwhile, it achieves a low overpotential of 73 mV at a current density of 10 mA cm-2 with the Tafel slope of 87 mV dec-1 for HER. In addition, for water electrolysis device, the electrocatalytic performance of ZFO-Co0.5||ZFO-Co0.5 surpasses that of commercial IrO2||Pt/C. Our work reveals that the hydrophilic morphology regulation combined with metallic doping strategy is a facile and effective approach to synthesize spinel TMOs as excellent bifunctional electrocatalyst for water splitting.

The apple MdGA2ox7 modulates the balance between growth and stress tolerance in an anthocyanin-dependent manner.

Apple (Malus domestica Borkh.) is a widely cultivated fruit crop worldwide but often suffers from abiotic stresses such as salt and cold. Gibberellic acid (GA) plays a pivotal in controlling plant development, environmental adaptability, and secondary metabolism. The GA2-oxidase (GA2ox) is responsible for the deactivation of bioactive GA. In this study, seventeen GA2-oxidase genes were identified in the apple genome, and these members could be clustered into four clades based on phylogenetic relationships and conserved domain structures. MdGA2ox7 exhibited robust expression across various tissues, responded to cold and salt treatments, and was triggered in apple fruit peels via light-induced anthocyanin accumulation. Subcellular localization prediction and experiments confirmed that MdGA2ox7 was located in the cytoplasm. Overexpression of MdGA2ox7 in Arabidopsis caused a lower level of active GA and led to GA-deficient phenotypes, such as dwarfism and delayed flowering. MdGA2ox7 alleviated cold and salt stress damage in both Arabidopsis and apple in concert with melatonin (MT). Additionally, MdGA2ox7 enhanced anthocyanin biosynthesis in apple calli and activated genes involved in anthocyanin synthesis. These findings provide new insights into the functions of apple GA2ox in regulating development, stress tolerance, and secondary metabolism.

Metabolomics and Transcriptomics Provide Insights into Lipid Biosynthesis in the Embryos of Walnut (Juglans regia L.).

Walnut (Juglans regia L.) is an important woody oilseed tree species due to its commercial value. However, the regulation mechanism of walnut oil accumulation is still poorly understood, which restricted the breeding and genetic improvement of high-quality oil-bearing walnuts. In order to explore the metabolic mechanism that regulates the synthesis of walnut oil, we used transcriptome sequencing technology and metabolome technology to comprehensively analyze the key genes and metabolites involved in oil synthesis of the walnut embryo at 60, 90, and 120 days after pollination (DAP). The results showed that the oil and protein contents increased gradually during fruit development, comprising 69.61% and 18.32% of the fruit, respectively, during ripening. Conversely, the contents of soluble sugar and starch decreased gradually during fruit development, comprising 2.14% and 0.84%, respectively, during ripening. Transcriptome sequencing generated 40,631 unigenes across 9 cDNA libraries. We identified 51 and 25 candidate unigenes related to the biosynthesis of fatty acid and the biosynthesis of triacylglycerol (TAG), respectively. The expression levels of the genes encoding Acetyl-CoA carboxylase (ACCase), long-chain acyl-CoA synthetases (LACS), 3-oxoacyl-ACP synthase II (KASII), and glycerol-3-phosphate acyl transfer (GPAT) were upregulated at 60 DAP relative to the levels at 90 and 120 DAP, while the stearoyl-ACP-desaturase (SAD) and fatty acid desaturase 2 (FAD2) genes were highly abundantly expressed during all walnut developmental periods. We found that ABSCISIC ACID INSENSEITIVE3 (ABI3), WRINKLEDl (WRI1), LEAFY COTYLEDON1 (LEC1), and FUSCA3 (FUS3) may be key transcription factors involved in lipid synthesis. Additionally, the metabolomics analysis detected 706 metabolites derived from 18 samples, among which, 4 are implicated in the TAG synthesis, 2 in the glycolysis pathway, and 5 in the tricarboxylic acid cycle (TCA cycle) pathway. The combined analysis of the related genes and metabolites in TAG synthesis showed that phospholipid:diacylglycerol acyltransferase (PDAT) genes were highly abundantly expressed across walnut fruit developmental periods, and their downstream metabolite TAG gradually accumulated with the progression of fruit development. The FAD2 gene showed consistently higher expression during fruit development, and its downstream metabolites 18:2-PC and 18:3-PC gradually accumulated. The ACCase, LACS, SAD, FAD2, and PDAT genes may be crucial genes required for walnut oil synthesis. Our data will enrich public databases and provide new insights into functional genes related to lipid metabolism in walnut.

Genome-Wide Characterization of the Mitogen-Activated Protein Kinase Gene Family and Their Expression Patterns in Response to Drought and Colletotrichum Gloeosporioides in Walnut (Juglans regia).

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr (serine/threonine) protein kinases that play very important roles in plant responses to biotic and abiotic stressors. However, the MAPK gene family in the important crop walnut (Juglans regia L.) has been less well studied compared with other species. We discovered 25 JrMAPK members in the Juglans genome in this study. The JrMAPK gene family was separated into four subfamilies based on phylogenetic analysis, and members of the same subgroup had similar motifs and exons/introns. A variety of cis-acting elements, mainly related to the light response, growth and development, stress response, and hormone responses, were detected in the JrMAPK gene promoters. Collinearity analysis showed that purification selection was the main driving force in JrMAPK gene evolution, and segmental and tandem duplications played key roles in the expansion of the JrMAPK gene family. The RNA-Seq (RNA Sequencing) results indicated that many of the JrMAPK genes were expressed in response to different levels of Colletotrichum gloeosporioides infection. JrMAPK1, JrMAPK3, JrMAPK4, JrMAPK5, JrMAPK6, JrMAPK7, JrMAPK9, JrMAPK11, JrMAPK12, JrMAPK13, JrMAPK17, JrMAPK19, JrMAPK20, and JrMAPK21 were upregulated at the transcriptional level in response to the drought stress treatment. The results of this study will help in further investigations of the evolutionary history and biological functions of the MAPK gene family in walnut.

KNOTTED1-like homeobox (KNOX) transcription factors - Hubs in a plethora of networks: A review.

KNOX (KNOTTED1-like HOMEOBOX) belongs to a class of important homeobox genes, which encode the homeodomain proteins binding to the specific element of target genes, and widely participate in plant development. Advancements in genetics and molecular biology research generate a large amount of information about KNOX genes in model and non-model plants, and their functions in different developmental backgrounds are gradually becoming clear. In this review, we summarize the known and presumed functions of the KNOX gene in plants, focusing on horticultural plants and crops. The classification and structural characteristics, expression characteristics and regulation, interacting protein factors, functions, and mechanisms of KNOX genes are systematically described. Further, the current research gaps and perspectives were discussed. These comprehensive data can provide a reference for the directional improvement of agronomic traits through KNOX gene regulation.

Composition and Diversity of Endophytic Rhizosphere Microbiota in Apple Tree with Different Ages.

In order to determine the underlying mechanism of the senescence occurring in older apple trees, the effects of tree age on the community structure and dominant genus of endophytic rhizosphere bacteria in apple were investigated. The diversity and structure of the bacterial communities and corresponding changes in the dominant genera of endophytic rhizosphere bacteria of apple at different ages (2, 8, 16, 22 years) were compared based on 16S rRNA high-throughput sequencing technology. The results revealed that the longer the tree age, the less the number of ASV in the endophytic bacteria. Moreover, the number of ASV in the endophytic bacteria gradually decreased as the tree age increased, however no significant changes were observed in the alpha diversity. At the phyla level, the relative abundance of Actinobacteria increased, while that of Proteobateria decreased. At the genus level, the relative abundance of Mycobacterium, Chujaibacter, and other genera increased, while the relative abundance of Aquabacterium, Ralstonia, Streptomyces, Asticcacaulis, Hyphomicrobium, Pseudomonas, and Sphingomonas decreased. The reduced relative abundance of endophytic rhizosphere bacteria associated with plant growth and disease resistance may thus be the cause of tree senescence. This work acts as a reference to increases the understanding of plant-microbe interactions.