Comparative Evaluation of the Phytochemical Profiles and Antioxidant Potentials of Olive Leaves from 32 Cultivars Grown in China.

Olives (Olea europaea L.) are a significant part of the agroindustry in China. Olive leaves, the most abundant by-products of the olive and olive oil industry, contain bioactive compounds that are beneficial to human health. The purpose of this study was to evaluate the phytochemical profiles and antioxidant capacities of olive leaves from 32 cultivars grown in China. A total of 32 phytochemical compounds were identified using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, including 17 flavonoids, five iridoids, two hydroxycinnamic acids, six triterpenic acids, one simple phenol, and one coumarin. Specifically, olive leaves were found to be excellent sources of flavonoids (4.92-18.29 mg/g dw), iridoids (5.75-33.73 mg/g dw), and triterpenic acids (15.72-35.75 mg/g dw), and considerable variations in phytochemical content were detected among the different cultivars. All tested cultivars were classified into three categories according to their oil contents for further comparative phytochemicals assessment. Principal component analysis indicated that the investigated olive cultivars could be distinguished based upon their phytochemical profiles and antioxidant capacities. The olive leaves obtained from the low-oil-content (<16%) cultivars exhibited higher levels of glycosylated flavonoids and iridoids, while those obtained from high-oil-content (>20%) cultivars contained mainly triterpenic acids in their compositions. Correspondingly, the low-oil-content cultivars (OL3, Frantoio selection and OL14, Huaou 5) exhibited the highest ABTS antioxidant activities (758.01 ± 16.54 and 710.64 ± 14.58 mg TE/g dw, respectively), and OL9 (Olea europaea subsp. Cuspidata isolate Yunnan) and OL3 exhibited the highest ferric reducing/antioxidant power assay values (1228.29 ± 23.95 mg TE/g dw and 1099.99 ± 14.30 mg TE/g dw, respectively). The results from this study may be beneficial to the comprehensive evaluation and utilization of bioactive compounds in olive leaves.

Ectopic expression of GhCOBL9A, a cotton glycosyl-phosphatidyl inositol-anchored protein encoding gene, promotes cell elongation, thickening and increased plant biomass in transgenic Arabidopsis.

Cellulose is a major component of plant cell walls and is necessary for plant morphogenesis and biomass. COBL (COBRA-Like) proteins have been shown to be key regulators in the orientation of cell expansion and cellulose crystallinity status. To clarify the role of a cotton COBL gene, GhCOBL9A, we conducted the ectopic expression and functional analysis in Arabidopsis. Previous study showed that GhCOBL9A was preferentially expressed during secondary cell wall biosynthesis in cotton fibers, and showed a significant co-expression pattern with cellulose synthase genes. Here, we detected that overexpression of GhCOBL9A induced the up-regulation of genes related to cellulose synthesis and enhanced the cellulose deposition. As a result, GhCOBL9A transgenic plants displayed increased hypocotyl and root lengths in early development, and cell wall thickening at the SCW stage. Notably, overexpression of GhCOBL9A led to an erect, robust-stature phenotype and brought higher biomass in mature plants. In addition, overexpression of GhCOBL9A in Arabidopsis AtCOBL4 mutants, a paralogous gene of GhCOBL9A, also led to a stronger growth potential, but the Atcobl4 mutant phenotype could not be rescued, implying the functional divergence of GhCOBL9A and AtCOBL4 paralogs. Taken together, these results suggest that overexpression of GhCOBL9A contributes to plant cell elongation and thickening, and increased biomass, which provides references for further utilizing GhCOBL9A to improve yield and quality traits in cotton and other species.

Genome-wide analysis of CrRLK1L gene family in Gossypium and identification of candidate CrRLK1L genes related to fiber development.

Members of the CrRLK1L family, a subgroup of the receptor-like kinase (RLK) gene family, are thought to act as sensors for the integrity of the cell wall and regulators of polar elongation. To better understand the various functions in fiber development, we conducted genome-wide identification and characterization analyses of CrRLK1L family in cotton. Here 44, 40, and 79 CrRLK1L genes were identified from three cotton species: diploid G. raimondii (D5), diploid G. arboreum (A2), and tetraploid G. hirsutum TM-1 (AD1), respectively. The 44 CrRLK1Ls in G. raimondii were anchored to the 12 chromosomes unevenly and were classified into six groups (I-VI), with group II and group IV being further divided into two subgroups (groups IIa and IIb, and IVa and IVb, respectively). These CrRLK1Ls displayed a highly regular pattern of developmental and spatial regulation in cotton. Using the transcriptome data of five chromosomal segment introgression lines (CSILs) and the physical integration of CrRLK1Ls with the quantitative trait loci (QTLs) related to fiber quality traits, we revealed that six CrRLK1L genes were highly associated with fiber development. This study brings new insights into the integrated genome-wide identification of CrRLK1Ls in cotton and provides references for the genetic improvement of cotton fiber.

GhPSY, a phytoene synthase gene, is related to the red plant phenotype in upland cotton (Gossypium hirsutum L.).

Carotenoids are important accessory pigments in plants that are essential for photosynthesis. Phytoene synthase (PSY), a rate-controlling enzyme in the carotenoid biosynthesis pathway, has been widely characterized in rice, maize, and sorghum, but at present there are no reports describing this enzyme in cotton. In this study, GhPSY was identified as a candidate gene for the red plant phenotype via a combined strategy using: (1) molecular marker data for loci closely linked to R1; (2) the whole-genome scaffold sequence from Gossypium raimondii; (3) gene expression patterns in cotton accessions expressing the red plant and green plant phenotypes; and (4) the significant correlation between a single nucleotide polymorphisms (SNP) in GhPSY and leaf phenotypes of progeny in the (Sub16 × T586) F2 segregating population. GhPSY was relatively highly expressed in leaves, and the protein was localized to the plastid where it appeared to be mostly attached to the surface of thylakoid membranes. GhPSY mRNA was expressed at a significantly higher level in T586 and SL1-7-1 red plants than TM-1 and Hai7124 green plants. SNP analysis in the GhPSY locus showed co-segregation with the red and green plant phenotypes in the (Sub16 × T586) F2 segregating population. A phylogenetic analysis showed that GhPSY belongs to the PSY2 subfamily, which is related to photosynthesis in photosynthetic tissues. Using a reverse genetics approach based on Tobacco rattle virus-induced gene silencing, we showed that the knockdown of GhPSY caused a highly uniform bleaching of the red color in newly-emerged leaves in both T586 and SL1-7-1 plants with a red plant phenotype. These findings indicate that GhPSY is important for engineering the carotenoid metabolic pathway in pigment production.

Alterations of phenotype, physiology, and functional substances reveal the chilling-tolerant mechanism in two common Olea Europaea cultivars.

Olive suffers from cold damage when introduced to high-latitude regions from its native warm climes. Therefore, this study aims to improve the adaption of olive to climates in which it is cold for part of the year. The phenotype, physiological performance, nutrient content, and gene expression of olive leaves (from two widely planted cultivars) were examined after cultivation in normal and cold stress conditions. The results showed that the cold-tolerant cultivar possessed stronger photosynthesis efficiency and higher anti-oxidase activity after cold treatment than the cold-sensitive cultivar. Alteration of gene expression and metabolites in the amino acid metabolism, glycerolipid metabolism, diterpenoid biosynthesis, and oleuropein metabolism pathways played an important role in the cold responses of olive. Furthermore, the construction of the network of genes for ubiquitination and metabolites suggested that polyubiquitination contributes most to the stable physiology of olive under cold stress. Altogether, the results of this study can play an important role in helping us to understand the cold hardiness of olive and screen cold-resistant varieties for excellent quality and yield.

Antioxidant Capacity of Free and Bound Phenolics from Olive Leaves: In Vitro and In Vivo Responses.

Olive leaves are rich in phenolic compounds. This study explored the chemical profiles and contents of free phenolics (FPs) and bound phenolics (BPs) in olive leaves, and further investigated and compared the antioxidant properties of FPs and BPs using chemical assays, cellular antioxidant evaluation systems, and in vivo mouse models. The results showed that FPs and BPs have different phenolic profiles; 24 free and 14 bound phenolics were identified in FPs and BPs, respectively. Higher levels of phenolic acid (i.e., sinapinic acid, 4-coumaric acid, ferulic acid, and caffeic acid) and hydroxytyrosol were detected in the BPs, while flavonoids, triterpenoid acids, and iridoids were more concentrated in the free form. FPs showed a significantly higher total flavonoid content (TFC), total phenolic content (TPC), and chemical antioxidant properties than those of BPs (p < 0.05). Within the range of doses (20-250 µg/mL), both FPs and BPs protected HepG2 cells from H2O2-induced oxidative stress injury, and there was no significant difference in cellular antioxidant activity between FPs and BPs. The in vivo experiments suggested that FP and BP treatment inhibited malondialdehyde (MDA) levels in a D-galactose-induced oxidation model in mice, and significantly increased antioxidant enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and the total antioxidant capacity (T-AOC). Mechanistically, FPs and BPs exert their antioxidant activity in distinct ways; FPs ameliorated D-galactose-induced oxidative stress injury partly via the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway, while the BP mechanisms need further study.

Comprehensive evaluation of the response to aluminum stress in olive tree (Olea europaea L.).

Olive (Olea europaea L.) is an ancient tree species in the Mediterranean, but the lack of knowledge about aluminum-resistant varieties limits its introduction to acidic soil. The objective of this study was to have a comprehensive evaluation of the response to aluminum stress in olive tree at germplasm, metabolome, and transcriptome levels. In this experiment, seedlings of 97 olive germplasm with 1.0-3.0 cm roots and two leaves were treated with 50 µM Al3+ (pH = 5.0). By factor analysis of the traits of defoliation rate, rooting rate, length of extended root, and length of new root, 97 germplasm were classified into five different groups according to their diverse responses to aluminum stress: 5 highly resistant (5.15%), 30 moderately resistant (30.93%), 31 general (31.96%), 23 moderately sensitive (23.71%), and 8 highly sensitive (8.25%) germplasm. The three most sensitive and three most resistant germplasm were further used for metabolome and transcriptome analysis. Exposed to aluminum stress, 96 differentially accumulated metabolites (DAMs)/4,845 differentially expressed genes (DEGs) and 66 DAMs/2,752 DEGs were identified in highly sensitive and resistant germplasm, respectively. Using multi-omics technology, the pathways and related DAMs/DEGs involved in cell wall/cytoplasm receptors, reactive oxygen species balance, hormone induction, synthesis of organic acids, Al3+ transport, and synthesis of metabolites were identified to mainly regulate the response to aluminum stress in olive. This study provides a theoretical guide and prior germplasm and genes for further genetic improvement of aluminum tolerance in the olive tree.

Changes in Phytochemical Profiles and Biological Activity of Olive Leaves Treated by Two Drying Methods.

Olive leaves, which are the most abundant byproducts of the olive industry, offer multiple health benefits. The investigation of the phytochemical profiles and relevant biological activities is an essential step toward transforming these low-value byproducts into value-added ones. This study systematically investigated the phytochemical profiles, antioxidant capacity, and inhibition rates of olive leaves from four cultivars on the α-glucosidase, α-amylase, and angiotensin-converting enzyme (ACE). The leaves were prepared using two common drying methods, namely, hot air-drying and freeze-drying. A total of 33 bioactive compounds were identified in the olive leaves, namely, 19 flavonoids, 2 phenylethanoids, 2 coumarins, 2 hydroxycinnamic acids, 2 iridoids, and 6 triterpenic acids. Quantification of the bioactive compounds revealed high amounts of polyphenols, especially flavonoids [2,027-8,055 mg/kg dry weight (DW)], iridoids (566-22,096 mg/kg DW), and triterpenic acids (13,824-19,056 mg/kg DW) in the olive leaves. The hot air-dried leaves showed significantly (P < 0.05) higher iridoid (oleuropein and secoxyloganin) content than the fresh leaves, while freeze-drying resulted in significantly (P < 0.05) higher flavonoid aglycone and hydroxytyrosol content. Additionally, freeze-drying led to samples with the highest radical scavenging, α-amylase, α-glucosidase, and ACE inhibition abilities. The flavonoid (e.g., quercetin, luteolin, eriodictyol, kaempferol-7-O-glucoside, and luteolin-7-O-glucoside), hydroxytyrosol, and oleanolic acid contents in the olive leaves were positively correlated (P < 0.05) with their bioactive potentials.

Genome-Wide Identification and Functional Differentiation of Fatty Acid Desaturase Genes in Olea europaea L.

Olive (Olea europaea L.) is a world-famous woody oil tree and popular for redundant unsaturated fatty acids. Fatty acid desaturase (FAD) genes are responsible for fatty acid desaturation and stress regulation but have not yet been identified in olive at the whole genome level. This study identified 40 and 27 FAD genes in the cultivated olive O. europaea cv. Farga and the wild olive O. europaea var. Sylvestris, respectively. Phylogenetic analysis showed that all the FAD genes could be classified into the soluble FAB2/SAD clade and membrane-bound clade, including ADS/FAD5, DES, FAD4, SLD, ω-6 and ω-3, with the high consistency of subcellular localization, motif composition and exon-intron organization in each group. FAD genes in olive showed the diverse functional differentiation in morphology of different tissues, fruit development and stress responses. Among them, OeFAB2.8 and OeFAD2.3 were up-regulated and OeADS.1, OeFAD4.1 and OeFAD8.2 were down-regulated under the wound, Verticillium dahliae and cold stresses. This study presents a comprehensive analysis of the FAD genes at the whole-genome level in olives and will provide guidance for the improvement of oil quality or stress tolerance of olive trees.

Cotton Fiber Development Requires the Pentatricopeptide Repeat Protein GhIm for Splicing of Mitochondrial nad7 mRNA.

Pentatricopeptide repeat (PPR) proteins encoded by nuclear genomes can bind to organellar RNA and are involved in the regulation of RNA metabolism. However, the functions of many PPR proteins remain unknown in plants, especially in polyploidy crops. Here, through a map-based cloning strategy and Clustered regularly interspaced short palindromic repeats/cas9 (CRISPR/cas9) gene editing technology, we cloned and verified an allotetraploid cotton immature fiber (im) mutant gene (GhImA) encoding a PPR protein in chromosome A03, that is associated with the non-fluffy fiber phenotype. GhImA protein targeted mitochondrion and could bind to mitochondrial nad7 mRNA, which encodes the NAD7 subunit of Complex I. GhImA and its homolog GhImD had the same function and were dosage-dependent. GhImA in the im mutant was a null allele with a 22 bp deletion in the coding region. Null GhImA resulted in the insufficient GhIm dosage, affected mitochondrial nad7 pre-mRNA splicing, produced less mature nad7 transcripts, and eventually reduced Complex I activities, up-regulated alternative oxidase metabolism, caused reactive oxygen species (ROS) burst and activation of stress or hormone response processes. This study indicates that the GhIm protein participates in mitochondrial nad7 splicing, affects respiratory metabolism, and further regulates cotton fiber development via ATP supply and ROS balance.

Chloroplast Genome Variation and Evolutionary Analysis of Olea europaea L.

Olive (Olea europaea L.) is a very important woody tree and favored by consumers because of the fruit's high-quality olive oil. Chloroplast genome analysis will provide insights into the chloroplast variation and genetic evolution of olives. The complete chloroplast genomes of three accessions (O. europaea subsp. cuspidata isolate Yunnan, O. europaea subsp. europaea var. sylvestris, and O. europaea subsp. europaea var. frantoio) were obtained by next-generation sequencing technology. A total of 133 coding regions were identified in the three chloroplast genomes without rearrangement. O. europaea subsp. europaea var. sylvestris and O. europaea subsp. europaea var. frantoio had the same sequences (155,886 bp), while O. europaea subsp. cuspidata isolate Yunnan (155,531 bp) presented a large gap between rps16 and trnQ-UUG genes with six small gaps and fewer microsatellites. The whole chloroplast genomes of 11 O. europaea were divided into two main groups by a phylogenetic tree and O. europaea subsp. cuspidata formed a separate group (Cuspidata group) with the other subspecies (Mediterranean/North African group). Identification of consistency and diversity among O. europaea subspecies will benefit the exploration of domestication events and facilitate molecular-assisted breeding for O. europaea.

Genetic Diversity Analysis of Olive Germplasm (Olea europaea L.) With Genotyping-by-Sequencing Technology.

Olive (Olea europaea L.) is a very important edible oil crop and has been cultivated for about 4,000 years in the Mediterranean area. Due to its nutritional and economic importance, researches on germplasm characterization received extensive attention. In this study, using the genotyping-by-sequencing (GBS) technology, we carried out genetic diversity analysis on 57 olive cultivars with different geographical origins. In total, 73,482 high-quality single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) > 5%, call rate > 50%, and heterozygosity rate < 10% were obtained at the whole genome level. Genetic structure and phylogenetic analysis showed that the 57 olive cultivars could be classified into two groups (Group I and Group II). No clear geographical distributions of cultivars were observed generally between the two groups. The average nucleotide diversities (π) specific for Group I and Group II were 0.317 and 0.305. The fixation index (F ST) between Group I and Group II was 0.033. In Group II, cultivars could be further divided into two subgroups (Group IIa and Group IIb), which seem to be associated with their fruit sizes. The five Chinese-bred cultivars were all clustered in Group II, showing a closer genetic relationship with those from the central Mediterranean region and limited genetic background. It is therefore necessary for Chinese olive breeding programs to incorporate other genetic basis by utilizing germplasm from the other regions particularly from the east Mediterranean region as breeding parents. The results showed that GBS is an effective marker choice for cultivar characterization and genetic diversity analysis in olive and will help us better understand the genetic backgrounds of the crop.

Genome-Wide Association Studies Reveal Genetic Variation and Candidate Genes of Drought Stress Related Traits in Cotton (Gossypium hirsutum L.).

Cotton is an important industrial crop worldwide and upland cotton (Gossypium hirsutum L.) is most widely cultivated in the world. Due to ever-increasing water deficit, drought stress brings a major threat to cotton production. Thus, it is important to reveal the genetic basis under drought stress and develop drought tolerant cotton cultivars. To address this issue, in present study, 319 upland cotton accessions were genotyped by 55,060 single nucleotide polymorphisms (SNPs) from high-density CottonSNP80K array and phenotyped nine drought tolerance related traits. The two datasets were used to identify quantitative trait nucleotides (QTNs) for the above nine traits using multi-locus random-SNP-effect mixed linear model method. As a result, a total of 20 QTNs distributed on 16 chromosomes were found to be significantly associated with six drought tolerance related traits. Of the 1,326 genes around the 20 QTNs, 205 were induced after drought stress treatment, and 46 were further mapped to Gene ontology (GO) term "response to stress." Taken genome-wide association study (GWAS) analysis, RNA-seq data and qRT-PCR verification, four genes, RD2 encoding a response to desiccation 2 protein, HAT22 encoding a homeobox-leucine zipper protein, PIP2 encoding a plasma membrane intrinsic protein 2, and PP2C encoding a protein phosphatase 2C, were proposed to be potentially important for drought tolerance in cotton. These results will deepen our understanding of the genetic basis of drought stress tolerance in cotton and provide candidate markers to accelerate the development of drought-tolerant cotton cultivars.

Identification of genes related to salt stress tolerance using intron-length polymorphic markers, association mapping and virus-induced gene silencing in cotton.

Intron length polymorphisms (ILPs), a type of gene-based functional marker, could themselves be related to the particular traits. Here, we developed a genome-wide cotton ILPs based on orthologs annotation from two sequenced diploid species, A-genome Gossypium arboreum and D-genome G. raimondii. We identified 10,180 putative ILP markers from 5,021 orthologous genes. Among these, 535 ILP markers from 9 gene families related to stress were selected for experimental verification. Polymorphic rates were 72.71% between G. arboreum and G. raimondii and 36.45% between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. Furthermore, 14 polymorphic ILP markers were detected in 264 G. hirsutum accessions. Coupled with previous simple sequence repeats (SSRs) evaluations and salt tolerance assays from the same individuals, we found a total of 25 marker-trait associations involved in nine ILPs. The nine genes, temporally named as C1 to C9, showed the various expressions in different organs and tissues, and five genes (C3, C4, C5, C7 and C9) were significantly upregulated after salt treatment. We verified that the five genes play important roles in salt tolerance. Particularly, silencing of C4 (encodes WRKY DNA-binding protein) and C9 (encodes Mitogen-activated protein kinase) can significantly enhance cotton susceptibility to salt stress.

Comprehensive Analysis of the COBRA-Like (COBL) Gene Family in Gossypium Identifies Two COBLs Potentially Associated with Fiber Quality.

COBRA-Like (COBL) genes, which encode a plant-specific glycosylphosphatidylinositol (GPI) anchored protein, have been proven to be key regulators in the orientation of cell expansion and cellulose crystallinity status. Genome-wide analysis has been performed in A. thaliana, O. sativa, Z. mays and S. lycopersicum, but little in Gossypium. Here we identified 19, 18 and 33 candidate COBL genes from three sequenced cotton species, diploid cotton G. raimondii, G. arboreum and tetraploid cotton G. hirsutum acc. TM-1, respectively. These COBL members were anchored onto 10 chromosomes in G. raimondii and could be divided into two subgroups. Expression patterns of COBL genes showed highly developmental and spatial regulation in G. hirsutum acc. TM-1. Of them, GhCOBL9 and GhCOBL13 were preferentially expressed at the secondary cell wall stage of fiber development and had significantly co-upregulated expression with cellulose synthase genes GhCESA4, GhCESA7 and GhCESA8. Besides, GhCOBL9 Dt and GhCOBL13 Dt were co-localized with previously reported cotton fiber quality quantitative trait loci (QTLs) and the favorable allele types of GhCOBL9 Dt had significantly positive correlations with fiber quality traits, indicating that these two genes might play an important role in fiber development.