Diagnosing arsenic-mediated biochemical responses in rice cultivars using Raman spectroscopy.

Rice (Oryza sativa) is the primary crop for nearly half of the world's population. Groundwater in many rice-growing parts of the world often has elevated levels of arsenite and arsenate. At the same time, rice can accumulate up to 20 times more arsenic compared to other staple crops. This places an enormous amount of people at risk of chronic arsenic poisoning. In this study, we investigated whether Raman spectroscopy (RS) could be used to diagnose arsenic toxicity in rice based on biochemical changes that were induced by arsenic accumulation. We modeled arsenite and arsenate stresses in four different rice cultivars grown in hydroponics over a nine-day window. Our results demonstrate that Raman spectra acquired from rice leaves, coupled with partial least squares-discriminant analysis, enabled accurate detection and identification of arsenic stress with approximately 89% accuracy. We also performed high-performance liquid chromatography (HPLC)-analysis of rice leaves to identify the key molecular analytes sensed by RS in confirming arsenic poisoning. We found that RS primarily detected a decrease in the concentration of lutein and an increase in the concentration of vanillic and ferulic acids due to the accumulation of arsenite and arsenate in rice. This showed that these molecules are detectable indicators of biochemical response to arsenic accumulation. Finally, a cross-correlation of RS with HPLC and ICP-MS demonstrated RS's potential for a label-free, non-invasive, and non-destructive quantification of arsenic accumulation in rice.

The influence of zwitterionic and anionic phospholipids on protein aggregation.

The progressive aggregation of misfolded proteins is the underlying molecular cause of numerous pathologies including Parkinson's disease and injection and transthyretin amyloidosis. A growing body of evidence indicates that protein deposits detected in organs and tissues of patients diagnosed with such pathologies contain fragments of lipid membranes. In vitro experiments also showed that lipid membranes could strongly change the aggregation rate of amyloidogenic proteins, as well as alter the secondary structure and toxicity of oligomers and fibrils formed in their presence. In this review, the effect of large unilamellar vesicles (LUVs) composed of zwitterionic and anionic phospholipids on the aggregation rate of insulin, lysozyme, transthyretin (TTR) and α- synuclein (α-syn) will be discussed. The manuscript will also critically review the most recent findings on the lipid-induced changes in the secondary structure of protein oligomers and fibrils, as well as reveal the extent to which lipids could alter the toxicity of protein aggregates formed in their presence.

Species identification of adult ixodid ticks by Raman spectroscopy of their feces.

BACKGROUND: Ticks and tick-borne diseases pose significant challenges to cattle production, thus the species identification of ticks and knowledge on their presence, abundance, and dispersal are necessary for the development of effective control measures. The standard method of inspection for the presence of ticks is the visual and physical examination of restrained animals, but the limitations of human sight and touch can allow larval, nymphal, and unfed adult ticks to remain undetected due to their small size and site of attachment. However, Raman spectroscopy, an analytical tool widely used in agriculture and other sectors, shows promise for the identification of tick species in infested cattle. Raman spectroscopy is a non-invasive and efficient method that employs the interaction between molecules and light for the identification of the molecular constituents of specimens. METHODS: Raman spectroscopy was employed to analyze the structure and composition of tick feces deposited on host skin and hair during blood-feeding. Feces of 12 species from a total of five genera and one subgenus of ixodid ticks were examined. Spectral data were subjected to partial least squares discriminant analysis, a machine-learning model. We also used Raman spectroscopy and the same analytical procedures to compare and evaluate feces of the horn fly Haematobia irritans after it fed on cattle. RESULTS: Five genera and one sub-genus at overall true prediction rates ranging from 92.3 to 100% were identified from the Raman spectroscopy data of the tick feces. At the species level, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis at overall true prediction rates of 100, 99.3 and 100%, respectively, were identified. There were distinct differences between horn fly and tick feces with respect to blood and guanine vibrational frequencies. The overall true prediction rate for the separation of tick and horn fly feces was 98%. CONCLUSIONS: Our findings highlight the utility of Raman spectroscopy for the reliable identification of tick species from their feces, and its potential application for the identification of ticks from infested cattle in the field.

Long-Chain Polyunsaturated Fatty Acids Accelerate the Rate of Insulin Aggregation and Enhance Toxicity of Insulin Aggregates.

Long-chain polyunsaturated fatty acids (LCPUFAs) are essential components of a human diet. These molecules are critically important for cognitive attention and memory, mood states, coronary circulation, and cirrhosis. However, recently reported findings demonstrated that docosahexaenoic (DHA) and arachidonic acids (ARA), ω-3 and ω-6 LCPUFAs, accelerated the aggregation rates of insulin and α-synuclein, proteins that are directly linked to diabetes type 2 and Parkinson's disease, respectively. Furthermore, both DHA and ARA uniquely altered the structure and toxicity of the corresponding protein aggregates. Our objective is to ascertain whether other LCPUFAs, alongside long-chain unsaturated fatty acid (LCUFA) proteins, exhibit similar effects on amyloidogenic proteins. To explore this matter, we investigated the effect of 10 different LCPUFAs and LCUFAs on the rate of insulin aggregation. We found that all of the analyzed fatty acids strongly accelerated insulin aggregation. Moreover, we found that protein aggregates that were formed in the presence of these fatty acids exerted significantly higher cell toxicity compared with insulin fibrils grown in the lipid-free environment. These findings show that interactions between amyloid-associated proteins and LCPUFAs can be the underlying molecular cause of neurodegenerative diseases.

Nanoscale structural characterization of transthyretin aggregates formed at different time points of protein aggregation using atomic force microscopy-infrared spectroscopy.

Transthyretin (TTR) amyloidosis is a progressive disease characterized by an abrupt aggregation of misfolded protein in multiple organs and tissues TTR is a tetrameric protein expressed in the liver and choroid plexus. Protein misfolding triggers monomerization of TTR tetramers. Next, monomers assemble forming oligomers and fibrils. Although the secondary structure of TTR fibrils is well understood, there is very little if anything is known about the structural organization of TTR oligomers. To end this, we used nano-infrared spectroscopy, also known as atomic force microscopy infrared (AFM-IR) spectroscopy. This emerging technique can be used to determine the secondary structure of individual amyloid oligomers and fibrils. Using AFM-IR, we examined the secondary structure of TTR oligomers formed at the early (3-6 h), middle (9-12 h), and late (28 h) of protein aggregation. We found that aggregating, TTR formed oligomers (Type 1) that were dominated by α-helix (40%) and ß-sheet (~30%) together with unordered protein (30%). Our results showed that fibril formation was triggered by another type of TTR oligomers (Type 2) that appeared at 9 h. These new oligomers were primarily composed of parallel ß-sheet (55%), with a small amount of antiparallel ß-sheet, α-helix, and unordered protein. We also found that Type 1 oligomers were not toxic to cells, whereas TTR fibrils formed at the late stages of protein aggregation were highly cytotoxic. These results show the complexity of protein aggregation and highlight the drastic difference in the protein oligomers that can be formed during such processes.

Cholesterol and Sphingomyelin Uniquely Alter the Rate of Transthyretin Aggregation and Decrease the Toxicity of Amyloid Fibrils.

Transthyretin (TTR) is a small tetrameric protein that aggregates, forming highly toxic oligomers and fibrils. In the blood and cerebrospinal fluid, TTR can interact with various biomolecules, phospho- and sphingolipids, and cholesterol on the red blood cell plasma membrane. However, the role of these molecules in TTR aggregation remains unclear. In this study, we investigated the extent to which phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Cho), important components of plasma membranes, could alter the rate of TTR aggregation. We found that PC and SM inhibited TTR aggregation whereas Cho strongly accelerated it. The presence of these lipids during the stage of protein aggregation uniquely altered the morphology and secondary structure of the TTR fibrils, which changed the toxicity of these protein aggregates. These results suggest that interactions of TTR with red blood cells, whose membranes are rich with these lipids, can trigger irreversible aggregation of TTR and cause transthyretin amyloidosis.

Saturation of fatty acids in phosphatidic acid uniquely alters transthyretin stability changing morphology and toxicity of amyloid fibrils.

Transthyretin (TTR) is a small, ß-sheet-rich tetrameric protein that transports thyroid hormone thyroxine and retinol. Phospholipids, including phosphatidic acid (PA), can uniquely alter the stability of amyloidogenic proteins. However, the role of PA in TTR aggregation remains unclear. In this study, we investigated the effect of saturation of fatty acids (FAs) in PA on the rate of TTR aggregation. We also reveal the extent to which PAs with different length and saturation of FAs altered the morphology and secondary structure of TTR aggregates. Our results showed that TTR aggregation in the equimolar presence of PAs with different length and saturation of FAs yielded structurally and morphologically different fibrils compared to those formed in the lipid-free environment. We also found that PAs drastically lowered the toxicity of TTR aggregates formed in the presence of this phospholipid. These results shed light on the role of PA in the stability of TTR and transthyretin amyloidosis.

The toxicities of A30P and A53T α-synuclein fibrils can be uniquely altered by the length and saturation of fatty acids in phosphatidylserine.

Progressive degeneration of dopaminergic neurons in the midbrain, hypothalamus, and thalamus is a hallmark of Parkinson's disease (PD). Neuronal death is linked to the abrupt aggregation of α-synuclein (α-syn), a small protein that regulates vesicle trafficking in synaptic clefts. Studies of families with a history of PD revealed several mutations in α-syn including A30P and A53T that are linked to the early onset of this pathology. Numerous pieces of evidence indicate that lipids can alter the rate of protein aggregation, as well as modify the secondary structure and toxicity of amyloid oligomers and fibrils. However, the role of lipids in the stability of α-syn mutants remains unclear. In this study, we investigate the effect of phosphatidylserine (PS), an anionic lipid that plays an important role in the recognition of apoptotic cells by macrophages, in the stability of WT, A30P, and A53T α-syn. We found PS with different lengths and saturation of fatty acids accelerated the rate of WT and A30P aggregation. At the same time, the opposite effect was observed for most PS on A53T. We also found that PS with different lengths and saturation of fatty acids change the secondary structure and toxicities of WT, A30P, and A53T fibrils. These results indicate that lipids can play an important role in the onset and spread of familial PD.

Secondary structure and toxicity of transthyretin fibrils can be altered by unsaturated fatty acids.

Transthyretin amyloidosis is a severe pathology characterized by the progressive accumulation of transthyretin (TTR) in various organs and tissues. This highly conserved through vertebrate evolution protein transports thyroid hormone thyroxine. In our bodies, TTR can interact with a large number of molecules, including ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) that are broadly used as food supplies. In this study, we investigated the effect of ω-3 and ω-6 PUFAs, as well as their fully saturated analog, on TTR aggregation. Our results showed that both ω-3 and ω-6 PUFAs strongly decreased the rate of TTR aggregation. We also found that in the presence of PUFAs, TTR formed morphologically different fibrils compared to the lipid-free environment. Nano-Infrared imaging revealed that these fibrils had drastically different secondary structures compared to the secondary structure of TTR aggregates formed in the PUFAs-free environment. Furthermore, TTR fibrils formed in the presence of ω-3 and ω-6 PUFAs exerted significantly lower cell toxicity compared to the fibrils formed in the absence of fatty acids.

Role of Saturation and Length of Fatty Acids of Phosphatidylserine in the Aggregation of Transthyretin.

The progressive accumulation of transthyretin (TTR), a small protein that transports thyroxine, in various organs and tissues is observed upon transthyretin amyloidosis, a severe pathology that affects the central, peripheral, and autonomic nervous systems. Once expressed in the liver and choroid plexus, TTR is secreted into the bloodstream and cerebrospinal fluid. In addition to thyroxine, TTR interacts with a large number of molecules, including retinol-binding protein and lipids. In this study, we examined the extent to which phosphatidylserine (PS), a phospholipid that is responsible for the recognition of apoptotic cells by macrophages, could alter the stability of TTR. Using thioflavin T assay, we investigated the rates of TTR aggregation in the presence of PS with different lengths and saturation of fatty acids (FAs). We found that all analyzed lipids decelerated the rate of TTR aggregation. We also used a set of biophysical methods to investigate the extent to which the presence of PS altered the morphology and secondary structure of TTR aggregates. Our results showed that the length and saturation of fatty acids in PS uniquely altered the morphology and secondary structure of TTR fibrils. As a result, TTR fibrils that were formed in the presence of PS with different lengths and saturation of FAs exerted significantly lower cell toxicity compared with the TTR aggregates grown in the lipid-free environment. These findings help to reveal the role of PS in transthyretin amyloidosis and determine the role of the length and saturation of FAs in PS on the morphology and secondary structure of TTR fibrils.

Elucidation of Secondary Structure and Toxicity of α-Synuclein Oligomers and Fibrils Grown in the Presence of Phosphatidylcholine and Phosphatidylserine.

Abrupt aggregation of α-synuclein (α-Syn) in the midbrain hypothalamus and thalamus is a hallmark of Parkinson's disease (PD), the fastest growing neurodegenerative pathology, projected to strike 12 million people by 2040 worldwide. In this study, we examine the effect of two phospholipids that are present in neuronal membranes, phosphatidylcholine (PC) and phosphatidylserine (PS), on the rate of α-Syn aggregation. We found that PS accelerated α-Syn aggregation, whereas PC strongly inhibited α-Syn aggregation. We also utilized the nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to investigate whether PC and PS only change the rates or also modify the secondary structure of α-Syn aggregates. We found that both phospholipids uniquely altered the secondary structure of α-Syn aggregates present at the lag and growth phase, as well as the late stage of protein aggregation. In addition, compared to the α-Syn aggregates formed in the lipid-free environment, α-Syn:PC and α-Syn:PS aggregates demonstrated higher cellular toxicity to N27 rat neurons. Interestingly, both α-Syn:PC and α-Syn:PS aggregates showed similar levels of oxidative stress, but α-Syn:PC aggregates exhibited a greater degree of mitochondrial dysfunction compared to α-Syn:PS aggregates.

Length and saturation of fatty acids in phosphatidylserine determine the rate of lysozyme aggregation simultaneously altering the structure and toxicity of amyloid oligomers and fibrils.

Abrupt aggregation of misfolded proteins is the underlying molecular cause of numerous severe pathologies including Alzheimer's and Parkinson's diseases. Protein aggregation yields small oligomers that can later propagate into amyloid fibrils, ß-sheet-rich structures with a variety of topologies. A growing body of evidence suggests that lipids play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the roles of length and saturation of fatty acids (FAs) in phosphatidylserine (PS), an anionic lipid that is responsible for the recognition of apoptotic cells by macrophages, in lysozyme aggregation. We found that both the length and saturation of FAs in PS contribute to the aggregation rate of insulin. PS with 14-carbon-long FAs (14:0) enabled a much stronger acceleration of protein aggregation compared to PS with 18-carbon-long FAs (18:0). Our results demonstrate that the presence of double bonds in FAs accelerated the rate of insulin aggregation relative to PS with fully saturated FAs. Biophysical methods revealed morphological and structural differences in lysozyme aggregates grown in the presence of PS with varying lengths and FA saturation. We also found that such aggregates exerted diverse cell toxicities. These results demonstrate that the length and saturation of FAs in PS can uniquely alter the stability of misfolded proteins on lipid membranes.

Testing Raman spectroscopy as a diagnostic approach for Lyme disease patients.

Lyme disease (LD), the leading tick-borne disease in the Northern hemisphere, is caused by spirochetes of several genospecies of the Borreliella burgdorferi sensu lato complex. LD is a multi-systemic and highly debilitating illness that is notoriously challenging to diagnose. The main drawbacks of the two-tiered serology, the only approved diagnostic test in the United States, include poor sensitivity, background seropositivity, and cross-reactivity. Recently, Raman spectroscopy (RS) was examined for its LD diagnostic utility by our earlier proof-of-concept study. The previous investigation analyzed the blood from mice that were infected with 297 and B31 strains of Borreliella burgdorferi sensu stricto (s.s.). The selected strains represented two out of the three major clades of B. burgdorferi s.s. isolates found in the United States. The obtained results were encouraging and prompted us to further investigate the RS diagnostic capacity for LD in this study. The present investigation has analyzed blood of mice infected with European genospecies, Borreliella afzelii or Borreliella garinii, or B. burgdorferi N40, a strain of the third major class of B. burgdorferi s.s. in the United States. Moreover, 90 human serum samples that originated from LD-confirmed, LD-negative, and LD-probable human patients were also analyzed by RS. The overall results demonstrated that blood samples from Borreliella-infected mice were identified with 96% accuracy, 94% sensitivity, and 100% specificity. Furthermore, human blood samples were analyzed with 88% accuracy, 85% sensitivity, and 90% specificity. Together, the current data indicate that RS should be further explored as a potential diagnostic test for LD patients.

Solid-to-Liposome Conformational Transition of Phosphatidylcholine and Phosphatidylserine Probed by Atomic Force Microscopy, Infrared Spectroscopy, and Density Functional Theory Calculations.

Liposomes are emerging therapeutic formulations for site-specific delivery of chemotherapeutic drugs. The efficiency and selectivity of drug delivery by these carriers largely rely on their surface properties, shape, and size. There is a growing demand for analytical approaches that can be used for structural and morphological characterization of liposomes at the single-vesicle level. AFM-IR is a modern optical nanoscopic technique that combines the advantages of scanning probe microscopy and infrared spectroscopy. Our findings show that AFM-IR can be used to probe conformational changes in phospholipids that take place upon their assembly into liposomes. Such conclusions can be made based on the corresponding changes in intensities of the lipid vibrational bands as the molecules transition from a solid state into large unilamellar vesicles (LUVs). This spectroscopic analysis of LUV formation together with density functional theory calculations also reveals the extent to which the molecular conformation and local environment of the functional groups alter the AFM-IR spectra of phospholipids. Using melittin as a test protein, we also examined the extent to which LUVs can be used for protein internalization. We found that melittin enters LUVs nearly instantaneously, which protects it from possible structural modifications that are caused by a changing environment. This foundational work empowers AFM-IR analysis of liposomes and opens new avenues for determination of the molecular mechanisms of liposome-drug interactions.

Phosphatidylcholine and Phosphatidylserine Uniquely Modify the Secondary Structure of α-Synuclein Oligomers Formed in Their Presence at the Early Stages of Protein Aggregation.

Abrupt aggregation of α-synuclein (α-Syn) leads to a formation of highly toxic protein oligomers. These aggregates are the underlying molecular cause of an onset of the irreversible degeneration of dopaminergic neurons in midbrain, hypothalamus, and thalamus, a pathology known as Parkinson's disease. The transient nature of oligomers, as well as their structural and morphological heterogeneity, limits the use of cryo-electron microscopy and solid-state NMR, classical tools of structural biology, for elucidation of their secondary structure. Despite this limitation, numerous pieces of experimental evidence suggest that phospholipids can uniquely alter the structure and toxicity of oligomers. In this study, we utilize an innovative nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to examine the structure of individual α-Syn oligomers grown in the presence of phosphatidylcholine (α-Syn:PC) and phosphatidylserine (α-Syn:PS). We determined the amount of the parallel and the antiparallel ß-sheets, as well as the amount the α-helix and the unordered protein, in the secondary structure of α-Syn:PC and α-Syn:PS formed at day 2 (D2), 8 (D8), and 15 (D15) after initiation of protein aggregation. We found a gradual decrease in the amount of the parallel ß-sheet in both α-Syn:PC and α-Syn:PS from D2 to D15 together with an increase in the α-helix and the unordered protein secondary structure. We infer that this is due to the presence of lipids in the structure of oligomers that prevent an expansion of the parallel ß-sheet upon interaction of the oligomers with monomeric α-Syn.

Characterization of Substrates and Surface-Enhancement in Atomic Force Microscopy Infrared Analysis of Amyloid Aggregates.

Atomic force microscopy infrared (AFM-IR) spectroscopy is an emerging analytical technique that can be used to probe the structural organization of specimens with nanometer spatial resolution. A growing body of evidence suggests that nanoscale structural analysis of very small (<10 nm) biological objects, such as viruses and amyloid aggregates, requires substrates that must fit strict criteria of low surface roughness and low IR background, simultaneously. In this study, we examine the suitability of a broad range of substrates commonly used in AFM and IR fields, and we determined that silicon, zinc sulfide, and calcium fluoride are the most ideal substrates for nanoscale imaging of amyloid oligomers, protein aggregates that are directly linked to the onset and progression of neurodegenerative diseases. Our data show that these substrates provide the lowest roughness and the lowest background in the 800-1800 cm-1 spectral window from all examined AFM and IR substrates. We also investigate a contribution of surface enhancement in AFM-IR by the direct comparison of signal intensities from oligomers located on silicon and gold-coated silicon surfaces. We found that metallization of such substrates provides a factor of ~7 enhancements to the IR signal and induces an equivalent enhancement of the sample background in the 950-1250 cm-1 spectral region.

Raman Spectroscopy Detects Changes in Carotenoids on the Surface of Watermelon Fruits During Maturation.

A non-invasive and non-destructive technique, Raman spectroscopy, was explored to distinguish different maturity stages (20, 30, 40, and 50 days after anthesis) of watermelon (Citrullus lanatus) fruits from four cultivars: Fascination, Orange Crisp, Amarillo and Crimson Sweet. Spectral acquisition from the fruit surface was carried out at the wavelength range of 400-2,000 cm-1 using a handheld Raman spectrometer equipped with 830 nm laser excitation source. The spectra were normalized at 1,438 cm-1 which was assigned to CH2 and CH3 vibration. Detecting changes in the spectral features of carotenoids on the surface of watermelon fruits can be used as a marker to monitor the maturity of the fruit. The spectral analysis confirmed the presence of two major carotenoids, lutein and ß-carotene, and their intensity decreased upon maturity on the fruit surface. Identification of these pigments was further confirmed by resonance Raman spectra and high-performance liquid chromatography analysis. Results of partial least square discriminant analysis of pre-processed spectra have demonstrated that the method can successfully predict the maturity of watermelon samples with more than 85% accuracy. Analysis of Variance of individual Raman bands has revealed a significant difference among the stages as the level of carotenoids was declined during the ripening of the fruits. Thus, Raman spectral signatures can be used as a versatile tool for the non-invasive determination of carotenoid changes on the watermelon fruits' surface during ripening, thereby enabling effective monitoring of nutritional quality and maturity indices before harvesting the watermelon.

Raman-based identification of tick species (Ixodidae) by spectroscopic analysis of their feces.

Ticks are blood-feeding parasites that vector a large number of pathogens of medical and veterinary importance. There are strong connections between tick and pathogen species. Timely detection of certain tick species on cattle can cease the spread of numerous devastating diseases such as Bovine babiesiosis and anaplasmosis. Detection of ticks is currently performed by slow and laborious scout-based inspection of cattle. In this study, we investigated the possibility of identification of tick species (Ixodidae) based on spectroscopic signatures of their feces. We collected Raman spectra from individual grains of feces of seven different species of ticks. Our results show that Raman spectroscopy (RS) allows for highly accurate (above 90%) differentiation between tick species. Furthermore, RS can be used to predict the tick developmental stage and differentiate between nymphs, meta-nymphs and adult ticks. We have also demonstrated that diagnostics of tick species present on cattle can be achieved using a hand-held Raman spectrometer. These findings show that RS can be used for non-invasive, non-destructive and confirmatory on-site analysis of tick species present on cattle.

Raman-Based Diagnostics of Stalk Rot Disease of Maize Caused by Colletotrichum graminicola.

Stalk rot caused by Colletotrichum graminicola is a disease of worldwide importance. Stalk rot is difficult to detect at the early stages of infection because the fungus colonizes the tissues inside the maize stem. Current diagnostic methods are time-consuming, laborious, and destructive to the stem tissue. We utilized Raman spectroscopy to follow the development of stalk rot in three different maize genotypes grown either in the field or the greenhouse. We then used the acquired spectra to calibrate statistical models to differentiate amongst the different disease timepoints and the genotypes themselves. This non-invasive spectroscopic method enabled high-accuracy identification of stalk rot based on both stalk and leaf spectra. We additionally found that leaf spectra were favorable for identifying maize by genotype. Finally, we identified Raman bands that showed correlation with the sizes of stalk rot-associated lesions in the stems. We demonstrated that Raman spectroscopy is a viable tool for detection of stalk rot disease, as well as potent for the differentiation of maize genotypes.

Biochemical Origin of Raman-Based Diagnostics of Huanglongbing in Grapefruit Trees.

Biotic and abiotic stresses cause substantial changes in plant biochemistry. These changes are typically revealed by high-performance liquid chromatography (HPLC) and mass spectroscopy-coupled HPLC (HPLC-MS). This information can be used to determine underlying molecular mechanisms of biotic and abiotic stresses in plants. A growing body of evidence suggests that changes in plant biochemistry can be probed by Raman spectroscopy, an emerging analytical technique that is based on inelastic light scattering. Non-invasive and non-destructive detection and identification of these changes allow for the use of Raman spectroscopy for confirmatory diagnostics of plant biotic and abiotic stresses. In this study, we couple HPLC and HPLC-MS findings on biochemical changes caused by Candidatus Liberibacter spp. (Ca. L. asiaticus) in citrus trees to the spectroscopic signatures of plant leaves derived by Raman spectroscopy. Our results show that Ca. L. asiaticus cause an increase in hydroxycinnamates, the precursors of lignins, and flavones, as well as a decrease in the concentration of lutein that are detected by Raman spectroscopy. These findings suggest that Ca. L. asiaticus induce a strong plant defense response that aims to exterminate bacteria present in the plant phloem. This work also suggests that Raman spectroscopy can be used to resolve stress-induced changes in plant biochemistry on the molecular level.