Yasser Nehela

Phytohormone profiling of the sweet orange (Citrus sinensis (L.) Osbeck) leaves and roots using GC–MS-based method

Phytohormones mainly affect plant development and trigger varied responses to biotic and abiotic stresses. The sensitivity of methods used to profile phytohormones is a vital factor that affects the results. We used an improved GC-MS-based method in the selective ion-monitoring (SIM) mode to study the phytohormone profiling in citrus tissues. One extraction solvent mixture and two derivatization reagents were used, methyl chloroformate (MCF) and N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The method showed a low limit of detection and low limit of quantification with high extraction recovery percentage and reproducibility. Overall, we detected 13 phytohormones belonging to six different groups. Auxins, SAs, tJA, and ABA were detected after derivatization with MCF while cytokinins and GAs were detected after derivatization with MSTFA. Cytokinins, SAs, and gibberellins were found in all tissues while auxins and tJA were observed only in the leaves. ABA was found in leaves and roots, but not in root tips. The method we used is efficient, precise, and appropriate to study citrus phytohormonal profiles to understand their crosstalk and responses to environmental and biological stresses.

Possible role of plant volatiles in tolerance against huanglongbing in citrus

abstract Volatile organic compounds (VOC) play an important role in protecting plants from insect and pathogen attack. In this study, we investigated the leaf volatile profiles of 14 citrus varieties. The VOC in citrus leaves were extracted with n-hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Overall, 4six volatile compounds were identified in the n-hexane extract from citrus leaves. Most of the detected compounds belonged to 3 main groups (monoterpenes, sesquiterpenes, and aliphatic aldehydes). Principle component analysis was used to examine the relative distribution of the studied varieties to each other. Interestingly, volatile profiles of varieties that are tolerant to Candidatus Liberibacter asiaticus (CLas) were different from those of the susceptible ones. Tolerant and moderately-tolerant cultivars contained relatively higher amounts of volatiles than susceptible varieties. In addition, tolerant varieties were also higher in specific compounds which are known for their antimicrobial activities. These compounds include Aldehydes (undecanal, neral, geranial, and citronellal) and some monoterpenes such as linalool, d-limonene, myrcene, α- and β- phellandrene. In addition, some sesquiterpene compounds including t-caryophellene, γ-elemene, β-elemene, germacrene D, and geranyl acetate were higher in tolerant and moderately tolerant cultivars. Severinia buxifolia which is known for its tolerance to CLas and many other pathogens contained higher levels of santalenes and coumarins. Our results indicated that citrus leaf volatiles might play a role in citrus tolerance to CLas. The results of this study may help in understanding of the mechanism of citrus tolerance against CLas.

A plant pathogenic bacterium exploits the tricarboxylic acid cycle metabolic pathway of its insect vector

ABSTRACT Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.

Infection with phytopathogenic bacterium inhibits melatonin biosynthesis, decreases longevity of its vector, and suppresses the free radical-defense

Vector‐borne phytopathogenic bacteria may alter the reproductive fitness, survival, behavior, and metabolism of their vectors. Candidatus Liberibacter asiaticus (CLas) is associated with the Huanglongbing (also known as citrus greening disease), one of the most destructive citrus diseases worldwide, and transmitted by Asian citrus psyllid, Diaphorina citri (Insecta, Hemiptera, Liviidae). The genome sequencing of CLas revealed that it does not have the ability to synthesize tryptophan, the precursor of melatonin, and it must acquire it from its host plant or insect vector to achieve its biologic processes, such as growth and multiplication. Herein, we aimed to develop a GC‐MS‐SIM‐based method to detect the endogenous melatonin from small insects such as D. citri, and to explore the hidden relationship between melatonin content and D. citri‐adult survival. Then, we studied the ability of exogenous melatonin supplementation to reverse the negative effects of CLas‐infection. Our findings showed that CLas‐infection reduced the levels of melatonin and its biosynthetic genes (DcTPHs, DcAAAD, DcSNAT, and DcASMT) of D. citri compared to uninfected insects. In addition, CLas decreased the longevity of its vector, D. citri via the suppression of the free radical‐defense associated genes (SODs, GSTs, PODs, and PHGPXs). On the other hand, melatonin supplementation could reverse the negative effects of CLas‐infection. Melatonin supplementation enhanced the endogenous melatonin content, melatonin biosynthetic genes, free radical‐defense associated genes, and the longevity of both healthy and CLas‐infected D. citri. Furthermore, melatonin supplementation decreased the CLas bacterial population within the D. citri psyllids. Based on these findings, we hypothesize that melatonin plays multi‐layered defensive roles in D. citri. These roles include acting as a natural antioxidant or as an antibacterial compound.

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

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.