An in silico study of the citrus dioxygenases CCD4 family substrates.

The coloration of Citrus fruits is related with the concentration of carotenoids, isoprenoid pigments of 40 carbon atoms (C40). Rodrigo et al. and Ma et al. reported a CCD4-type citrus dioxygenase responsible for the generation of C30 apocarotenoids providing a reddish-orange pigmentation to the peel of many mandarins and oranges. Among them, CCD4b was the first case described of a dioxygenase that cleaves carotenoids C40 in the double bond 7', 8' or 7, 8, generating ß-citraurin or 8-ß-apocarotenal. Here we report the three-dimensional structures of CCD4a and CCD4b, modeled by sequence homology (2BIW) and validated by molecular dynamics (MD). Docking calculations were performed in CCD4a and CCD4b structures with thousands of rotated initial carotenoid conformations and all the possible poses in the active site were found. The interaction energy was measured by means of ASE scoring, Amber99 refinement and London ΔG rescoring. For the case of CCD4a model, the results showed London ΔG score of -19, -17 and -15 kcal/mol for zeaxanthin, ß-cryptoxanthin and ß-carotene, respectively. The same sequence in the estimated interaction strength for the three ligands was obtained using MD. The interaction energy of CCD4b indicated that, in agreement with experimental data, zeaxanthin and ß-cryptoxanthin could be cleaved by the enzyme, ß- and α-carotene have chances to be oxidized and lycopene has not good interaction energy to be predicted as substrate. These findings will be discussed considering the potential in vivo substrates and products, and the physiological role in Citrus fruits. Communicated by Ramaswamy H. Sarma.

Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation.

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus.

Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb.

The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs.

Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco.

9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

Effects of grafting on key photosynthetic enzymes and gene expression in the citrus cultivar Huangguogan.

Grafting influences scion photosynthetic capacity and fruit quality. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which strongly affects photosynthetic rate, and Rubisco activase (RCA), which regulates Rubisco activity, are two key photosynthetic enzymes. However, little information is available regarding the effect of grafting on the concentration and expression of Rubisco and RCA in the citrus cultivar Huangguogan. The objective of this study was to investigate the effect of grafting Huangguogan plants onto trifoliate orange, tangerine, and orange on: 1) the concentration of Rubisco and RCA; 2) the mRNA levels of rbcL, rbcS, and rca; and 3) fruit quality. Overall, the results showed that when Huangguogan plants budded on tangerine and orange, they had better fruit quality, while on trifoliate orange they had higher Rubisco concentration. Tangerine and orange are probably the most suitable rootstocks for Huangguogan plants given the environmental conditions of Sichuan Province, China.

Citrus pulp and enzyme complex for growing and finishing pigs / Polpa cítrica e complexo enzimático para suínos na fase de crescimento e terminação

The purpose is to evaluate the effects of levels of citrus pulp (CP), over the aperformance and the carcass characteristics, with or without the addition of an enzyme complex in the feed. Seventy-two pigs were used, at 25.71 } 7.51kg initial body weight (BW) and 98.81 } 10.64kg final BW. The diets were formulated for the stages: 1 - 70 to 101; 2 - 102 to 130; 3 - 131 to 143 days of age. Because there was no redistribution of the animals in blocks at the beginning of stages 2 and 3, the data were evaluated at the periods: 1 - 70 to 101; 2 - 70 to 130; and 3 - 70 to 143 days of age. The inclusion of up to 15% of CP, with or without the addition of an enzyme complex in the diets, decreased the daily feed intake in Periods 1 and 2, and improved feed:gain in Period 2; however, the animals showed adaptive responses in Period 3. In the absence of the enzyme complex, the carcass yield and the fat/meat ratio decreased linearly as the levels of CP increased. In the presence of the enzyme complex, the fat/meat ratio had a quadratic behavior, and the level of 7.78% of CP determined the lowest ratio. The inclusion of up to 15% of CP with or without the addition of the enzyme complex in diets no differences in performance; therewith, it can be a feed alternative, without effect on the carcass characteristics.

Objetivou-se avaliar os efeitos de níveis de polpa cítrica (PC), com ou sem adição de um complexo enzimático nas rações, sobre o desempenho dos suínos e as características de carcaça. Utilizou-se 72 suínos, com 25,71 ± 7,51 e 98,81 ± 10,64kg de pesos inicial e final, respectivamente. As dietas foram formuladas para as fases: 1 - 70 aos 101; 2 - 102 aos 130; 3 - 131 aos 143 dias de idade. Como não houve redistribuição dos animais nos blocos no início das fases 2 e 3, os dados foram avaliados nos períodos: 1 - 70 aos 101; 2 - 70 aos 130 e 3 - 70 aos 143 dias de idade. A inclusão de até 15% de PC, com ou sem a adição do complexo enzimático nas dietas, diminuiu o consumo diário de ração nos Períodos 1 e 2, e melhorou a conversão alimentar no Período 2, porém, os animais apresentaram respostas adaptativas no Período 3. O rendimento de carcaça e a relação gordura/carne diminuíram linearmente, na medida em que aumentaram os níveis de PC, na ausência do complexo enzimático. Na presença do produto, a relação gordura/carne apresentou comportamento quadrático, e o nível de 7,78% de PC determinou a menor relação. A inclusão de até 15% de PC com ou sem a adição do complexo enzimático nas dietas para suínos, não proporciona diferenças no desempenho e pode ser uma alternativa alimentar, sem afetar as características de carcaça.

Citrus pulp and enzyme complex for growing and finishing pigs / Polpa cítrica e complexo enzimático para suínos na fase de crescimento e terminação

The purpose is to evaluate the effects of levels of citrus pulp (CP), over the aperformance and the carcass characteristics, with or without the addition of an enzyme complex in the feed. Seventy-two pigs were used, at 25.71 } 7.51kg initial body weight (BW) and 98.81 } 10.64kg final BW. The diets were formulated for the stages: 1 - 70 to 101; 2 - 102 to 130; 3 - 131 to 143 days of age. Because there was no redistribution of the animals in blocks at the beginning of stages 2 and 3, the data were evaluated at the periods: 1 - 70 to 101; 2 - 70 to 130; and 3 - 70 to 143 days of age. The inclusion of up to 15% of CP, with or without the addition of an enzyme complex in the diets, decreased the daily feed intake in Periods 1 and 2, and improved feed:gain in Period 2; however, the animals showed adaptive responses in Period 3. In the absence of the enzyme complex, the carcass yield and the fat/meat ratio decreased linearly as the levels of CP increased. In the presence of the enzyme complex, the fat/meat ratio had a quadratic behavior, and the level of 7.78% of CP determined the lowest ratio. The inclusion of up to 15% of CP with or without the addition of the enzyme complex in diets no differences in performance; therewith, it can be a feed alternative, without effect on the carcass characteristics.(AU)

Objetivou-se avaliar os efeitos de níveis de polpa cítrica (PC), com ou sem adição de um complexo enzimático nas rações, sobre o desempenho dos suínos e as características de carcaça. Utilizou-se 72 suínos, com 25,71 ± 7,51 e 98,81 ± 10,64kg de pesos inicial e final, respectivamente. As dietas foram formuladas para as fases: 1 - 70 aos 101; 2 - 102 aos 130; 3 - 131 aos 143 dias de idade. Como não houve redistribuição dos animais nos blocos no início das fases 2 e 3, os dados foram avaliados nos períodos: 1 - 70 aos 101; 2 - 70 aos 130 e 3 - 70 aos 143 dias de idade. A inclusão de até 15% de PC, com ou sem a adição do complexo enzimático nas dietas, diminuiu o consumo diário de ração nos Períodos 1 e 2, e melhorou a conversão alimentar no Período 2, porém, os animais apresentaram respostas adaptativas no Período 3. O rendimento de carcaça e a relação gordura/carne diminuíram linearmente, na medida em que aumentaram os níveis de PC, na ausência do complexo enzimático. Na presença do produto, a relação gordura/carne apresentou comportamento quadrático, e o nível de 7,78% de PC determinou a menor relação. A inclusão de até 15% de PC com ou sem a adição do complexo enzimático nas dietas para suínos, não proporciona diferenças no desempenho e pode ser uma alternativa alimentar, sem afetar as características de carcaça.(AU)

Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit.

Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

TAL effectors target the C-terminal domain of RNA polymerase II (CTD) by inhibiting the prolyl-isomerase activity of a CTD-associated cyclophilin.

Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri.

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by the bacterial phytopathogen, Xanthomonas citri subsp. citri (Xcc). GenBank houses a large collection of Expressed Sequence Tags (ESTs) enriched with transcripts generated during the defence response against this pathogen; however, there are currently no strategies in citrus to assess the function of candidate genes. This has greatly limited research as defence signalling genes are often involved in multiple pathways. In this study, we demonstrate the efficacy of RNA interference (RNAi) as a functional genomics tool to assess the function of candidate genes involved in the defence response of Citrus limon against the citrus canker pathogen. Double-stranded RNA expression vectors, encoding hairpin RNAs for citrus host genes, were delivered to lemon leaves by transient infiltration with transformed Agrobacterium. As proof of principle, we have established silencing of citrus phytoene desaturase (PDS) and callose synthase (CalS1) genes. Phenotypic and molecular analyses showed that silencing vectors were functional not only in lemon plants but also in other species of the Rutaceae family. Using silencing of CalS1, we have demonstrated that plant cell wall-associated defence is the principal initial barrier against Xanthomonas infection in citrus plants. Additionally, we present here results that suggest that H2O2 accumulation, which is suppressed by xanthan from Xcc during pathogenesis, contributes to inhibition of xanthan-deficient Xcc mutant growth either in wild-type or CalS1-silenced plants. With this work, we have demonstrated that high-throughput reverse genetic analysis is feasible in citrus.

Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae.

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

IP3 production in the hypersensitive response of lemon seedlings against Alternaria alternata involves active protein tyrosine kinases but not a G-protein

IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR) of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK) are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC) not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.

The development of Alternaria alternata is prevented by chitinases and beta-1,3-glucanases from Citrus limon seedlings.

In addition to phytoalexin synthesis, the defense response of intact Citrus limon seedlings against Alternaria alternata involves both constitutive and induced enzyme activities such as chitinases (Ch) and beta-1,3-glucanases (Glu). A alternata conidial germination was prevented by protein extracts from inoculated lemon seedlings, but also by extracts from mock-inoculated specimens. On the other hand, degradation of mycelia was accomplished only by protein extracts from inoculated seedlings. The presence of six Ch isoenzymes and of four Glu isoenzymes was detected in protein extracts from mock-inoculated seedlings. As a result of fungal inoculation, the isoenzyme pattern of Ch and Glu changed, making possible the detection of a new Ch isoenzyme and of three new Glu. Also, some constitutive Ch and Glu increased their enzyme activity, and those Ch that increased their activity also showed a broadening of their substrate specificity. These changes were prevented by alpha-amanitin and cycloheximide, suggesting that the presence of new Ch and Glu isoenzymes was due to de novo synthesis of proteins. Results suggest that constitutive Ch and Glu could act as pre-formed defense molecules in Citrus limon preventing A. alternata germination, while those induced after fungal inoculation of lemon seedlings could act along with the former, to produce lysis of fungal mycelia, resulting in a more efficient control of A. alternata development.