Bud mutation is a vital method of citrus. ‘Wuzi Ougan’ (mutant type, MT) as a bud variant of ‘Ougan’ (wild type, WT) was first found in 1996 and has become popular because of its male sterility and seedless character. Previous analysis of its cytological sections and transcriptome revealed that the abnormal microsporogenesis that occurs before the tetrad stage of anther development might be the result of down-regulated oxidation-reduction biological processes in MT. To reveal the mechanism behind the male sterility in MT at the post-transcriptional stage, proteome profiling and integrative analysis on previously obtained transcriptome and proteome data were performed in two strains. The proteome profiling was performed by iTRAQ (isobaric Tags for relative and absolute quantitation) analysis and 6201 high-confidence proteins were identified, among which there were 487 differentially expressed proteins (DEPs) in one or more developmental stages of anthers between MT and WT. The main functional subcategories associated with the main category biological process into which the DEPs were classified were sporopollenin biosynthesis process and pollen exine formation. The enriched pathways were phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism. Moreover, there were eight pathways linked in terms of being related to phenylpropanoid metabolism. Eighteen important genes related to phenylpropanoid metabolism were also analysized by qRT-PCR (quantitative real time PCR). An integrative analysis of the fold change at the transcript (log2 FPKM ratios) and protein (log1.2 iTRAQ ratios) levels was performed to reveal the consistency of gene expression at transcriptional and proteomic level. In general, the expression of genes and proteins tended to be positively correlated, in which the correlation coefficients were 0.3414 (all genes and all proteins) and 0.5686 (DEPs and according genes). This study is the first to offer a comprehensive understanding of the gene regulation in ‘Wuzi Ougan’ and its wild type, especially during the microsporocyte to meiosis stage. Specifically, the involved genes include those in phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism, as determined by integrative transcriptome and proteome analysis.

Background: Chimeras synthesized artificially by grafting are crucial to the breeding of perennial woody plants. ‘Hongrou Huyou’ (Citrus changshan-huyou + Citrus unshiu) is a new graft chimera originating from the junction where aCitrus changshan-huyou(“C”) scion was top-grafted onto a stock Satsuma mandarin ‘Owari’ (C. unshiu, “O”). The chimera was named OCC because the cell layer constitutions were O for Layer 1(L1) and C for L2 and L3. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues in OCC and the two donors were investigated, with the aim of determining the relationship between the layer donors and metabolites.Results: The comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and each of the two donors. The absence or presence of specific metabolites (such as the carotenoids violaxanthin andβ-cryptoxanthin, the volatile hydrocarbon germacrene D, and the primary metabolites citric acid and sorbose) in each tissue was identified in the three phenotypes. According to principal component analysis (PCA), overall, the metabolites in the peel of the chimera were derived from donor C, whereas those in the juice sac of the chimera came from donor O.Conclusion: The profiles of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically compared. The content and composition of metabolites were different between the tissues and between OCC and the each of the two donors. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC and was basically consistent with the layer origin; the peel of the chimera was derived from C, and the juice sacs of the chimera came from O. These profiles provide potential chemical markers for genotype differentiation, citrus breeding assessment, and donor selection during artificial chimera synthesis.

Background: Polyploids in citrus are generally used to improve crop varieties. Changshan Huyou (Citrus changshan-huyou) is a native citrus species in China that is highly adaptable and has pharmaceutical value. However, the influence in Changshan Huyou following polyploidization remains unclear. Here we evaluated the adult tetraploid scions of Changshan Huyou with contemporary diploid scions as the control in the phenotypic variations, metabolic alterations of fruits and associated transcriptomic changes. Result: The tetraploid scions had rounder and thicker leaves, larger floral organs and fruits, and satisfactory viability of pollen grains and ovules. The tetraploid fruits accumulated lower levels of soluble solids but similar levels of organic acids. Metabolic profiling of three tissues of fruits revealed that most of 2064 differentially accumulated metabolites (DAMs), including flavonoids, lignans, and coumarins, were downregulated. In contrast, the upregulated DAMs mainly included alkaloids (clausine K and 2-(1-pentenyl)quinoline), amino acids (L-asparagine and L-ornithine), and terpenoids (deacetylnomilin and evodol) in tetraploid peels, as well as, flavonoids (neohesperidin and quercetin-5-O-β-D-glucoside) and organic acids (2-methylsuccinic acid and dimethylmalonic acid) in juice sacs. The upregulated genes were associated with phenylpropanoid biosynthesis, secondary metabolite biosynthesis, and the biosynthesis of various alkaloid pathways. Pearson Correlation Analysis showed that the upregulated genes encoding PEROXIDASE and CYTOCHROME P450 (CYP450) were closely related to the higher accumulation of amino acids and alkaloids in tetraploid peels, and up-regulated neohesperidin and quercetin glucoside were positively associated with FERULATE-5-HYDROXYLASE (F5H), CYP450 81Q32, FLAVONOID 3'-MONOOXYGENASE (F3'H), 4-COUMARATE–CoA LIGASE 1 (4CL1), and UDP-GLUCOSE FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFOG), as well as, some transcription factors in tetraploid juice sacs. Conclusion: The tetraploid Changshan Huyou investigated here may be used in triploids breeding to produce seedless citrus, and for fruit processing on pharmaceutical purpose due to the alteration of metabolites following polyploidization.

Determining the responses of candidate plants to salt stress is a prerequisite for selecting and breeding suitable plants with high salt tolerance to grow in coastal mudflat areas with high salinity. Here, 2-year cutting seedlings of Ilex purpurea Hassk. (local species) and I. integra Thunb. (introduced species) were grown in pots in a glasshouse and irrigated with a Hoagland-NaCl solution at 0, 24, and 48 h. Root samples were collected at 0, 1, 6, 24, and 72 h, and concentration of Na⁺ ion; content of proline, soluble carbohydrate, malondialdehyde (MDA), H₂O₂ and ascorbate; and activity of three key antioxidative enzymes were measured. Roots of I. integra accumulated relatively less Na⁺ and had less membrane lipid peroxidation and H₂O₂ during salt stress, thus indicating a relatively higher salt tolerance than roots of I. purpurea. Values for ascorbate content and antioxidant enzymatic activity suggest that the antioxidant ascorbate and antioxidative catalase may play substantial roles for scavenging reactive oxygen species in I. integra roots during salt treatment. Thus, I. integra is apparently more suitable for growing in local highly saline coastal mudflats.

Vegetable oil is an indispensable nutritional resource for human health and mainly characterized by the composition and content of fatty acids (FAs). As a commercial species of gymnosperm, Torreya grandis produces oil-rich nuts with high unsaturated fatty acids content in the mature kernels. In this study, two cultivars, T. grandis ‘Xifei’ and T. grandis ‘Dielsii,’ with distinct oil content were employed to compare the profiles of FAs accumulation during kernel development. The accumulation rate of oil content was significantly different between ‘Xifei’ and ‘Dielsii.’ Besides, the final oil content of ‘Xifei’ (52.87%) was significantly higher than that of ‘Dielsii’ (41.62%) at maturity. The significant differences in main FAs were observed at almost each kernel development stages between the two cultivars. C_16:0, C_18:1, and C_20:3 FA exhibited different accumulation patterns between cultivars. The content and the initiation of accumulation of C_20:3 FA were different between the two cultivars. To explore the molecular mechanism associated with different content of oil and FAs between two cultivars, de novo transcriptome of kernels was compared between ‘Xifei’ (high oil) and ‘Dielsii’ (low oil) at three stages of oil accumulation, respectively. Totally 142,213 unigenes were assembled and 16,379 unigenes with a length of over 1,000 nt were successfully annotated, including 139 unigenes related to FA biosynthesis, elongation, and metabolism. Compared with ‘Dielsii,’ totally 1,476, 2,140, and 1,145 differentially expressed genes (DEGs) were upregulated in ‘Xifei’ at the stage of the initiative, the rapid rise, and the stationary oil accumulation, respectively; the number of downregulated DEGs reached 913, 1,245, and 904, respectively. Relative expressions of 11 DEGs involved in FAs biosynthesis and metabolism were confirmed by RT-qPCR. Abundant differentially expressed transcription factors and pathway DEGs were correlated to oil and FAs according to Pearson’s correlation analysis between transcriptome and metabolites (oil and FAs), suggesting their contributions to the differential oil and FAs between the two cultivars during kernel development of T. grandis. To conclude, our findings can provide novel insights into the developmental differences in metabolites and de novo transcriptome correlated to lipid accumulation and FA synthesis of kernels between cultivars with contrasting oil deposits and demystify the regulatory mechanism of high oil accumulation in T. grandis.

Chimeras occur spontaneously or artificially and are valuable in horticultural crop breeding. A new diploid citrus chimera, named 'Hongrou Huyou' (abbreviated HH) (Citrus changshan-huyou + C. unshiu), was found during a bud sports investigation in China. The morphology, flesh carotenoid content, and molecular markers were evaluated in HH and the two grafted donors. The results showed that characteristics derived from L2/L3, such as the fruit size, winged leaf, seed, pollen, and rind aroma, were similar to those of C. changshan-huyou (CH), whereas the juice sac and stomatal characteristics originating from L1 were the same as those of the satsuma mandarin. High-performance liquid chromatography analysis of carotenes from the flesh of HH showed that the content was the same as that of the satsuma mandarin, with β-cryptoxanthin producing the main carotenoid spectrum, whereas lutein and violaxanthin were the main carotenoids in CH. Nuclear simple sequence repeat, chloroplast simple sequence repeat, and mitochondrial simple sequence repeat analyses showed that the leaves, outer pericarp (epidermis and flavedo), segment wall, and juice sac of HH contained the nuclear, chloroplast, and mitochondrial genomes of both donors; however, the albedo of HH only contained the genetic material of CH. Thus, HH is confirmed to be a periclinal chimera that consists of L1 from C. unshiu and L2/L3 from CH.