Shelley E. Jones

An HPLC-MS Characterization of the Changes in Sweet Orange Leaf Metabolite Profile following Infection by the Bacterial Pathogen Candidatus Liberibacter asiaticus

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri, the vector of huanglongbing

3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old ‘Valencia’ and ‘Hamlin’ trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated ‘Hamlin’ and ‘Valencia’ trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from ‘Hamlin’ and ‘Valencia’ sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.

Profiling of volatile organic compounds released from individual intact juvenile and mature citrus leaves

The Asian citrus psyllid Diaphorina citri Kuwayama is currently threatening the citrus industry by transmitting the causative agent Candidatus Liberibacter asiaticus (CLas) of huanglongbing. Multiplication of CLas in haemolymph of D. citri indicates that it contains the necessary nutrients for CLas. Although many studies examine D. citri, the haemolymph composition of this dangerous pest remains to be investigated. In the present study, the haemolymph of D. citri is collected using a nanolitre syringe after the removal of one of its forelegs. The haemolymph is either derivatized with methyl chloroformate (MCF) or trimethylsilyl (TMS) derivatizing reagent and analyzed with gas chromatography–mass spectrometry. Nineteen amino acids, two organic acids and seven fatty acids are detected in the haemolymph after MCF derivatization. More metabolites are detected after TMS derivatization. Sugars are the most abundant metabolites in the haemolymph. Glucose and fructose are the main monosaccharides. Trehalose and sucrose are the major disaccharides. Furthermore, three inositol isomers (myo‐inositol, scyllo‐inositol and chiro‐inositol) are detected in the haemolymph. Organic acids are found in low amounts, whereas phosphoric acid is found at a higher concentration. Twenty‐four nucleotides and sugar nucleotides, including ATP, ADP and AMP, are detected using high‐performance anion‐exchange chromatography. Adenine nucleotides are the most abundant nucleotides followed by uridine and guanosine. The adenylate energy charge for the haemolymph is 0.77. Our results show that many metabolites found in the citrus phloem sap are also found in the haemolymph of D. citri.

Metabolic alterations in the nymphal instars of Diaphorina citri induced by Candidatus Liberibacter asiaticus, the putative pathogen of huanglongbing

Plants release volatiles to communicate with each other and to attract or repel insects. The methods used to collect volatiles are varied. Here, we describe a simple solvent-less, solid phase microextraction-based method to collect the volatiles released from intact citrus leaves. We were able to collect up to 39 volatiles from both juvenile and mature leaves. Our results indicated that juvenile leaves produced both monoterpenes and sesquiterpenes, and while mature leaves continued to produce a variety of monoterpenes, their release of sesquiterpenes decreased dramatically. The finding that juvenile leaves emitted higher levels of sesquiterpenes while mature leaves released mostly monoterpenes suggests that younger leaves of plants may be involved in a more complex chemical communication system.

Metabolically engineered anthocyanin-producing lime provides additional nutritional value and antioxidant potential to juice

Currently, huanglongbing is the most damaging disease of citrus causing huge economic losses. The disease is caused by the Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). The pathogen is transmitted in a persistent propagative circulative manner within its vector, the Asian citrus psyllid, Diaphorina citri. Exploring the metabolic alteration in the vector may lead to a better understanding of the nutritional needs of CLas and to designing an artificial medium for culturing the pathogen. It has been shown that the nymphal stages have a greater role in transmission mainly because they feed on plants more actively than adults. In this study, we carried out an untargeted comparative metabolomic analysis for healthy and CLas-infected 4th / 5th instar nymphs. The metabolic analysis was performed using trimethylsilylation and methyl chloroformate derivatization followed by Gas Chromatography-Mass Spectrometry (GC-MS). Overall, the changes in the nymph metabolism due to the infection with CLas were more pronounced than in adults, as we previously published. Nymphs reared on CLas-infected Valencia sweet orange were higher in many metabolites, mainly those of the TCA cycle, C16 and C18 fatty acids, glucose, sucrose, L-proline, L-serine, pyroglutamic acid, saccharic acid, threonic acid and myo-inositol than those reared on healthy plants. In contrast, CLas-infected nymphs were lower in putrescine, glycine, L -phenylalanine, L -tyrosine, L -valine, and chiro-inositol. The information provided from this study may contribute in acceleration of the availability of CLas in culture and consequent screening of antibacterial compounds to discover a definitive solution for huanglongbing.

High hydrostatic pressure protection of a pectinase cocktail against thermal inactivation

Anthocyanins are synthesized via the flavonoid pathway through a complex expression of several genes such as MYB transcription factors. Anthocyanins protect plants against biotic and abiotic stresses. Herein, we studied the effect of expression of MYB (VvmybA1 cloned from the red grape and Ruby cloned from ‘Moro’ blood orange) transcription factors in “Mexican” lime on juice quality and leaf pigments, leaf metabolites, and phytohormones. Anthocyanins, furanocoumarins, flavonoids, and hydroxycinnamates were analyzed with high-performance liquid chromatography–mass spectrometry, whereas chlorophylls, carotenoids, and xanthophylls were analyzed using HPLC coupled with photodiode array detector (PDA). The rest of metabolites were analyzed using gas chromatography–mass spectrometry. Overexpression of VvmybA1 and Ruby resulted in accumulation of anthocyanins in leaves, flowers, and fruits of the transgenic plants. However, the level of anthocyanins in Ruby plants was significantly lower than that in VvmybA1 plants. The level of anthocyanins and the gene expression of VvmybA1 and Ruby in young leaves were higher than mature leaves. On the other hand, the level of several furanocoumarins, and hydroxycinnamates decreased in mature VvmybA1 leaves, indicating a drainage of p-coumaric acid due to the induction of anthocyanins biosynthesis. The level of chlorophyll decreased in mature VvmybA1 leaves, whereas zeaxanthin level increased, indicated a photoprotection role for anthocyanins. Most of polar and volatile metabolites also decreased VvmybA1 leaves, indicating a decrease in the photosynthetic efficiency. Benzoic acid and salicylic acid increased, whereas auxins decreased. The level of abscisic acid was not affected by the overexpression of VvmybA1 and the plants showed normal growth and development. Overexpression of VvmybA1 highly increased the antioxidant activity of the transgenic juice and leaves, whereas overexpression of Ruby showed only a slight increase. The pH, °Brix value, and TA of the transgenic juice were not affected by the expression of VvmybA1 or Ruby.