Volatile compounds in citrus in adaptation to water deficit and to herbivory by Diaphorina citri: how the secondary metabolism of the plant is modulated under concurrent stresses. A review.

Citrus plants are grown in diverse regions of the world, from subtropical to semi-arid and humid tropical areas. Through mechanisms essential for their survival, they adapt to the environmental conditions to which they are subjected. Although there is vast literature on adaptation of citrus plants to individual stresses, plant responses to interaction among different types of stresses have not been clearly examined. Abiotic or biotic stresses, or a combination of these stresses, result in reorganization of plant energy resources for defense, whether it be for resistance, tolerance, or prevention of stress. Plants generally respond to these stress factors through production of secondary metabolites, such as volatile compounds, derived from different biosynthesis and degradation pathways, which are released through distinct routes. Volatile compounds vary among plant species, meeting the specific needs of the plant. Simultaneous exposure to the stress factors of water deficit and herbivory leads to responses such as qualitative and quantitative changes in the emission of secondary metabolites, and compounds may accumulate within the leaves or predispose the plant to more quickly respond to the stress brought about by the herbivore. The genetic makeup of citrus plants can contribute to a better response to stress factors; however, studies on the emission of volatile compounds in different citrus genotypes under simultaneous stresses are limited. This review examines the effects of abiotic stress due to water deficit and biotic stress due to herbivory by Diaphorina citri in citrus plants and examines their connection with volatile compounds. A summary is made of advances in knowledge regarding the performance of volatile compounds in plant defense against both stress factors, as well as the interaction between them and possible findings in citrus plants. In addition, throughout this review, we focus on how genetic variation of the citrus species is correlated with production of volatile compounds to improve stress tolerance.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Trichoderma stromaticum spores induce autophagy and downregulate inflammatory mediators in human peripheral blood-derived macrophages.

Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1ß, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.

Irradiance and light quality affect two annatto (Bixa orellana L.) cultivars with contrasting bixin production.

Light is a key factor influencing growth and development in plants. Specific irradiance and light quality can improve development and production of secondary compounds such as carotenoids during plant tissue culture. Bixin and norbixin, two apocarotenoids obtained from the seeds of Bixa orellana L. (annatto), are used as natural dyes in various industries. While annatto tissue culture has been successful, the effect of light in this species remains poorly understood. Here, we analyze for the first time the effect of irradiance regime (50, 150, 50 + 150, 200, 50 + 200 µmol m-2 s-1) and light spectral quality (fluorescent, white, blue/red LED) on in vitro development of apexes and bixin content in two contrasting bixin-producing varieties of B. orellana, namely 'Piave Vermelha' and 'UESB74'. The number of leaves per plant, stomatal density, leaf area, leaf expansion, chlorophylls and carotenoids content, malondialdehyde and bixin content were analyzed in the leaves of both cultivars. 'Piave Vermelha' produced 1.6-fold more bixin than 'UESB74'. Stomata cells of both cultivars had a paracytic arrangement with peltate trichomes along the adaxial and abaxial leaf surfaces. 'Piave Vermelha' preferred blue/red LED light; whereas fluorescent light was optimal for 'UESB74'. Under fluorescent light, an irradiance of 50 µmol m-2 s-1 is indicated for both cultivars. LED light increased bixin content only in 'Piave Vermelha', suggesting that the dye biosynthetic pathway is genotype-dependent. The present findings suggest the possibility of using light to modulate the bixin biosynthetic pathway.

Recurrent water deficit causes alterations in the profile of redox proteins in citrus plants.

Plant acclimation to recurrent stress involves profound alterations in multiple genetic, metabolic and physiological processes. Stressful conditions usually implicate imbalance in reactive oxygen species (ROS) production and removal rates, which may lead to oxidative stress. However, the primary cellular targets of oxidative stress and their relevance in plant acclimation to abiotic stresses remains poorly characterized. By comparing redox proteomic and sugar profiles in citrus Valencia (VO) scions grafted onto two rootstocks with different soil water extraction capacities - Rangpur Lime (RL) and Sunki Maravilha (SM) - here we demonstrate that both ROS-mediated post-translational protein modification and changes in sugar composition are associated with acclimation to recurrent drought in citrus. The redox proteomic analysis of the distinct scion/rootstock combinations exposed to one (WD1), two (WD2) or three (WD3) water deficit episodes revealed a total of 32 and 55 redox protein spots present in VO/RL and VO/SM plants, respectively. Mass spectrometry analysis of these protein spots revealed essential targets of ROS-mediated posttranslational protein modification in citrus plants challenged by recurrent drought. The oxidation of cysteine thiol groups into glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was shown to increase in WD3 samples of the VO/RL combination, whereas the opposite was observed for the VO/SM combination. Similarly, recurrent drought promoted the oxidation of catalase thiol groups in VO/SM, but not in VO/RL. Carbohydrate profiling revealed that glucose, fructose and galactose may also contribute to the phenotypic differences observed between the citrus genotypes exposed to drought. These findings reveal for the first time that recurrent drought differentially affects the profile of redox proteomics of citrus, suggesting that this alteration may be part of the stress memory in perennial plants.

Recurrent water deficit causes epigenetic and hormonal changes in citrus plants.

The present study evaluated the physiological, molecular and hormonal parameters from scion/rootstock interaction of citrus plants during recurrent water deficit. Responses of the Valencia (VO) scion variety grafted on two rootstocks with different soil water extraction capacities, Rangpur Lime (RL) and Sunki Maravilha (SM), during three successive periods of water deficit: plants exposed to a single episode of water deficit (WD1) and plants exposed to two (WD2) and three (WD3) recurrent periods of WD were compared. The combinations VO/RL and VO/SM presented polymorphic alterations of epigenetic marks and hormonal (i.e. abscisic acid, auxins and salicylicacid) profiles, which were particularly prominent when VO/SM plantswere exposed toWD3 treatment. Upon successive drought events, the VO/SM combination presented acclimatization characteristics that enable higher tolerance to water deficit by increasing transpiration (E), stomatal conductance (g s ) and photosynthetic rate (A), which in turn may have facilitated the whole plant survival. Besides providing comprehensive data on the scion/rootstock interactions upon successive stress events, this study brings the first dataset suggesting that epigenetic alterations in citrus plants triggered by recurrent water deficit lead to improved drought tolerance in this crop species.

Differential accumulation of flavonoids and phytohormones resulting from the canopy/rootstock interaction of citrus plants subjected to dehydration/rehydration.

Water scarcity can elicit drastic changes in plant metabolic and hormonal regulation, which may be of fundamental importance to stress tolerance. The study of plant the metabolic alterations in response to water deficit, especially the effects of the rootstocks level, is important to elucidate the mechanisms associated to drought tolerance. To verify the influence of rootstock and grafting on the tolerance to drought in citrus plants, we analyzed the growth, phytohormone levels and flavonoid profiles in grafted and ungrafted citrus plants subjected to different soil water regimes on plant status (well-watered, moderate drought and severe drought and rehydrated) under field conditions. The experiments were conducted under field conditions in the Brazilian Agricultural Research Corporation (EMBRAPA), Cruz das Almas, BA, Brazil. Water deficit reduced the total leaf area per plant in all canopy/rootstock combinations. Self-grafting reduce root volume, area and length when compared to ungrafted plants. Drought-induced increases in salicylic acid and abscisic acid associated with concomitant reductions in indoleacetic acid were observed in most canopy/rootstock combinations. However, plants with 'Sunki Maravilha' rootstocks exhibited the most pronounced changes in hormonal levels upon drought stress. Associated to these hormonal changes, drought also significantly affected flavonoid content and profile in both leaves and roots of the distinct citrus combinations. Glycosylated (GFs) and polimethoxylated flavonoids were predominantly found in leaves, whereas prenylated coumarins were found in the roots. Leaf levels of GFs (vicenin, F11, rutin and rhoifolin) were particularly modulated by drought in plants with 'Rangpur Santa Cruz' lime rootstock, whereas root levels of prenylated coumarins were most regulated by drought in plants with the 'Sunki Maravilha' root system. Taken together, these data indicate that the impacts of water deficit restriction on growth, hormonal balance and flavonoid profiles significantly varies depending on the canopy/rootstock combinations.

Combining ability, heritability and genotypic relations of different physiological traits in cacao hybrids.

Selecting parents and evaluating progenies is a very important step in breeding programs and involves approaches such as understanding the initial stages of growth and characterizing the variability among genotypes for different parameters, such as physiological, growth, biomass partitioning and nutrient translocation to the aerial part. In these cases, facilitating tools can be used to understand the involved gene dynamics, such as diallel crosses and genetic and phenotypic correlations. Our main hypothesis is that the contrasting phenotypes of these parental genotypes of cocoa used are due to genetic factors, and progenies derived from crosses of these parental genotypes are useful for breeding programs related to plant architecture, physiological parameters and translocation of mineral nutrients. We aimed to evaluate the combining abilities in progenies of cacao (Theobroma cacao L) originating from contrasting parents for canopy vigor. Emphasis was given to the evaluation of morphological and physiological parameters and the phenotypic and genotypic correlations to understand the dynamics of the action of the genes involved, as well as in expression profile from genes of gibberellins biosynthesis pathway in the parents. Fifteen F1 progenies were obtained from crosses of six clones (IMC 67, P4B, PUCALA, SCA 6, SCA 24 and SJ 02) that were evaluated in a randomized complete block design with four replicates of 12 plants per progeny, in a balanced half table diallel scheme. It is possible to identify and select plants and progenies of low, medium and high height, as there is expressive genetic variability for the evaluated parameters, some of these on higher additive effects, others on larger nonadditive effects and others under a balance of these effects. Most physiological parameters evaluated show that for selection of plants with the desired performance, no complex breeding methods would be necessary due to the high and medium heritability observed. Strong genetic components were observed from many of the correlations, which indicate the possibility to formulate selection indices for multi-traits, such as dwarfism or semidwarfism, tolerance to increase of leaf sodium concentrations and maintenance of the photosynthetic apparatus integrity under these conditions. Additionally, plants with higher carbon fixation, better water use, higher carboxylation efficiency and greater magnesium accumulation in leaves can be selected.

Late Embryogenesis Abundant (LEA) Constitutes a Large and Diverse Family of Proteins Involved in Development and Abiotic Stress Responses in Sweet Orange (Citrus sinensis L. Osb.).

Late Embryogenesis Abundant (LEA) proteins are an ubiquitous group of polypeptides that were first described to accumulate during plant seed dehydration, at the later stages of embryogenesis. Since then they have also been recorded in vegetative plant tissues experiencing water limitation and in anhydrobiotic bacteria and invertebrates and, thereby, correlated with the acquisition of desiccation tolerance. This study provides the first comprehensive study about the LEA gene family in sweet orange (Citrus sinensis L. Osb.), the most important and widely grown fruit crop around the world. A surprisingly high number (72) of genes encoding C. sinensis LEAs (CsLEAs) were identified and classified into seven groups (LEA_1, LEA_2, LEA_3 and LEA_4, LEA_5, DEHYDRIN and SMP) based on their predicted amino acid sequences and also on their phylogenetic relationships with the complete set of Arabidopsis thaliana LEA proteins (AtLEAs). Approximately 60% of the CsLEAs identified in this study belongs to the unusual LEA_2 group of more hydrophobic LEA proteins, while the other LEA groups contained a relatively small number of members typically hydrophilic. A correlation between gene structure and motif composition was observed within each LEA group. Investigation of their chromosomal localizations revealed that the CsLEAs were non-randomly distributed across all nine chromosomes and that 33% of all CsLEAs are segmentally or tandemly duplicated genes. Analysis of the upstream sequences required for transcription revealed the presence of various stress-responsive cis-acting regulatory elements in the promoter regions of CsLEAs, including ABRE, DRE/CRT, MYBS and LTRE. Expression analysis using both RNA-seq data and quantitative real-time RT-PCR (qPCR) revealed that the CsLEA genes are widely expressed in various tissues, and that many genes containing the ABRE promoter sequence are induced by drought, salt and PEG. These results provide a useful reference for further exploration of the CsLEAs functions and applications on crop improvement.

Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (Citrus sinensis L. Osb.).

The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast (TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb.), the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs) were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs) based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety "Westin".

Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.

The activity of TcCYS4 modified by variations in pH and temperature can affect symptoms of witches' broom disease of cocoa, caused by the fungus Moniliophthora perniciosa.

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches' broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches' broom disease in cocoa.

Increased resistance against citrus canker mediated by a citrus mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAPK) play crucial roles in plant immunity. We previously identified a citrus MAPK (CsMAPK1) as a differentially expressed protein in response to infection by Xanthomonas aurantifolii, a bacterium that causes citrus canker in Mexican lime but a hypersensitive reaction in sweet oranges. Here, we confirm that, in sweet orange, CsMAPK1 is rapidly and preferentially induced by X. aurantifolii relative to Xanthomonas citri. To investigate the role of CsMAPK1 in citrus canker resistance, we expressed CsMAPK1 in citrus plants under the control of the PR5 gene promoter, which is induced by Xanthomonas infection and wounding. Increased expression of CsMAPK1 correlated with a reduction in canker symptoms and a decrease in bacterial growth. Canker lesions in plants with higher CsMAPK1 levels were smaller and showed fewer signs of epidermal rupture. Transgenic plants also revealed higher transcript levels of defense-related genes and a significant accumulation of hydrogen peroxide in response to wounding or X. citri infection. Accordingly, nontransgenic sweet orange leaves accumulate both CsMAPK1 and hydrogen peroxide in response to X. aurantifolii but not X. citri infection. These data, thus, indicate that CsMAPK1 functions in the citrus canker defense response by inducing defense gene expression and reactive oxygen species accumulation during infection.

High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration.

An improved method for the Agrobacterium infiltration of epicotyl segments of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and 'Swingle' citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method ('dipping' method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for 'Pineapple' sweet orange and 11.2% for 'Swingle' citrumelo) are the highest ones reported for both cultivars.