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Methyl jasmonate (MeJA) is an airborne signaling phytohormone that can induce changes in endogenous jasmonates (JAs) and cause photosynthetic responses. However, the response of these two aspects of citrus plants at different MeJA concentrations is still unclear. Four MeJA concentrations were used in two citrus varieties, Huangguogan (C. reticulata × C. sinensis) and Shiranuhi [C. reticulata × (C. reticulata × C. sinensis)], to investigate the effects of MeJA dose on the endogenous JAs pathway and photosynthetic capacity. We observed that MeJA acted in a dose-dependent manner, and its stimulation in citrus leaves showed a bidirectional character at different concentrations. This work demonstrates that MeJA at only a concentration of 2.2 mM or less contributed to the activation of magnesium protoporphyrin IX methyltransferase (ChlM, EC 2.1.1.11) and protochlorophyllide oxidoreductase (POR, EC 1.3.1.11) and the simultaneous accumulation of Chl a and Chl b, which in turn contributed to an improved photosynthetic capacity and PSII photochemistry efficiency of citrus. Meanwhile, the inhibition of endogenous JAs synthesis by exogenous MeJA was observed. This was achieved by reducing the ratio of monogalactosyl diacylglycerol (MGDG) to diagalactosyl diacylglycerol (DGDG) and inhibiting the activities of key enzymes in JAs synthesis, especially 12-oxo-phytodienoic acid reductase (OPR, EC 1.3.1.42). Another noteworthy finding is that there may exist a JA-independent pathway that could regulate 12-oxo-phytodienoic acid (OPDA) synthesis. This study jointly analyzed the internal hormone regulation mechanism and the external physiological response, as well as revealed the effects of exogenous MeJA on promoting the photosynthesis and inhibiting the endogenous JAs synthesis.

The nomilin and limonin content in citrus fruits of different varieties was determined at fruit growth and maturation stages by HPLC. The results showed that the two limonoids can be separated, identified, and quantified in citrus fruits within 10 min by the developed method. The method exhibited good precision, repeatability, stability, and recovery rate. The content of limonin and nomilin in most citrus fruits presented an increasing trend initially, and then decreased during fruit growth and maturation; a peak was observed at the young fruit or fruit expansion stage. The dropped fruits also contained some amount of limonoids, suggesting their industrial application. The variation and cluster analyses results revealed that the orange varieties contained the highest amount of limonoids at the mature stage. The results of this study will enable better use of citrus limonoids.

Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) rootstock is effective in Citrus production; however, when Cj rootstock was used, sugar content in Kiyomi tangor fruit was significantly lower than that in the fruit produced using Poncirus trifoliata (L.) Raf. rootstock (Pt). Therefore, using K. tangor, we explored the cause of this difference, determining sugar accumulation, sucrose-metabolism enzyme activities, and gene expression. Before ripening, sugar content in fruits with Cj rootstock was significantly lower than that in fruits with Pt rootstock, due to low fructose and sucrose content. Sucrose phosphate synthase (SPS) activity of Pt was higher than that of Cj in the early growth stage (at 90–210 days after anthesis), however it was opposite at 240–300 days after anthesis. Additionally, neutral invertase (NI) activity of Pt was higher than that of Cj. Gene expression in Pt was higher than that in Cj, but is was essentially the same at maturity. SPS and NI activities and CitSPS1 expression were positively correlated with sucrose, fructose, and glucose content, but CSCW1 expression was negatively correlated with the sugars. Overall, the weak flavour of K. tangor fruit with Cj rootstock was regulated by the sucrose metabolism-related enzymes and gene expression.

Bitterness, which is mainly attributed to limonoids, seriously affects the taste of pomelo fruits and hinders their competitiveness in the market. Therefore, a high-performance liquid chromatography method was developed for simultaneous isolation and determination of limonin, nomilin, and limonin glucoside contents in pomelo fruit, and the method showed good precision (RSD ≤ 2.85 %), repeatability (RSD ≤ 3.37 %) and high sample recovery rate (95.8 %–99.7 %). Four pomelo varieties were used to investigate the accumulation and distribution of limonoids in different stages of fruit development and in different plant tissues and varieties, as well as to reveal the synthesis mechanism and provide a basis for the utilisation of pomelo resources in different industries. Results showed that flavedo was the tissue richest in limonoids, and the maximum limonoid content could reach 5959 μg/g fresh weight. During fruit growth and development, the limonin and nomilin contents in juice sacs and albedo first increased and then decreased. Although there were some fluctuations, the limonin and nomilin contents in the flavedo generally showed a decreasing trend with fruit development. Our investigation of the LGT gene expression pattern provided the basis for exploring natural debittering mechanisms in pomelo. Expression of the LGT gene was positively correlated with limonin glucoside content.

Citrus species are among the most economically important fruit crops. Physiological characteristics and molecular mechanisms associated with de-etiolation have been partially revealed. However, little is known about the mechanisms controlling the expression and function of genes associated with photosynthesis and chlorophyll biosynthesis in variegated citrus seedlings. The lower biomass, chlorophyll contents, and photosynthetic parameter values recorded for the variegated seedlings suggested that chlorophyll biosynthesis was partially inhibited. Additionally, roots of the variegated seedlings were longer than the roots of green seedlings. We obtained 567.07 million clean reads and 85.05 Gb of RNA-sequencing data, with more than 94.19% of the reads having a quality score of Q30 (sequencing error rate = 0.1%). Furthermore, we detected 4,786 and 7,007 differentially expressed genes (DEGs) between variegated and green Shiranuhi and Huangguogan seedlings. Thirty common pathways were differentially regulated, including pathways related to photosynthesis (GO: 0015979) and the chloroplast (GO: 0009507). Photosynthesis (44 and 63 DEGs), photosynthesis-antenna proteins (14 and 29 DEGs), and flavonoid biosynthesis (16 and 29 DEGs) pathways were the most common KEGG pathways detected in two analyzed libraries. Differences in the expression patterns of PsbQ, PetF, PetB, PsaA, PsaN, PsbP, PsaF, Cluster-2274.8338 (ZIP1), Cluster-2274.38688 (PTC52), and Cluster-2274.78784 might be responsible for the variegation in citrus seedlings. We completed a physiological- and transcriptome-level comparison of the Shiranuhi and Huangguogan cultivars that differ in terms of seedling variegation. We performed mRNA-seq analyses of variegated and green Shiranuhi and Huangguogan seedlings to explore the genes and regulatory pathways involved in the inhibition of chlorophyll biosynthesis and decreases in Chl a and Chl b contents. The candidate genes described herein should be investigated in greater detail to further characterize variegated citrus seedlings.

Considering the known effects of xyloglucan endotransglycosylase (XET) on plant growth and development, we aimed to determine whether XETs help to regulate the growth and elongation of Huangguogan shoots and roots. We confirmed a possible role for XET during seedling etiolation. Our results revealed that the roots of etiolated seedlings (H-E) were longer than those of green seedlings (H-G). However, shoot length exhibited the opposite pattern. We also observed positive and negative effects on the xyloglucan-degrading activity of XET in the root sub-apical region and shoots of etiolated Huangguogan seedling, respectively. There was a significant down-regulation in CitXET expression in the etiolated shoots at 15 days after seed germination. On the contrary, it was significantly increased in the root sub-apical region of etiolated and multicolored seedlings at 15 days after seed germination. The XET coding sequence (i.e., CitXET) was cloned from Huangguogan seedlings using gene-specific primers. The encoded amino acid sequence was predicted by using bioinformatics-based methods. The 990-bp CitXET gene was highly homologous to other XET genes. The CitXET protein was predicted to contain 319 amino acids, with a molecular mass of 37.45 kDa and an isoelectric point of 9.05. The predicted molecular formula was C1724H2548N448O466S14, and the resulting protein included only one transmembrane structure. The CitXET secondary structure consisted of four main structures (i.e., 21% α-helix, 30.72% extended strand, 9.09% β-turn, and 39.18% random coil). Analyses involving the NCBI Conserved Domains Database (NCBI-CDD), InterPro, and ScanProsite revealed that CitXET was a member of the glycosyl hydrolase family 16 (GH16), and included the DEIDFEFLG motif. Our results indicate that the differed degrees of etiolation influenced the CitXET expression pattern and XET activity in Huangguogan seedlings. The differential changes in XET activity and CitXET expression levels in Huangguogan seedlings may influence the regulation of root and shoot development, and may be important for seedling etiolation.