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This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.
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Carica , Genotipo , Técnicas de Embriogénesis Somática de Plantas , Carica/genética , Carica/embriología , Técnicas de Embriogénesis Somática de Plantas/métodos , Transformación Genética , Plantas Modificadas Genéticamente/genética , Regeneración/genética , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
A genome-wide analysis for two families of key transcription factors (TF; WRKY and NAC) involved in drought response revealed 46 WRKY and 66 NAC members of the Carica papaya genome. A phylogenetic analysis grouped the CpWRKY proteins into three groups (I, II a, b, c, d, e and III), while the CpNAC proteins were clustered into 15 groups. The conserved domains, chromosomal localization and promoter cis-acting elements were also analyzed. In addition, from a previous transcriptome study of two contrasting genotypes in response to 14 days of water deficit stress (WDS), we found that 29 of the 46 CpWRKYs genes and 25 of the 66 CpNACs genes were differentially expressed in response to the WDS. In the present paper, the native wild genotype (WG) (collected in its center of origin) consistently showed a higher expression (transcripts per million; TPM and fold change; FC) than the commercial genotype (CG) in almost all the members of the CpWRKY and CpNAC gene families. To corroborate this, we selected CpWRKY50 and CpNAC83.1 for further evaluation by RT-qPCR. Consistently, the WG showed higher relative expression levels (REL) after 14 days of WDS than the CG, in both the leaves and roots. The results suggest that the CpWRKY and CpNAC TF families are important for drought tolerance in this species. The results may also suggest that, during the domestication process, the ability of the native (wild) C. papaya genotypes to respond to drought (including the overexpression of the CpWRKY and CpNAC genes) was somehow reduced in the current commercial genotypes.
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Huanglongbing (HLB) is a vascular disease of Citrus caused by three species of the α-proteobacteria "Candidatus Liberibacter", with "Candidatus Liberibacter asiaticus" (CLas) being the most widespread and the one causing significant economic losses in citrus-producing regions worldwide. However, Persian lime (Citrus latifolia Tanaka) has shown tolerance to the disease. To understand the molecular mechanisms of this tolerance, transcriptomic analysis of HLB was performed using asymptomatic and symptomatic leaves. RNA-Seq analysis revealed 652 differentially expressed genes (DEGs) in response to CLas infection, of which 457 were upregulated and 195 were downregulated. KEGG analysis revealed that after CLas infection, some DEGs were present in the plant-pathogen interaction and in the starch and sucrose metabolism pathways. DEGs present in the plant-pathogen interaction pathway suggests that tolerance against HLB in Persian lime could be mediated, at least partly, by the ClRSP2 and ClHSP90 genes. Previous reports documented that RSP2 and HSP90 showed low expression in susceptible citrus genotypes. Regarding the starch and sucrose metabolism pathways, some genes were identified as being related to the imbalance of starch accumulation. On the other hand, eight biotic stress-related genes were selected for further RT-qPCR analysis to validate our results. RT-qPCR results confirmed that symptomatic HLB leaves had high relative expression levels of the ClPR1, ClNFP, ClDR27, and ClSRK genes, whereas the ClHSL1, ClRPP13, ClPDR1, and ClNAC genes were expressed at lower levels than those from HLB asymptomatic leaves. Taken together, the present transcriptomic analysis contributes to the understanding of the CLas-Persian lime interaction in its natural environment and may set the basis for developing strategies for the integrated management of this important Citrus disease through the identification of blanks for genetic improvement.
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Citrus , Rhizobiaceae , Citrus/genética , Citrus/microbiología , Transcriptoma , Perfilación de la Expresión Génica , Liberibacter , Sacarosa , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Rhizobiaceae/fisiologíaRESUMEN
Resilience of growing in arid and semiarid regions and a high capacity of accumulating sugar-rich biomass with low lignin percentages have placed Agave species as an emerging bioenergy crop. Although transcriptome sequencing of fiber-producing agave species has been explored, molecular bases that control wall cell biogenesis and metabolism in agave species are still poorly understood. Here, through RNAseq data mining, we reconstructed the cellulose biosynthesis pathway and the phenylpropanoid route producing lignin monomers in A. tequilana, and evaluated their expression patterns in silico and experimentally. Most of the orthologs retrieved showed differential expression levels when they were analyzed in different tissues with contrasting cellulose and lignin accumulation. Phylogenetic and structural motif analyses of putative CESA and CAD proteins allowed to identify those potentially involved with secondary cell wall formation. RT-qPCR assays revealed enhanced expression levels of AtqCAD5 and AtqCESA7 in parenchyma cells associated with extraxylary fibers, suggesting a mechanism of formation of sclerenchyma fibers in Agave similar to that reported for xylem cells in model eudicots. Overall, our results provide a framework for understanding molecular bases underlying cell wall biogenesis in Agave species studying mechanisms involving in leaf fiber development in monocots.
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Among the most serious problems in papaya production are the viruses associated with papaya ringspot and papaya sticky disease (PSD). PSD concerns producers worldwide because its symptoms are extremely aggressive and appear only after flowering. As no resistant cultivar is available, several disease management strategies have been used in affected countries, such as the use of healthy seeds, exclusion of the pathogen, and roguing. In the 1990s, a dsRNA virus, papaya meleira virus (PMeV), was identified in Brazil as the causal agent of PSD. However, in 2016 a second virus, papaya meleira virus 2 (PMeV2), with an ssRNA genome, was also identified in PSD plants. Only PMeV is detected in asymptomatic plants, whereas all symptomatic plants contain both viral RNAs separately packaged in particles formed by the PMeV capsid protein. PSD also affects papaya plants in Mexico, Ecuador, and Australia. PMeV2-like viruses have been identified in the affected plants, but the partner virus(es) in these countries are still unknown. In Brazil, PMeV and PMeV2 reside in laticifers that promote spontaneous latex exudation, resulting in the affected papaya fruit's sticky appearance. Genes modulated in plants affected by PSD include those involved in reactive oxygen species and salicylic acid signaling, proteasomal degradation, and photosynthesis, which are key plant defenses against PMeV complex infection. However, the complete activation of the defense response is impaired by the expression of negative effectors modulated by the virus. This review presents a summary of the current knowledge of the Carica papaya-PMeV complex interaction and management strategies.
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Carica , Virus de Plantas , Australia , Brasil , Ecuador , México , Virus de Plantas/genéticaRESUMEN
We investigated the transcriptional regulation of six genes involved in carotenoid biosynthesis, together with the carotenoid accumulation during postharvest ripening of three different papaya genotypes of contrasting pulp color. Red-pulp genotype (RPG) showed the lowest content of yellow pigments (YP), such as ß-cryptoxanthin, zeaxanthin, and violaxanthin, together with the lowest relative expression levels (REL) of CpLCY-ß2 and CpCHX-ß genes. On the contrary, the yellow-pulp genotype (YPG) showed the highest content of YP and the highest REL of CpLCY-ß2 and CpCHX-ß genes. Interestingly, the orange-pulp genotype (OPG) showed intermediate content of YP and intermediate REL of CpLCY-ß2 and CpCHX-ß genes. The highest content of ß-carotene shown by OPG despite having an intermediate REL of the CpLCY-ß2 genes, suggests a post-transcriptional regulation. Thus, the transcriptional level of the genes, directing the carotenoid biosynthesis pathway, can partially explain the accumulation of carotenoids during the postharvest ripening in C. papaya genotypes of contrasting pulp color.
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Carica/genética , Carica/metabolismo , Citrus sinensis/genética , Citrus sinensis/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , beta Caroteno/genética , beta Caroteno/metabolismo , beta-Criptoxantina/genética , beta-Criptoxantina/metabolismo , Carica/clasificación , Carotenoides/análisis , Carotenoides/genética , Carotenoides/metabolismo , Citrus sinensis/clasificación , Color , Frutas/química , Frutas/genética , Regulación de la Expresión Génica de las Plantas/genética , Genotipo , Licopeno , Pigmentación , Proteínas de Plantas/genética , ARN de Planta/aislamiento & purificación , Xantófilas/genética , Xantófilas/metabolismo , Zeaxantinas/genética , Zeaxantinas/metabolismo , beta Caroteno/análisisRESUMEN
KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.
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Agave/genética , Agave/fisiología , Metilación de ADN/genética , Técnicas de Cultivo de Tejidos/métodos , Agave/anatomía & histología , Agave/ultraestructura , Carotenoides/metabolismo , Clorofila/metabolismo , Segregación Cromosómica , Células Clonales , Malatos/metabolismo , Fenotipo , Fotoperiodo , Estomas de Plantas/anatomía & histología , Estomas de Plantas/metabolismo , Estomas de Plantas/ultraestructuraRESUMEN
Salvinia minima Baker accumulates a fair amount of lead in its tissues; however, no studies have investigated the effect of lead on the physiological processes that affect photosynthesis in this species. The objective of the present study was to assess whether the high amounts of lead accumulated by S. minima can affect its photosynthetic apparatus. The physiological changes in the roots and leaves in response to lead accumulation were analyzed. An exposure to 40 µM Pb(NO3)2 for 24 h (first stage) was sufficient to reduce the photosynthetic rate (Pn) by 44%. This reduction in Pn was apparently the result of processes at various levels, including damage to the cell membranes (mainly in roots). Interestingly, although the plants were transferred to fresh medium without lead for an additional 24 h (second stage), Pn not only remained low, but was reduced even further, which was apparently related to stomatal closure, and may have led to reduced CO2 availability. Therefore, it can be concluded that lead exposure first decreases the photosynthetic rate by damaging the root membrane and then induces stomatal closure, resulting in decreased CO2 availability.
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Plomo/metabolismo , Plomo/toxicidad , Fotosíntesis/efectos de los fármacos , Estomas de Plantas/efectos de los fármacos , Tracheophyta/efectos de los fármacos , Tracheophyta/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hojas de la Planta/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
An experiment was designed to assess the capacity of Salvinia minima Baker to uptake and accumulate nickel in its tissues and to evaluate whether or not this uptake can affect its physiology. Our results suggest that S. minima plants are able to take up high amounts of nickel in its tissues, particularly in roots. In fact, our results support the idea that S. minima might be considered a hyper-accumulator of nickel, as it is able to accumulate 16.3 mg g(-1) (whole plant DW basis). Our results also showed a two-steps uptake pattern of nickel, with a fast uptake of nickel at the first 6 to 12h of being expose to the metal, followed by a slow take up phase until the end of the experiment at 144 h. S. minima thus, may be considered as a fern useful in the phytoremediation of residual water bodies contaminated with this metal. Also from our results, S. minima can tolerate fair concentrations of the metal; however, at concentrations higher than 80 µM Ni (1.5 mg g(-1) internal nickel concentration), its physiological performance can be affected. For instance, the integrity of cell membranes was affected as the metal concentration and exposure time increased. The accumulation of high concentrations of internal nickel did also affect photosynthesis, the efficiency of PSII, and the concentration of photosynthetic pigments, although at a lower extent.
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Helechos/efectos de los fármacos , Helechos/metabolismo , Níquel/metabolismo , Níquel/farmacología , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/farmacología , Biodegradación Ambiental , Clorofila/metabolismo , Relación Dosis-Respuesta a Droga , Luz , Metales/metabolismo , Metales/farmacología , Níquel/administración & dosificación , Fotosíntesis/efectos de los fármacos , Raíces de Plantas/metabolismoRESUMEN
The effects of copper toxicity on the photosynthetic activities of Avicennia germinans was investigated using two CuSO4 concentrations (0.062 and 0.33 M) added in Hoagland's solution in an aerated hydroponic system. Photosynthesis and chlorophyll fluorescence were measured after 30 h of copper stress. Results obtained in this study show that increasing levels of Cu(+2) of 0.062 and 0.33 M Cu(+2) resulted in a general reduction of the stomatal conductance (28 and 18%, respectively) and 100% of inhibition of net photosynthesis. Additionally, at these concentrations of Cu(+2), reductions of chlorophyll fluorescence parameters were also observed. These changes suggested that the photosynthetic apparatus of Avicennia germinans was the primary target of the Cu(+2) action. It is concluded that Cu(+2) ions causes a drastic decline in photosynthetic gas exchange and Chlorophyll fluorescence parameters in A. germinans leaves.
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Avicennia/efectos de los fármacos , Clorofila/metabolismo , Cobre/toxicidad , Fotosíntesis/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , FluorescenciaRESUMEN
The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.
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Secuencia de Aminoácidos , Arabidopsis/genética , Carica/genética , Expresión Génica , Genes de Plantas , Proteínas de Plantas/genética , Factores de Transcripción/genética , Arabidopsis/metabolismo , Carica/metabolismo , Clonación Molecular , Flores/metabolismo , Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/genética , Inmunidad de la Planta/genética , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Salicílico/farmacología , Alineación de Secuencia , Homología de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Fruit ripening in papaya Carica papaya cultivars varies widely in terms of skin color changes, pulp firmness and shelf life. Most papaya ripening studies have been done using Solo varieties. No objective maturity indices have been developed for Maradol papaya, and studies describing color changes during fruit ripening only cover the initial and final maturity stages. Changes in the main quality attributes of Maradol papaya were documented during the ripening process to identify maturity stages and define objective maturity indices to be applied as harvest indices and quality standards. Six maturity stages were identified and quality attribute value ranges proposed as quality standards. Skin color can be considered an appropriate maturity index, b* values are good indicators for early maturity stages, while a* value are better for late stages.
Las variedades de papaya muestran una amplia variación en la maduración del fruto en términos de ablandamiento, cambios del color de la cáscara y vida de anaquel. La mayoría de los estudios sobre maduración de papaya se han reportado en variedades Solo. No se han desarrollado índices de maduración objetivos para la variedad Maradol, los estudios que describen el cambio de color en la maduración del fruto cubren sólo a los estados de maduración inicial y final. Se evaluaron los cambios en los principales atributos de calidad de papaya Maradol durante la maduración para identificar estados de maduración y definir índices de maduración objetivos que puedan ser sugeridos como índices de cosecha y estándares de calidad. El color de la cáscara puede ser considerado como un índice de madurez apropiado, los valores de b* son buenos indicadores para los estados tempranos de madurez, mientras que los valores de a* son mejores para los últimos estados.
As variedades de papaya mostram uma ampla variação no amadurecimento do fruto em termos de amolecimento, mudança da cor da casca e vida nas gôndolas. A maioria dos estudos sobre amadurecimento de papaya tem sido relatado na variedade Solo. Não tem se desenvolvido índices de amadurecimento objetivos para a variedade Maradol, os estudos que descrevem a mudança de cor no amadurecimento do fruto abrangem somente aos estados de amadurecimento inicial e final. Avaliaram-se as mudanças nos principais atributos de qualidade de papaya Maradol durante o amadurecimento para identificar estados de amadurecimento e definir índices de amadurecimento objetivos que possam ser sugeridos como índices de colheita e estándares de qualidade. A cor da casca pode ser considerada como um índice de maturação apropriado, os valores de b* são bons indicadores para os estágios recentes de maturação, enquanto que os valores de a* são melhores para os últimos estágios.
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The toxic effects of cadmium on the photosynthetic apparatus of Avicennia germinans were evaluated by means of the chlorophyll fluorescence transient O-J-I-P. The chlorophyll fluorescence transients were recorded in vivo with high time resolution and analyzed according to the OJIP-test that can quantify the performance of photosystem II. Cadmium-treated plants showed a decrease in yield for primary photochemistry, TR0/ABS. The performance index of photosystem II (PSII), PI(ABS), decreased due to cadmium treatment. This performance index is the combination of the indexes of three independent parameters: (1) total number of active reaction centers per absorption (RC/ABS), (2) yield of primary photochemistry (TR0/ABS), and (3) efficiency with which a trapped exciton can move an electron into the electron transport chain (ET0/TR0). Additionally, the F0/Fv registered the highest sensitivity to the metal, thus indicating that the water-splitting apparatus of the oxidizing side of PSII is the primary site of action of cadmium. In summary, cadmium affects several targets of photosystem II. More specifically the main targets of cadmium, according to the OJIP-test, can be listed as a decrease in the number of active reaction centers and damage to the activity of the water-splitting complex.