Proteomic and Transcriptomic Analyses Provide New Insights into the Mechanism Underlying Lipid Deterioration in Pecan Kernels during Storage.

Pecan nuts are rich in lipids that tend to deteriorate during storage. Tandem mass-tag-based quantitative proteomics and transcriptomics were used to investigate the changes in the protein and gene profiles of stored pecan kernels for the first time. Our previous lipidomic data were jointly analyzed to elucidate the coordinated changes in lipid molecules and related proteins/genes. The mechanism underlying lipid deterioration in pecan kernels during storage was revealed by multiomics analyses. Lipid metabolism-related pathways were activated during pecan storage. Phospholipases, triacylglycerol lipases, lipoxygenases, and oil body-related proteins/genes were highly expressed during storage, revealing their involvement in lipid deterioration. These data provide rich information and will be valuable for future genetic or chemical research to alleviate lipid deterioration in pecans.

Targeted metabolomics reveals key phenolic changes in pecan nut quality deterioration under different storage conditions.

Pecan nuts are highly enriched in phenolic compounds, which contribute to the health benefits of pecans. Phenolic compounds represent the main oxidation reaction substrates, thus leading to quality deterioration, namely pellicle browning or a decrease in beneficial effects during pecan storage. Hence, four different storage conditions were performed for 180 d to simulate real production situations. Targeted metabolomics was chosen to identify the specific phenolic compounds involved in quality deterioration under different storage conditions in 0, 90, and 180 d samples. A total of 118 phenolic compounds were detected, nine of which were identified for the first time in pecan. The total phenolic content (TPC) and antioxidant capacities initially demonstrated high scores, after which they tended to decrease during the storage process. The significantly modified phenolic compounds during storage were selected as the metabolite markers of pecan quality deterioration, including catechin, procyanidin (PA) trimer, PA tetramer, trigalloyl hexahydroxydiphenoyl (HHDP) glucose, and tetragalloyl hexoside. Fresh pecan kernels resulted in more pronounced changes in hydrolysable tannins (HTs), whereas dry kernels resulted in the most accentuated changes in condensed tannins (CTs). To the best of our knowledge, this is the first attempt to study individual phenolic changes during storage of pecan in such massive amounts. The results can offer a valuable theoretical basis for future control of pecan quality deterioration through phenolics during storage.

The chloroplast genome of Prunus zhengheensis: Genome comparative and phylogenetic relationships analysis.

Prunus zhengheensis is a novel species originated in Fujian province, China. However, there is no further information available on its classification and molecular biology study. In this study, we first report the complete chloroplast (cp) genome sequence of P. zhengheensis. The cp genome of P. zhengheensis is 158,106 bp and GC content is 36.73%, is a circular structure composed of LSC (large single copy), SSC (small single copy), and IR (inverted repeat) regions, with the size of the three regions being 86,321 bp, 18,999 bp and 26,393 bp, respectively. The cp genome of P. zhengheensis contains 130 genes, and 242 SSRs are identified in the cp genome. The comparative analysis of cp genomes in eight Prunus plants demonstrates the subtle divergences occur in the protein-coding gene rps18, rps12, psbF, rpl33, matK, and rbcL, and that the KA/KS nucleotide substitution ratio of the ndhF of P. zhengheensis and P. armeniaca is 1.79636. The phylogenetic results indicate that the P. zhengheensis is closely related to P. mume, compared to other species of Prunus. Our research results provide the important genomic information for molecular phylogeny of P. zhengheensis.

Characterization of the complete chloroplast genome of Carya hunanensis W. C. Cheng & R. H. Chang.

Carya hunanensis W. C. Cheng & R. H. Chang is a tree of great economic importance in China. In this study, the complete chloroplast genome of C. hunanensis was determined and analyzed phylogenetically. The whole genome was determined to be 159,892 bp in length, presenting a typical quadripartite structure with a large single copy (LSC) region (89,466 bp), a small single copy (SSC) region (18,714 bp), and a pair of inverted repeat (IR) regions (25,856 bp each). There were 132 genes annotated in the genome, including 84 protein-coding genes, 40 tRNA genes, and eight rRNA genes. The overall GC content of C. hunanensis chloroplast genome was 36.24%. Phylogenetic analysis indicated that C. hunanensis was closely related to C. kweichowensis and Annamocarya sinensis.

Characterization of the complete chloroplast genome of Carya laciniosa (F.Michx.) G.Don (Juglandaceae).

Carya laciniosa (F.Michx.) G.Don is a slow-growing valuable tree in the family Juglandaceae. In this research, we first reported the chloroplast genome sequence of C. laciniosa. The chloroplast genome was a circular form with 160,832 bp in size, comprising four subregions: a large single-copy (LSC) region of 90,065 bp, a small single-copy (SSC) region of 18,791 bp, and two copies of inverted repeats (IRs; IRa and IRb) of 25,988 bp each. A total of 132 genes were annotated in the chloroplast genome of C. laciniosa, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of the whole genome was 36.14%. Phylogenetic analysis suggested that C. laciniosa was closely related to C. ovata.

Identification and Analysis of NBS-LRR Genes in Actinidia chinensis Genome.

Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes represent the most important disease resistance genes in plants. The genome sequence of kiwifruit (Actinidia chinensis) provides resources for the characterization of NBS-LRR genes and identification of new R-genes in kiwifruit. In the present study, we identified 100 NBS-LRR genes in the kiwifruit genome and they were grouped into six distinct classes based on their domain architecture. Of the 100 genes, 79 are truncated non-regular NBS-LRR genes. Except for 37 NBS-LRR genes with no location information, the remaining 63 genes are distributed unevenly across 18 kiwifruit chromosomes and 38.01% of them are present in clusters. Seventeen families of cis-acting elements were identified in the promoters of the NBS-LRR genes, including AP2, NAC, ERF and MYB. Pseudomonas syringae pv. actinidiae (pathogen of the kiwifruit bacterial canker) infection induced differential expressions of 16 detected NBS-LRR genes and three of them are involved in plant immunity responses. Our study provides insight of the NBS-LRR genes in kiwifruit and a resource for the identification of new R-genes in the fruit.

Metabolic profiling revealed the organ-specific distribution differences of tannins and flavonols in pecan.

Carya illinoinensis is rich in phenolic metabolites such as tannins and flavonols, but both the composition and the distribution of these nutritional constituents in most pecan organs were still unclear. In this experiment, a comprehensive qualification and quantification of phenolic metabolites in eight organs of pecan were conducted for the first time. Ninety-seven phenolic metabolites were identified, in which twelve were identified for the first time in pecan, including a series of ellagitannins with high molecular weight. Hydrolysable tannin was the dominant kind of phenolic metabolites in pecan. The metabolic profiles of tannins in pecan were extended. Thirty-three phenolic metabolites were quantified, among them the highest content was ellagic acid pentose in testa. From this experiment, we can see that the distribution of phenolic metabolites in pecan was organ-specific, tannins tend to accumulate in pecan testa with both diverse structures and high contents, while flavonols tend to accumulate in organs such as branch, bark, or leaf. Among all organs, testa contained the highest content of phenolics, which might play important roles in protecting pecan kernel from diseases and insects. A massive phenolic metabolites' matrix in different pecan organs was built in this experiment, which should be useful for related researches in the future and help provide a theoretical basis for using these organs as functional foods.

Photosystem Disorder Could be the Key Cause for the Formation of Albino Leaf Phenotype in Pecan.

Pecan is one of the most famous nut species in the world. The phenotype of mutants with albino leaves was found in the process of seeding pecan, providing ideal material for the study of the molecular mechanisms leading to the chlorina phenotype in plants. Both chlorophyll a and chlorophyll b contents in albino leaves (ALs) were significantly lower than those in green leaves (GLs). A total of 5171 differentially expression genes (DEGs) were identified in the comparison of ALs vs. GLs using high-throughput transcriptome sequencing; 2216 DEGs (42.85%) were upregulated and 2955 DEGs (57.15%) were downregulated. The expressions of genes related to chlorophyll biosynthesis (HEMA1, encoding glutamyl-tRNA reductase; ChlH, encoding Mg-protoporphyrin IX chelatase (Mg-chelatase) H subunit; CRD, encoding Mg-protoporphyrin IX monomethylester cyclase; POR, encoding protochlorophyllide reductase) in ALs were significantly lower than those in GLs. However, the expressions of genes related to chlorophyll degradation (PAO, encoding pheophorbide a oxygenase) in ALs were significantly higher than those in GLs, indicating that disturbance of chlorophyll a biosynthesis and intensification of chlorophyll degradation lead to the absence of chlorophyll in ALs of pecan. A total of 72 DEGs associated with photosynthesis pathway were identified in ALs compared to GLs, including photosystem I (15), photosystem II (19), cytochrome b6-f complex (3), photosynthetic electron transport (6), F-type ATPase (7), and photosynthesis-antenna proteins (22). Moreover, almost all the genes (68) mapped in the photosynthesis pathway showed decreased expression in ALs compared to GLs, declaring that the photosynthetic system embedded within the thylakoid membrane of chloroplast was disturbed in ALs of pecan. This study provides a theoretical basis for elucidating the molecular mechanism underlying the phenotype of chlorina seedlings of pecan.

Characterization and phylogenetic relationships analysis of the complete chloroplast genome of Carya ovata.

Carya ovata is a slow-growing, long-lived deciduous species that belongs to section Carya of genus Carya. In this study, we de novo assembled the complete chloroplast genome of C. ovata, and analyzed its phylogenetic relationship. The circular genome was 160,765 bp in length, comprising a large single-copy region (89,975 bp), a small single-copy region (18,788 bp), and a pair of inverted repeat regions (26,001 bp each). The chloroplast genome was predicted to contain 131 genes, including 83 protein-coding genes, 40 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Overall, the GC content of the chloroplast genome was 36.16%. Phylogenetic analysis suggested that C. ovata was closely related to C. illinoinensis, a representative of section Apocarya within the genus Carya.

AdRAP2.3, a Novel Ethylene Response Factor VII from Actinidia deliciosa, Enhances Waterlogging Resistance in Transgenic Tobacco through Improving Expression Levels of PDC and ADH Genes.

APETALA2/ethylene-responsive factor superfamily (AP2/ERF) is a transcription factor involved in abiotic stresses, for instance, cold, drought, and low oxygen. In this study, a novel ethylene-responsive transcription factor named AdRAP2.3 was isolated from Actinidia deliciosa 'Jinkui'. AdRAP2.3 transcription levels in other reproductive organs except for the pistil were higher than those in the vegetative organs (root, stem, and leaf) in kiwi fruit. Plant hormones (Salicylic acid (SA), Methyl-jasmonate acid (MeJA), 1-Aminocyclopropanecarboxylic Acid (ACC), Abscisic acid (ABA)), abiotic stresses (waterlogging, heat, 4 °C and NaCl) and biotic stress (Pseudomonas Syringae pv. Actinidiae, Psa) could induce the expression of AdRAP2.3 gene in kiwi fruit. Overexpression of the AdRAP2.3 gene conferred waterlogging stress tolerance in transgenic tobacco plants. When completely submerged, the survival rate, fresh weight, and dry weight of transgenic tobacco lines were significantly higher than those of wile type (WT). Upon the roots being submerged, transgenic tobacco lines grew aerial roots earlier. Overexpression of AdRAP2.3 in transgenic tobacco improved the pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme activities, and improved the expression levels of waterlogging mark genes NtPDC, NtADH, NtHB1, NtHB2, NtPCO1, and NtPCO2 in roots under waterlogging treatment. Overall, these results demonstrated that AdRAP2.3 might play an important role in resistance to waterlogging through regulation of PDC and ADH genes in kiwi fruit.

Genome-Wide Bioinformatics Analysis of MAPK Gene Family in Kiwifruit (Actinidia Chinensis).

Mitogen activated protein kinase (MAPK) cascades are universal signal transduction modules that play crucial roles in various biotic and abiotic stresses, hormones, cell division, and developmental processes in plants. Mitogen activated protein kinase (MAPK/MPK), being a part of this cascade, performs an important function for further appropriate cellular responses. Although MAPKs have been investigated in several model plants, no systematic analysis has been conducted in kiwifruit (Actinidia chinensis). In the present study, we identified 18 putative MAPKs in the kiwifruit genome. This gene family was analyzed bioinformatically in terms of their chromosome locations, sequence alignment, gene structures, and phylogenetic and conserved motifs. All members possess fully canonical motif structures of MAPK. Phylogenetic analysis indicated that AcMAPKs could be classified into five subfamilies, and these gene motifs in the same group showed high similarity. Gene structure analysis demonstrated that the number of exons in AcMAPK genes ranged from 2 to 29, suggesting large variation among kiwifruit MAPK genes. The expression profiles of these AcMAPK genes were further investigated using quantitative real-time polymerase chain reaction (qRT-PCR), which demonstrated that AcMAPKs were induced or repressed by various biotic and abiotic stresses and hormone treatments, suggesting their potential roles in the biotic and abiotic stress response and various hormone signal transduction pathways in kiwifruit. The results of this study provide valuable insight into the putative physiological and biochemical functions of MAPK genes in kiwifruit.

Chlorophyll, carotenoid and vitamin C metabolism regulation in Actinidia chinensis 'Hongyang' outer pericarp during fruit development.

Ascorbic acid (AsA), chlorophyll and carotenoid contents and their associated gene expression patterns were analysed in Actinidia chinensis 'Hongyang' outer pericarp. The results showed chlorophyll degradation during fruit development and softening, exposed the yellow carotenoid pigments. LHCB1 and CLS1 gene expressions were decreased, while PPH2 and PPH3 gene expressions were increased, indicating that downregulation of chlorophyll biosynthesis and upregulation of its degradation, caused chlorophyll degradation. A decrease in the expression of the late carotenoid biosynthesis and maintenance genes (LCYB1, LCYE1, CYP1, CYP2, ZEP1, VDE1, VDE2, and NCED2) and degradation gene (CCD1), showed biosynthesis and degradation of carotenoid could be regulatory factors involved in fruit development. Most genes expression data of L-galactose and recycling pathway were agreement with the AsA concentrations in the fruit, suggesting these are the predominant pathways of AsA biosynthesis. GMP1, GME1 and GGP1 were identified as the key genes controlling AsA biosynthesis in 'Hongyang' outer pericarp.

Dynamic Changes in Phenolics and Antioxidant Capacity during Pecan (Carya illinoinensis) Kernel Ripening and Its Phenolics Profiles.

Pecan (Carya illinoinensis) kernels have a high phenolics content and a high antioxidant capacity compared to other nuts-traits that have attracted great interest of late. Changes in the total phenolic content (TPC), condensed tannins (CT), total flavonoid content (TFC), five individual phenolics, and antioxidant capacity of five pecan cultivars were investigated during the process of kernel ripening. Ultra-performance liquid chromatography coupled with quadruple time-of-flight mass (UPLC-Q/TOF-MS) was also used to analyze the phenolics profiles in mixed pecan kernels. TPC, CT, TFC, individual phenolics, and antioxidant capacity were changed in similar patterns, with values highest at the water or milk stages, lowest at milk or dough stages, and slightly varied at kernel stages. Forty phenolics were tentatively identified in pecan kernels, of which two were first reported in the genus Carya, six were first reported in Carya illinoinensis, and one was first reported in its kernel. The findings on these new phenolic compounds provide proof of the high antioxidant capacity of pecan kernels.

Transcriptome Analysis of Kiwifruit in Response to Pseudomonas syringae pv. actinidiae Infection.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has brought about a severe threat to the kiwifruit industry worldwide since its first outbreak in 2008. Studies on other pathovars of P. syringae are revealing the pathogenesis of these pathogens, but little about the mechanism of kiwifruit bacterial canker is known. In order to explore the species-specific interaction between Psa and kiwifruit, we analyzed the transcriptomic profile of kiwifruit infected by Psa. After 48 h, 8255 differentially expressed genes were identified, including those involved in metabolic process, secondary metabolites metabolism and plant response to stress. Genes related to biosynthesis of terpens were obviously regulated, indicating terpens may play roles in suppressing the growth of Psa. We identified 283 differentially expressed resistant genes, of which most U-box domain containing genes were obviously up regulated. Expression of genes involved in plant immunity was detected and some key genes showed differential expression. Our results suggest that Psa induced defense response of kiwifruit, including PAMP (pathogen/microbe-associated molecular patterns)-triggered immunity, effector-triggered immunity and hypersensitive response. Metabolic process was adjusted to adapt to these responses and production of secondary metabolites may be altered to suppress the growth of Psa.

Functional Characterization of Waterlogging and Heat Stresses Tolerance Gene Pyruvate decarboxylase 2 from Actinidia deliciosa.

A previous report showed that both Pyruvatedecarboxylase (PDC) genes were significantly upregulated in kiwifruit after waterlogging treatment using Illumina sequencing technology, and that the kiwifruit AdPDC1 gene was required during waterlogging, but might not be required during other environmental stresses. Here, the function of another PDC gene, named AdPDC2, was analyzed. The expression of the AdPDC2 gene was determined using qRT-PCR, and the results showed that the expression levels of AdPDC2 in the reproductive organs were much higher than those in the nutritive organs. Waterlogging, NaCl, and heat could induce the expression of AdPDC2. Overexpression of kiwifruit AdPDC2 in transgenic Arabidopsis enhanced resistance to waterlogging and heat stresses in five-week-old seedlings, but could not enhance resistance to NaCl and mannitol stresses at the seed germination stage and in early seedlings. These results suggested that the kiwifruit AdPDC2 gene may play an important role in waterlogging resistance and heat stresses in kiwifruit.

Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.

Ethanolic fermentation is classically associated with waterlogging tolerance when plant cells switch from respiration to anaerobic fermentation. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we cloned a full-length PDC cDNA sequence from kiwifruit, named AdPDC1. We determined the expression of the AdPDC1 gene in kiwifruit under different environmental stresses using qRT-PCR, and the results showed that the increase of AdPDC1 expression during waterlogging stress was much higher than that during salt, cold, heat and drought stresses. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis enhanced the resistance to waterlogging stress but could not enhance resistance to cold stress at five weeks old seedlings. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis could not enhance resistance to NaCl and mannitol stresses at the stage of seed germination and in early seedlings. These results suggested that the kiwifruit AdPDC1 gene is required during waterlogging but might not be required during other environmental stresses. Expression of the AdPDC1 gene was down-regulated by abscisic acid (ABA) in kiwifruit, and overexpression of the AdPDC1 gene in Arabidopsis inhibited seed germination and root length under ABA treatment, indicating that ABA might negatively regulate the AdPDC1 gene under waterlogging stress.

[Study on Salt Tolerance of Echinacea purpurea].

OBJECTIVE: To explore the salt tolerance of Echiancea purpurea and its mechanism. METHODS: Echiancea purpurea was used as test material in this study and six salinity levels (0, 30, 60, 90, 120 and 150 mmol/L NaCl) were set. Effects on seed germination and salt tolerance relevant physiological and biochemical indexes of Echiancea purpurea were studied. RESULTS: Salt stress suppressed the germination of Echiancea purpurea seeds, induced osmotic adjustment substances proline, soluble sugar and K+ to increase, and activities of POD and SOD to rise, and meanwhile resulted in accumulation of Na+ and decrease of K+/Na+. CONCLUSION: Echiancea purpurea can tolerant salt stress to a certain degree, but in case of high salt concentrations, severe salt injury would remarkably suppress the growth of Echinacea purpurea.

Overexpression of the Malus hupehensis MhNPR1 gene increased tolerance to salt and osmotic stress in transgenic tobacco.

Earlier, we have reported that overexpression of Malus hupehensis Non-expressor of pathogenesis related gene 1 (MhNPR1) gene in tobacco could induce the expression of pathogenesis-related genes and enhance resistance to fungus Botrytis cinerea. In this study, we showed that MhNPR1 can be induced by NaCl, PEG6000, low temperature (4 °C), abscisic acid and apple aphids' treatments in M. hupehensis. Heterogonous expression of MhNPR1 gene in tobacco conferred enhanced resistance to NaCl at the stage of seed germination, and conferred resistance to mannitol at the stage of seed germination and to PEG6000 at the stage of seedlings. Furthermore, overexpression of MhNPR1 in transgenic tobacco led to higher expression levels of osmotic-stress related genes compared with wild-type plants. This was the first report of a novel function of NPR1 that overexpression of MhNPR1 gene has a positive effect on salt and osmotic stress in tobacco, which differs from the function that overexpressing of AtNPR1 gene has a negative effect on dehydration and salt stress in rice.

Comparative proteomic analysis of the stolon cold stress response between the C4 perennial grass species Zoysia japonica and Zoysia metrella.

Zoysiagrass, the most cold-tolerant grass among the warm-season turfgrasses, is often used as a model species for isolating cellular components related to cold stress. To understand the proteomic responses to cold stress in zoysiagrass stolons, we extracted stolon proteins from Zoysiajaponica, cv. Meyer (cold-tolerant) and Z. metrella, cv. Diamond (cold-sensitive), which were grown with or without cold treatment. Approximately 700 proteins were resolved on 2-DE gels, and 70 protein spots were differentially accumulated. We further observed that 45 of the identified proteins participate in 10 metabolic pathways and cellular processes. A significantly greater number of proteins accumulated in the Meyer than in the Diamond and 15 increased proteins were detected only in the Meyer cultivar under cold stress. Furthermore, we propose a cold stress-responsive protein network composed of several different functional components that exhibits a balance between reactive oxygen species (ROS) production and scavenging, accelerated protein biosynthesis and proteolysis, reduced protein folding, enhanced photosynthesis, abundant energy supply and enhanced biosynthesis of carbohydrates and nucleotides. Generally, the cold-tolerant Meyer cultivar showed a greater ROS scavenging ability, more abundant energy supply and increased photosynthesis and protein synthesis than did the cold-sensitive Diamond cultivar, which may partly explain why Meyer is more cold tolerant.

[Progresses on plant AP2/ERF transcription factors].

Plant AP2/ERF transcription factor with AP2/ERF domain containing 60-70 amino acids is a huge gene family present in all plant. AP2/ERF transcriptional factors are involved in various biological functions such as plant development, flower development, fruit and seed maturation, wounding, pathogen defense, high salty, drought, and so on. AP2/ERF transcription factor are involved in salicylic acid, jasmonic acid, ethylene, abscisic acid signal transduction pathways and among them. The transcription factors are cross-talk factor in stress signal pathway. This paper summarizes the most advanced researches on types, biological functions, and gene regulations of plant AP2/ERF transcription factors.