Transcriptome Analysis of Sweet Cherry (Prunus avium L.) Cultivar 'Lapins' upon Infection of Pseudomonas syringae pv. syringae.

Bacterial canker caused by Pseudomonas syringae pv. syringae (Pss) is responsible for substantial loss to the production of sweet cherry in Chile. To date, the molecular mechanisms of the Pss-sweet cherry interaction and the disease-related genes in the plant are poorly understood. In order to gain insight into these aspects, a transcriptomic analysis of the sweet cherry cultivar 'Lapins' for differentially expressed genes (DEGs) in response to Pss inoculation was conducted. Three Pss strains, A1M3, A1M197, and 11116_b1, were inoculated in young twigs, and RNA was extracted from tissue samples at the inoculation site and distal sections. RNA sequencing and transcriptomic expression analysis revealed that the three strains induced different patterns of responses in local and distal tissues. In the local tissues, A1M3 triggered a much more extensive response than the other two strains, enriching DEGs especially involved in photosynthesis. In the distal tissues, the three strains triggered a comparable extent of responses, among which 11116_b1 induced a group of DEGs involved in defense responses. Furthermore, tissues from various inoculations exhibited an enrichment of DEGs related to carbohydrate metabolism, terpene metabolism, and cell wall biogenesis. This study opened doors to future research on the Pss-sweet cherry interaction, immunity responses, and disease control.

The LeEIX Locus Determines Pathogen Resistance in Tomato.

The mechanisms underlying the ability of plants to differentiate between pathogens and commensals in their environment are currently unresolved. It has been suggested that spatiotemporal regulation of pattern-recognition receptor (PRR) content could be one of the components providing plants with the ability to distinguish between pathogens and nonpathogenic microbes. The LeEIX PRRs recognize xylanases derived from beneficial or commensal plant colonizers of Trichoderma species, including the xylanase known as EIX. Here, we investigated possible general roles of PRRs from the LeEIX locus in immunity and pathogen resistance in tomato. Mutating the inhibitory PRR LeEIX1, or overexpressing the activating PRR LeEIX2, resulted in resistance to a wide range of pathogens and increased basal and elicited immunity. LeEIX1 knockout caused increases in the expression level of several tested PRRs, including FLS2, as well as bacterial pathogen resistance coupled with an increase in flg22-mediated immunity. The wild tomato relative Solanum pennellii contains inactive LeEIX PRR variants. S. pennellii does not respond to elicitation with the LeEIX PRR ligand EIX. Given that EIX is derived from a mostly nonpathogenic microbe, the connection of its PRRs to disease resistance has not previously been investigated directly. Here, we observed that compared with S. lycopersicum cultivar M82, S. pennellii was more sensitive to several fungal and bacterial pathogens. Our results suggest that the LeEIX locus might determine resistance to fungal necrotrophs, whereas the resistance to biotrophs is effected in combination with a gene/quantitative trait locus not within the LeEIX locus.

Dynamin-Related Proteins Enhance Tomato Immunity by Mediating Pattern Recognition Receptor Trafficking.

Pattern recognition receptor (PRR) trafficking to the plasma membrane and endocytosis plays a crucial role in pattern triggered immunity (PTI). Dynamin-related proteins (DRPs) participate in endocytosis and recycling. In Arabidopsis, DRP1 and DRP2 are involved in plasma membrane scission during endocytosis. They are required for the PRR FLS2 endocytosis induction and PTI activation after elicitation with flg22, the MAMP recognized by FLS2. In tomato, SlDRP2A regulates the PRR LeEIX2 endocytosis and PTI activation in response to EIX, the MAMP recognized by LeEIX2. However, it is unknown if other DRPs participate in these processes. Taking advantage of bioinformatics tools, we selected SlDRP2B among the eight DRP2 tomato orthologues to study its functionality in trafficking and plant immunity. Through transient expression of SlDRP1B and its dominant-negative mutant on Nicotiana benthamiana and Nicotiana tabacum, we analyzed SlDRP1B function. We observed that SlDRP1B is physically associated with the LeEIX2 and modifies LeEIX2 trafficking, increasing its presence in endosomes. An enhancement of EIX-elicitated defense responses accompanies the role of SlDRP1B on LeEIX endocytosis. In addition, SlDRP1B overexpression enhanced flg22-elicited defense response. With these results, we conclude that SlDRP1B regulates PRR trafficking and, therefore, plant immunity, similarly to the SlDRP2A role.

Cytokinin Inhibits Fungal Development and Virulence by Targeting the Cytoskeleton and Cellular Trafficking.

Cytokinin (CK) is an important plant developmental regulator, having activities in many aspects of plant life and response to the environment. CKs are involved in diverse processes in the plant, including stem cell maintenance, vascular differentiation, growth and branching of roots and shoots, leaf senescence, nutrient balance, and stress tolerance. In some cases, phytopathogens secrete CKs. It has been suggested that to achieve pathogenesis in the host, CK-secreting biotrophs manipulate CK signaling to regulate the host cell cycle and nutrient allocation. CK is known to induce host plant resistance to several classes of phytopathogens from a few works, with induced host immunity via salicylic acid signaling suggested to be the prevalent mechanism for this host resistance. Here, we show that CK directly inhibits the growth, development, and virulence of fungal phytopathogens. Focusing on Botrytis cinerea (Bc), we demonstrate that various aspects of fungal development can be reversibly inhibited by CK. We also found that CK affects both budding and fission yeast in a similar manner. Investigating the mechanism by which CK influences fungal development, we conducted RNA next-generation sequencing (RNA-NGS) on mock- and CK-treated B. cinerea samples, finding that CK alters the cell cycle, cytoskeleton, and endocytosis. Cell biology experiments demonstrated that CK affects cytoskeleton components and cellular trafficking in Bc, lowering endocytic rates and endomembrane compartment sizes, likely leading to reduced growth rates and arrested developmental programs. Mutant analyses in yeast confirmed that the endocytic pathway is altered by CK. Our work uncovers a remarkably conserved role for a plant growth hormone in fungal biology, suggesting that pathogen-host interactions resulted in fascinating molecular adaptations on fundamental processes in eukaryotic biology. IMPORTANCE Cytokinins (CKs), important plant growth/developmental hormones, have previously been associated with host disease resistance. Here, we demonstrate that CK directly inhibits the growth, development, and virulence of B. cinerea (Bc) and many additional phytopathogenic fungi. Molecular and cellular analyses revealed that CK is not toxic to Bc, but rather, Bc likely recognizes CK and responds to it, resulting in cell cycle and individual cell growth retardation, via downregulation of cytoskeletal components and endocytic trafficking. Mutant analyses in yeast confirmed that the endocytic pathway is a CK target. Our work demonstrates a conserved role for CK in yeast and fungal biology, suggesting that pathogen-host interactions may cause molecular adaptations in fundamental processes in eukaryotic biology.

Cytokinin Modulates Cellular Trafficking and the Cytoskeleton, Enhancing Defense Responses.

The plant hormone cytokinin (CK) plays central roles in plant development and throughout plant life. The perception of CKs initiating their signaling cascade is mediated by histidine kinase receptors (AHKs). Traditionally thought to be perceived mostly at the endoplasmic reticulum (ER) due to receptor localization, CK was recently reported to be perceived at the plasma membrane (PM), with CK and its AHK receptors being trafficked between the PM and the ER. Some of the downstream mechanisms CK employs to regulate developmental processes are unknown. A seminal report in this field demonstrated that CK regulates auxin-mediated lateral root organogenesis by regulating the endocytic recycling of the auxin carrier PIN1, but since then, few works have addressed this issue. Modulation of the cellular cytoskeleton and trafficking could potentially be a mechanism executing responses downstream of CK signaling. We recently reported that CK affects the trafficking of the pattern recognition receptor LeEIX2, influencing the resultant defense output. We have also recently found that CK affects cellular trafficking and the actin cytoskeleton in fungi. In this work, we take an in-depth look at the effects of CK on cellular trafficking and on the actin cytoskeleton in plant cells. We find that CK influences the actin cytoskeleton and endomembrane compartments, both in the context of defense signaling-where CK acts to amplify the signal-as well as in steady state. We show that CK affects the distribution of FLS2, increasing its presence in the plasma membrane. Furthermore, CK enhances the cellular response to flg22, and flg22 sensing activates the CK response. Our results are in agreement with what we previously reported for fungi, suggesting a fundamental role for CK in regulating cellular integrity and trafficking as a mechanism for controlling and executing CK-mediated processes.

Co-occurrence Interaction Networks of Extremophile Species Living in a Copper Mining Tailing.

Copper mining tailings are characterized by high concentrations of heavy metals and an acidic pH, conditions that require an extreme adaptation for any organism. Currently, several bacterial species have been isolated and characterized from mining environments; however, very little is known about the structure of microbial communities and how their members interact with each other under the extreme conditions where they live. This work generates a co-occurrence network, representing the bacterial soil community from the Cauquenes copper tailing, which is the largest copper waste deposit worldwide. A representative sampling of six zones from the Cauquenes tailing was carried out to determine pH, heavy metal concentration, total DNA extraction, and subsequent assignment of Operational Taxonomic Units (OTUs). According to the elemental concentrations and pH, the six zones could be grouped into two sectors: (1) the "new tailing," characterized by neutral pH and low concentration of elements, and (2) the "old tailing," having extremely low pH (~3.5) and a high concentration of heavy metals (mainly copper). Even though the abundance and diversity of species were low in both sectors, the Pseudomonadaceae and Flavobacteriaceae families were over-represented. Additionally, the OTU identifications allowed us to identify a series of bacterial species with diverse biotechnological potentials, such as copper bioleaching and drought stress alleviation in plants. Using the OTU information as a template, we generated co-occurrence networks for the old and new tailings. The resulting models revealed a rearrangement between the interactions of members living in the old and new tailings, and highlighted conserved bacterial drivers as key nodes, with positive interactions in the network of the old tailings, compared to the new tailings. These results provide insights into the structure of the soil bacterial communities growing under extreme environmental conditions in mines.

Gene Editing of the Decoy Receptor LeEIX1 Increases Host Receptivity to Trichoderma Bio-Control.

Fungal and bacterial pathogens generate devastating diseases and cause significant tomato crop losses worldwide. Due to chemical pesticides harming the environment and human health, alternative disease control strategies, including microorganismal bio-control agents (BCAs), are increasingly sought-after in agriculture. Bio-control microorganisms such as Trichoderma spp. have been shown to activate induced systemic resistance (ISR) in the host. However, examples of highly active bio-control microorganisms in agricultural settings are still lacking, due primarily to inconsistency in bio-control efficacy, often leading to widespread disease prior to the required ISR induction in the host. As part of its plant colonization strategy, Trichoderma spp. can secrete various compounds and molecules, which can effect host priming/ISR. One of these molecules synthesized and secreted from several species of Trichoderma is the family 11 xylanase enzyme known as ethylene inducing xylanase, EIX. EIX acts as an ISR elicitor in specific plant species and varieties. The response to EIX in tobacco and tomato cultivars is controlled by a single dominant locus, termed LeEIX, which contains two receptors, LeEIX1 and LeEIX2, both belonging to a class of leucine-rich repeat cell-surface glycoproteins. Both receptors are able to bind EIX, however, while LeEIX2 mediates plant defense responses, LeEIX1 acts as a decoy receptor and attenuates EIX induced immune signaling of the LeEIX2 receptor. By mutating LeEIX1 using CRISPR/Cas9, here, we report an enhancement of receptivity to T. harzianum mediated ISR and disease bio-control in tomato.

SlRLK-like is a malectin-like domain protein affecting localization and abundance of LeEIX2 receptor resulting in suppression of EIX-induced immune responses.

The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe-associated molecular patterns. Plant PRRs are either receptor-like kinases (RLKs), which have an extracellular domain for ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor-like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene-inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two-hybrid screen and found a tomato protein that we termed SlRLK-like. The interaction of LeEIX2 with SlRLK-like was verified using co-immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK-like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK-like using the CRISPR/Cas9 system increased EIX-induced ethylene production and 1-aminocyclopropane-1-carboxylate synthase (SlACS2) gene expression in tomato. Co-expression of SlRLK-like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum-associated degradation process. Notably, truncation of SlRLK-like protein revealed that the malectin-like domain is sufficient and essential for its function. Moreover, SlRLK-like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)-induced burst of reactive oxygen species. In addition, SlRLK-like co-expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK-like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses.

Cytokinin response induces immunity and fungal pathogen resistance, and modulates trafficking of the PRR LeEIX2 in tomato.

Plant immunity is often defined by the immunity hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). These hormones are well known for differentially regulating defence responses against pathogens. In recent years, the involvement of other plant growth hormones such as auxin, gibberellic acid, abscisic acid, and cytokinins (CKs) in biotic stresses has been recognized. Previous reports have indicated that endogenous and exogenous CK treatment can result in pathogen resistance. We show here that CK induces systemic immunity in tomato (Solanum lycopersicum), modulating cellular trafficking of the pattern recognition receptor (PRR) LeEIX2, which mediates immune responses to Xyn11 family xylanases, and promoting resistance to Botrytis cinerea and Oidium neolycopersici in an SA- and ET-dependent mechanism. CK perception within the host underlies its protective effect. Our results support the notion that CK promotes pathogen resistance by inducing immunity in the host.

A gain of function mutation in SlNRC4a enhances basal immunity resulting in broad-spectrum disease resistance.

Plants rely on innate immunity to perceive and ward off microbes and pests, and are able to overcome the majority of invading microorganisms. Even so, specialized pathogens overcome plant defenses, posing a persistent threat to crop and food security worldwide, raising the need for agricultural products with broad, efficient resistance. Here we report a specific mutation in a tomato (S. lycopersicum) helper nucleotide-binding domain leucine-rich repeat H-NLR, SlNRC4a, which results in gain of function constitutive basal defense activation, in absence of PRR activation. Knockout of the entire NRC4 clade in tomato was reported to compromise Rpi-blb2 mediated immunity. The SlNRC4a mutant reported here possesses enhanced immunity and disease resistance to a broad-spectrum of pathogenic fungi, bacteria and pests, while lacking auto-activated HR or negative effects on plant growth and crop yield, providing promising prospects for agricultural adaptation in the war against plant pathogens that decrease productivity.

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus.

The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine-learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild-type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the 'richest' bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Tomato Dynamin Related Protein 2A Associates With LeEIX2 and Enhances PRR Mediated Defense by Modulating Receptor Trafficking.

The endocytic trafficking pathway is employed by the plant to regulate immune responses, and is often targeted by pathogen effectors to promote virulence. The model system of the tomato receptor-like protein (RLP) LeEIX2 and its ligand, the elicitor EIX, employs endocytosis to transmit receptor-mediated signals, with some of the signaling events occurring directly from endosomal compartments. Here, to explore the trafficking mechanism of LeEIX2-mediated immune signaling, we used a proteomic approach to identify LeEIX2-associating proteins. We report the identification of SlDRP2A, a dynamin related protein, as an associating partner for LeEIX2. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 in VHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune responses.

Organelle-Targeted BODIPY Photocages: Visible-Light-Mediated Subcellular Photorelease.

Photocaging facilitates non-invasive and precise spatio-temporal control over the release of biologically relevant small- and macro-molecules using light. However, sub-cellular organelles are dispersed in cells in a manner that renders selective light-irradiation of a complete organelle impractical. Organelle-specific photocages could provide a powerful method for releasing bioactive molecules in sub-cellular locations. Herein, we report a general post-synthetic method for the chemical functionalization and further conjugation of meso-methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)-, lysosome-, and mitochondria-targeted derivatives. We also demonstrate that 2,4-dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.

NRC proteins - a critical node for pattern and effector mediated signaling.

Plants are constantly exposed to numerous diverse microbes and pests. They lack an adaptive immune system and rely on innate immunity to perceive and ward off potential pathogens. The plant immune system enables plants to overcome invading microorganisms, and can be defined as highly successful in this regard. Nevertheless, specialized pathogens are able to overcome structural barriers, preformed defenses, innate immunity and are a persistent threat to crop and food supplies worldwide. The rapidly growing world population results in massive demands for agricultural products and reliable crop yields. Therefore, the ability to precisely manipulate plant immunity to resist diverse diseases holds significant promise for enhancing crop production.

The intracellular nucleotide-binding leucine-rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception.

Plant recognition and defence against pathogens employs a two-tiered perception system. Surface-localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR required for hypersensitive response-associated cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.

Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response.

Plants recognize microbial/pathogen associated molecular patterns (MAMP/PAMP) through pattern recognition receptors (PRRs) triggering an immune response against pathogen progression. MAMP/PAMP triggered immune response requires PRR endocytosis and trafficking for proper deployment. LeEIX2 is a well-known Solanum lycopersicum RLP-PRR, able to recognize and respond to the fungal MAMP/PAMP ethylene-inducing xylanase (EIX), and its function is highly dependent on intracellular trafficking. Identifying protein machinery components regulating LeEIX2 intracellular trafficking is crucial to our understanding of LeEIX2 mediated immune responses. In this work, we identified a novel trafficking protein, SlPRA1A, a predicted regulator of RAB, as an interactor of LeEIX2. Overexpression of SlPRA1A strongly decreases LeEIX2 endosomal localization, as well as LeEIX2 protein levels. Accordingly, the innate immune responses to EIX are markedly reduced by SlPRA1A overexpression, presumably due to a decreased LeEIX2 availability. Studies into the role of SlPRA1A in LeEIX2 trafficking revealed that LeEIX2 localization in multivesicular bodies/late endosomes is augmented by SlPRA1A. Furthermore, inhibiting vacuolar function prevents the LeEIX2 protein level reduction mediated by SlPRA1A, suggesting that SlPRA1A may redirect LeEIX2 trafficking to the vacuole for degradation. Interestingly, SlPRA1A overexpression reduces the amount of several RLP-PRRs, but does not affect the protein level of receptor-like kinase PRRs, suggesting a specific role of SlPRA1A in RLP-PRR trafficking and degradation.

Involvement of SchRabGDI1 from Solanum chilense in endocytic trafficking and tolerance to salt stress.

Physiological responses of plants to salinity stress requires the coordinated activation of many genes. A salt-induced gene was isolated from roots of the wild tomato species Solanum chilense and named SchRabGDI1 because it encodes a protein with high identity to GDP dissociation inhibitors of plants. These proteins are regulators of the RabGTPase cycle that play key roles in intracellular vesicular trafficking. The expression pattern of SchRabGDI1 showed an early up-regulation in roots and leaves under salt stress. Functional activity of SchRabGDI1 was shown by restoring the defective phenotype of the yeast sec19-1 mutant and the capacity of SchRabGDI1 to interact with RabGTPase was demonstrated through BiFC assays. Expression of SchRabGDI1 in Arabidopsis thaliana plants resulted in increased salt tolerance. Also, the root cells of transgenic plants showed higher rate of endocytosis under normal growth conditions and higher accumulation of sodium in vacuoles and small vesicular structures under salt stress than wild type. Our results suggest that in salt tolerant species such as S. chilense, bulk endocytosis is one of the early mechanisms to avoid salt stress, which requires the concerted expression of regulatory genes involved in vesicular trafficking of the endocytic pathway.

Regulation of protein trafficking: posttranslational mechanisms and the unexplored transcriptional control.

Endomembrane protein trafficking assures protein location through the endocytic and secretory routes. Trafficking pathways are diverse, depending on the proteins being trafficked, the final destination as well as their itinerary. Trafficking pathways are operated by machineries composed of a set of coordinately acting factors that transport proteins between compartments. Different machineries participate in each protein trafficking pathway, providing specificity and accuracy. Changes in the activity and abundance of trafficking proteins regulate protein flux. The preponderance of one pathway over another regulates protein location and relocation. Cellular requirements change during different processes and in response to stimuli; modulation of trafficking mechanisms must relocate proteins or alternatively increase/decrease the targeting rate of certain proteins. Conventionally, protein trafficking modulation has been explained as posttranslational modification of components of the relevant trafficking machinery. However, trafficking components are also transcriptionally regulated and several reports support that this regulation can modulate protein trafficking as well. This transcriptional modulation has an impact on plant physiology, and is a critical and fundamental mechanism. This scenario suggests a determinant mechanism that must be considered in the endomembrane protein trafficking research field.

Levels of lycopene ß-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene ß-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and ß-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and ß-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and ß-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.