AtALMT5 mediates vacuolar fumarate import and regulates the malate/fumarate balance in Arabidopsis.

Malate and fumarate constitute a significant fraction of the carbon fixed by photosynthesis, and they are at the crossroad of central metabolic pathways. In Arabidopsis thaliana, they are transiently stored in the vacuole to keep cytosolic homeostasis. The malate and fumarate transport systems of the vacuolar membrane are key players in the control of cell metabolism. Notably, the molecular identity of these transport systems remains mostly unresolved. We used a combination of imaging, electrophysiology and molecular physiology to identify an important molecular actor of dicarboxylic acid transport across the tonoplast. Here, we report the function of the A. thaliana Aluminium-Activated Malate Transporter 5 (AtALMT5). We characterised its ionic transport properties, expression pattern, localisation and function in vivo. We show that AtALMT5 is expressed in photosynthetically active tissues and localised in the tonoplast. Patch-clamp and in planta analyses demonstrated that AtALMT5 is an ion channel-mediating fumarate loading of the vacuole. We found in almt5 plants a reduced accumulation of fumarate in the leaves, in parallel with increased malate concentrations. These results identified AtALMT5 as an ion channel-mediating fumarate transport in the vacuoles of mesophyll cells and regulating the malate/fumarate balance in Arabidopsis.

Greater mesophyll conductance and leaf photosynthesis in the field through modified cell wall porosity and thickness via AtCGR3 expression in tobacco.

Mesophyll conductance (gm) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing gm is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase gm. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and gm measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing gm in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Mesophyll conductance is unaffected by expression of Arabidopsis PIP1 aquaporins in the plasmalemma of Nicotiana.

In plants with C3 photosynthesis, increasing the diffusion conductance for CO2 from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO2 assimilation and photosynthetic water use. In the diffusion pathway from substomatal cavity to chloroplast stroma, the plasmalemma and chloroplast envelope membranes impose a considerable barrier to CO2 diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO2, and increase photosynthesis in tobacco, we generated transgenic lines in Nicotiana tabacum L. cv Petite Havana carrying either the Arabidopsis PIP1;2 (AtPIP1;2) or PIP1;4 (AtPIP1;4) gene driven by the constitutive dual 2x35S CMV promoter. From a collection of independent T0 transgenics, two T2 lines from each gene were characterized, with western blots confirming increased total aquaporin protein abundance in the AtPIP1;2 tobacco lines. Transient expression of AtPIP1;2-mGFP6 and AtPIP1;4-mGFP6 fusions in Nicotiana benthamiana identified that both AtPIP1;2 and AtPIP1;4 localize to the plasmalemma. Despite achieving ectopic production and correct localization, gas exchange measurements combined with carbon isotope discrimination measurements detected no increase in mesophyll conductance or CO2 assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance gm through modified aquaporin activity.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

MicroRNA expression profile in bovine mammary gland parenchyma infected by coagulase-positive or coagulase-negative staphylococci.

MicroRNAs (miRNAs) are short, non-coding RNAs, 21-23 nucleotides in length which are known to regulate biological processes that greatly impact immune system activity. The aim of the study was to compare the miRNA expression in non-infected (H) mammary gland parenchyma samples with that of glands infected with coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS) using next-generation sequencing. The miRNA profile of the parenchyma was found to change during mastitis, with its profile depending on the type of pathogen. Comparing the CoPS and H groups, 256 known and 260 potentially new miRNAs were identified, including 32 that were differentially expressed (p ≤ 0.05), of which 27 were upregulated and 5 downregulated. Comparing the CoNS and H groups, 242 known and 171 new unique miRNAs were identified: 10 were upregulated (p ≤ 0.05), and 2 downregulated (p ≤ 0.05). In addition, comparing CoPS with H and CoNS with H, 5 Kyoto Encyclopedia of Genes and Genomes pathways were identified; in both comparisons, differentially-expressed miRNAs were associated with the bacterial invasion of epithelial cells and focal adhesion pathways. Four gene ontology terms were identified in each comparison, with 2 being common to both immune system processes and signal transduction. Our results indicate that miRNAs, especially miR-99 and miR-182, play an essential role in the epigenetic regulation of a range of cellular processes, including immunological systems bacterial growth in dendritic cells and disease pathogenesis (miR-99), DNA repair and tumor progression (miR-182).

Comparing the effects of different exogenous hormone combinations on seed-derived callus induction in Nicotiana tabacum.

Callus from Nicotiana tabacum is used as a model in plant developmental research. We tested several phytohormone (Indoleacetic acid - IAA; 2,4-Dichlorophenoxyacetic acid - 2,4-D; kinetin - KIN; 6-Benzylaminopurine - BAP) combinations to compare different approaches to callus induction directly from the seeds of Nicotiana tabacum. Callus formation was observed up to 4 weeks after sowing and the most effective were 0.5 mg/L of 2,4-D with 0.25 mg/L of BAP and 2 mg/L 2,4-D with 1 mg/L of BAP. The calli were green, photosynthetically active and after 6 weeks of growth, no stress symptoms (estimated on the basis of fluorescence of chlorophyll a in photosystem II) were noticed.

Plant Tissue Cultures.

Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium from which enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation and callus culture initiation and its maintenance as well as those for protoplast isolation and viability observation are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.

S-Nitroso-Proteome Revealed in Stomatal Guard Cell Response to Flg22.

Nitric oxide (NO) plays an important role in stomata closure induced by environmental stimuli including pathogens. During pathogen challenge, nitric oxide (NO) acts as a second messenger in guard cell signaling networks to activate downstream responses leading to stomata closure. One means by which NO's action is achieved is through the posttranslational modification of cysteine residue(s) of target proteins. Although the roles of NO have been well studied in plant tissues and seedlings, far less is known about NO signaling and, more specifically, protein S-nitrosylation (SNO) in stomatal guard cells. In this study, using iodoTMTRAQ quantitative proteomics technology, we analyzed changes in protein SNO modification in guard cells of reference plant Arabidopsis thaliana in response to flg22, an elicitor-active peptide derived from bacterial flagellin. A total of 41 SNO-modified peptides corresponding to 35 proteins were identified. The proteins cover a wide range of functions, including energy metabolism, transport, stress response, photosynthesis, and cell-cell communication. This study creates the first inventory of previously unknown NO responsive proteins in guard cell immune responses and establishes a foundation for future research toward understanding the molecular mechanisms and regulatory roles of SNO in stomata immunity against bacterial pathogens.

The apoplastic antioxidant system and altered cell wall dynamics influence mesophyll conductance and the rate of photosynthesis.

Mesophyll conductance (gm ), the diffusion of CO2 from substomatal cavities to the carboxylation sites in the chloroplasts, is a highly complex trait driving photosynthesis (net CO2 assimilation, AN ). However, little is known concerning the mechanisms by which it is dynamically regulated. The apoplast is considered as a 'key information bridge' between the environment and cells. Interestingly, most of the environmental constraints affecting gm also cause apoplastic responses, cell wall (CW) alterations and metabolic rearrangements. Since CW thickness is a key determinant of gm , we hypothesize that other changes in this cellular compartiment should also influence gm . We study the relationship between the antioxidant apoplastic system and CW metabolism and the gm responses in tobacco plants (Nicotiana sylvestris L.) under two abiotic stresses (drought and salinity), combining in vivo gas-exchange measurements with analyses of antioxidant activities, CW composition and primary metabolism. Stress treatments imposed substantial reductions in AN (58-54%) and gm (59%), accompanied by a strong antioxidant enzymatic response at the apoplastic and symplastic levels. Interestingly, apoplastic but not symplastic peroxidases were positively related to gm . Leaf anatomy remained mostly stable; however, the stress treatments significantly affected the CW composition, specifically pectins, which showed significant relationships with AN and gm . The treatments additionally promoted a differential primary metabolic response, and specific CW-related metabolites including galactose, glucosamine and hydroxycinnamate showed exclusive relationships with gm independent of the stress. These results suggest that gm responses can be attributed to specific changes in the apoplastic antioxidant system and CW metabolism, opening up more possibilities for improving photosynthesis using breeding/biotechnological strategies.

Chloride as a macronutrient increases water-use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO2.

Chloride (Cl- ) has been recently described as a beneficial macronutrient, playing specific roles in promoting plant growth and water-use efficiency (WUE). However, it is still unclear how Cl- could be beneficial, especially in comparison with nitrate (NO3- ), an essential source of nitrogen that shares with Cl- similar physical and osmotic properties, as well as common transport mechanisms. In tobacco plants, macronutrient levels of Cl- specifically reduce stomatal conductance (gs ) without a concomitant reduction in the net photosynthesis rate (AN ). As stomata-mediated water loss through transpiration is inherent in the need of C3 plants to capture CO2 , simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C3 crop productivity. Our results showed that Cl- -mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces gs and water consumption. Conversely, Cl- improves mesophyll diffusion conductance to CO2 (gm ) and photosynthetic performance due to a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, possibly as a consequence of the stimulation of chloroplast biogenesis. A key finding of this study is the simultaneous improvement of AN and WUE due to macronutrient Cl- nutrition. This work identifies relevant and specific functions in which Cl- participates as a beneficial macronutrient for higher plants, uncovering a sustainable approach to improve crop yield.

Leaf anatomy does not explain apparent short-term responses of mesophyll conductance to light and CO2 in tobacco.

Mesophyll conductance to CO2 (gm ), a key photosynthetic trait, is strongly constrained by leaf anatomy. Leaf anatomical parameters such as cell wall thickness and chloroplast area exposed to the mesophyll intercellular airspace have been demonstrated to determine gm in species with diverging phylogeny, leaf structure and ontogeny. However, the potential implication of leaf anatomy, especially chloroplast movement, on the short-term response of gm to rapid changes (i.e. seconds to minutes) under different environmental conditions (CO2 , light or temperature) has not been examined. The aim of this study was to determine whether the observed rapid variations of gm in response to variations of light and CO2 could be explained by changes in any leaf anatomical arrangements. When compared to high light and ambient CO2 , the values of gm estimated by chlorophyll fluorescence decreased under high CO2 and increased at low CO2 , while it decreased with decreasing light. Nevertheless, no changes in anatomical parameters, including chloroplast distribution, were found. Hence, the gm estimated by analytical models based on anatomical parameters was constant under varying light and CO2 . Considering this discrepancy between anatomy and chlorophyll fluorescence estimates, it is concluded that apparent fast gm variations should be due to artefacts in its estimation and/or to changes in the biochemical components acting on diffusional properties of the leaf (e.g. aquaporins and carbonic anhydrase).

Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC).

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.

Red Light-Induced Phosphorylation of Plasma Membrane H+-ATPase in Stomatal Guard Cells.

Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane (PM) H+-ATPase by phosphorylating its penultimate residue, threonine, via a blue light photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated PM H+-ATPase promotes the accumulation of osmolytes and, thus, the osmotic influx of water into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, how red light induces stomatal opening and whether PM H+-ATPase is involved in this process have remained unclear. In this study, we established an immunohistochemical technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces PM H+-ATPase phosphorylation in whole leaves. Red light-induced PM H+-ATPase phosphorylation in whole leaves was correlated with stomatal opening under red light and was inhibited by the plant hormone abscisic acid. In aha1-9, a knockout mutant of one of the major isoforms of PM H+-ATPase in guard cells, red light-dependent stomatal opening was delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited red light-induced PM H+-ATPase phosphorylation as well as red light-induced stomatal opening in whole leaves. Our results indicate that red light-induced PM H+-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.

Beryllium Stress-Induced Modifications in Antioxidant Machinery and Plant Ultrastructure in the Seedlings of Black and Yellow Seeded Oilseed Rape.

Beryllium (Be) could be a threatening heavy metal pollutant in the agroecosystem that may severely affect the performance of crops. The present study was conducted to evaluate the toxic effects of Be (0, 100, 200, and 400 µM) on physiological, ultrastructure, and biochemical attributes in hydroponically grown six-day-old seedlings of two cultivars of Brassica napus L., one tolerant (ZS 758, black seeded) and one sensitive (Zheda 622, yellow seeded). Higher Be concentrations reduced the plant growth, biomass production, chlorophyll contents, and the total soluble protein contents. A significant accumulation of ROS (H2O2, OH-) and MDA contents was observed in a dose-dependent manner. Antioxidant enzymatic activities including SOD, POD, GR, APX, and GSH (except CAT) were enhanced with the increase in Be concentrations in both cultivars. Relative transcript gene expression of above-mentioned antioxidant enzymes further confirmed the alterations induced by Be as depicted from higher involvement in the least susceptible cultivar ZS 758 as compared to Zheda 622. The electron microscopic study showed that higher level of Be (400 µM) greatly damaged the leaf mesophyll and root tip cells. More damage was observed in cultivar Zheda 622 as compared to ZS 758. The damage in leaf mesophyll cells was highlighted as the disruption in cell wall, immature nucleus, damaged mitochondria, and chloroplast structures. In root tip cells, disruption in Golgi bodies and damage in cell wall were clearly noticed. As a whole, the present study confirmed that more inhibitory effects were recorded in yellow seeded Zheda 622 as compared to black seeded ZS 758 cultivar, which is regarded as more sensitive cultivar.

Leaf blade structure of Verbesina macrophylla (Cass.) F. S. Blake (Asteraceae): ontogeny, duct secretion mechanism and essential oil composition.

Secretory structures are common in Asteraceae, where they exhibit a high degree of morphological diversity. The species Verbesina macrophylla, popularly known as assa-peixe, is native to Brazil where it is widely used for medicinal purposes. Despite its potential medical importance, there have been no studies of the anatomy of this species, especially its secretory structures and secreted compounds. This study examined leaves of V. macrophylla with emphasis on secretory structures and secreted secondary metabolites. Development of secretory ducts and the mechanism of secretion production are described for V. macrophylla using ultrastructure, yield and chemical composition of its essential oils. Verbesina macrophylla has a hypostomatic leaf blade with dorsiventral mesophyll and secretory ducts associated with vascular bundles of schizogenous origin. Histochemistry identified the presence of lipids, terpenes, alkaloids and mucopolysaccharides. Ultrastructure suggests that the secretion released into the duct lumen is produced in plastids of transfer cells, parenchymal sheath cells and stored in vacuoles in these cells and duct epithelial cells. The essential oil content was 0.8%, and its major components were germacrene D, germacrene D-4-ol, ß-caryophyllene, bicyclogermacrene and α-cadinol. Secretory ducts of V. macrophylla are squizogenous. Substances identified in tissues suggest that both secretions stored in the ducts and in adjacent parenchyma cells are involved in chemical defence. The essential oil is rich in sesquiterpenes, with germacrene D and its derivatives being notable components.

Co-Compartmentation of Terpene Biosynthesis and Storage via Synthetic Droplet.

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.

Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum.

Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants.

Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.