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Maduración, perdurabilidad y conservación en árbol de una variedad de clementina en marzo

  • Iglesias, Domingo J.
  • Usach, Antonio
  • Boix, A.
  • Almernar, A.
  • Blázquez, E.
  • López, I.
  • Sanchis, I.
  • Ibanez, Victoria
  • Ortega, A. H.
  • Buj, A.
  • Brumós, J.
  • Cercós, Manuel
  • Merelo, Paz
  • Tadeo, Francisco R.
  • Terol, Javier
  • Bono, Rafael
  • Talón, Manuel
Proyecto:


Proteins Related to Stress and Redox State of 'Moncada' Mandarin Leaves with Contrasting Fruit Load, Acta Horticulturae

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • Agusti, Manuel
  • Tarraga, Susana
  • Iglesias, Domingo J.
  • Primo-Millo, Eduardo
  • González-Mas, María C.
A proteomic study was used to know more about the Citrus redox state and stress response related to fruit load. To this end, we researched for differences in the accumulation of proteins related to these processes between on-crop and off-crop 'Moncada' mandarin leaves. Samples were collected in November, were immediately stored frozen at -80 degrees C, and proteins were extracted in less than two weeks. From 2D DIGE gel of these extracts, 12 related spots were isolated: only 1 showed increased accumulation in the off-crop samples compared to on-crop samples, while the other 11 showed increased accumulation in the on-crop samples against off-crop samples. These spots were identified by MALDI-MS or LC-MS-MS. The only protein with increased expression in the off-crop samples was putative monocopper oxidase precursor, while the up-expressed proteins in on-crop leaves were catalase, monodehydroascorbate reductase, Fe-superoxide dismutase, glutathione peroxidase, and three proteins related to stress response. Thus according to these results, generally, in the off-crop samples, proteins with oxidoreductase activity are down-regulated, while in the on-crop samples the proteins related to stress response and to oxidoreductase activity are up-regulated. Therefore, the redox state is different for off-crop and for on-crop Citrus leaves.
Proyecto:


Proteomic analysis of "Moncada" mandarin leaves with contrasting fruit load

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • Agusti, Manuel
  • Tarraga, Susana
  • Iglesias, Domingo J.
  • Primo-Millo, Eduardo
  • González-Mas, María C.
A proteomic approach was used to know more about the molecular mechanism related to Citrus alternate bearing. To this end, we researched protein expression differences between on-crop and off-crop "Moncada" [Clementine 'Oroval' (Citrus clementina Hort ex Tanaka) x 'Kara' mandarin (Citrus unshiu Marc. x Citrus nobilis Lou.)] mandarin leaves. This variety usually shows a remarkable behaviour in alternate production. Samples were collected in the period during which the fruit affect flowering induction. From 2D DIGE gel, 110 spots were isolated: 43 showed increased expression in the off-crop samples compared to on-crop samples, while 67 showed increased expression in the on-crop samples against off-crop samples. These spots were identified by MALDI-MS or LC-MS-MS. According to the up-expressed proteins in off-crop leaves such as proteins related to nutrient reservoir activity or to the pentose phosphate pathway, the primary metabolism was more active in off-crop trees than in on-crop trees. In contrast, the proteins up-expressed in on-crop samples such as catalase were related to the oxidoreductase activity and, therefore, the redox state seemed different for off-crop and for on-crop leaves. Other proteins with unknown functions were isolated, which could be also related to the alternate bearing and to the flowering induction. (c) 2012 Elsevier Masson SAS. All rights reserved.
Proyecto:


Gibberellic Acid Reduces Flowering Intensity in Sweet Orange [Citrus sinensis (L.) Osbeck] by Repressing CiFT Gene Expression

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • González-Mas, María C.
  • Iglesias, Domingo J.
  • Primo-Millo, Eduardo
  • Agusti, Manuel
In Citrus, gibberellic acid (GA(3)) applied at the floral bud inductive period significantly reduces flowering intensity. This effect is being used to improve the fruit set of parthenocarpic cultivars that tend to flower profusely. However, the molecular mechanisms involved in the process remain unclear. To contribute to the knowledge of this phenomenon, adult trees of 'Salustiana' sweet orange were sprayed at the floral bud inductive period with 40 mg L-1 of GA(3) and the expression pattern of flowering genes was examined up to the onset of bud sprouting. Trees sprayed with paclobutrazol (PBZ, 2,000 mg L-1), a gibberellin biosynthesis inhibitor, were used to confirm the effects, and untreated trees served as control. Bud sprouting, flowering intensity, and developed shoots were evaluated in the spring. GA(3) significantly reduced the number of flowers per 100 nodes by 72% compared to the control, whereas PBZ increased the number by 123%. Data of the expression pattern of flowering genes in leaves of GA(3)-treated trees revealed that this plant growth regulator inhibited flowering by repressing relative expression of the homolog of FLOWERING LOCUS T, CiFT, whereas PBZ increased flowering by boosting its expression. The activity of the homologs TERMINAL FLOWER 1, FLOWERING LOCUS C, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, and APETALA1 was not affected by the treatments. The number of flowers per inflorescence, in both leafy and leafless inflorescences, was not altered by GA(3) but increased with PBZ; the latter paralleled LEAFY relative expression. These results suggest that GA(3) inhibits flowering in Citrus by repressing CiFT expression in leaves.
Proyecto:


Fruit load modulates flowering-related gene expression in buds of alternate-bearing 'Moncada' mandarin

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • González-Mas, María C.
  • Primo-Millo, Eduardo
  • Agusti, Manuel
  • Iglesias, Domingo J.
Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering. First defruiting experiments were performed to manipulate blossoming intensity in oMoncada' mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages. Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 ) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage. These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.
Proyecto:


Fruit regulates seasonal expression of flowering genes in alternate-bearing 'Moncada' mandarin

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • González-Mas, María C.
  • Primo-Millo, Eduardo
  • Agusti, Manuel
  • Iglesias, Domingo J.
Background and Aims The presence of fruit has been widely reported to act as an inhibitor of flowering in fruit trees. This study is an investigation into the effect of fruit load on flowering of 'Moncada' mandarin and on the expression of putative orthologues of genes involved in flowering pathways to provide insight into the molecular mechanisms underlying alternate bearing in citrus. Methods The relationship between fruit load and flowering intensity was examined first. Defruiting experiments were further conducted to demonstrate the causal effect of fruit removal upon flowering. Finally, the activity of flowering-related genes was investigated to determine the extent to which their seasonal expression is affected by fruit yield. Key Results First observations and defruiting experiments indicated a significant inverse relationship between preceding fruit load and flowering intensity. Moreover, data indicated that when fruit remained on the tree from November onwards, a dramatic inhibition of flowering occurred the following spring. The study of the expression pattern of flowering-genes of on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (FT), SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1) and LEAFY (LFY) were negatively affected by fruit load. Thus, CiFT expression showed a progressive increase in leaves from off trees through the study period, the highest differences found from December onwards (10-fold). Whereas differences in the relative expression of SOC1 only reached significance from September to mid-December, CsAP1 expression was constantly higher in those trees through the whole study period. Significant variations in CsLFY expression only were found in late February (close to 20%). On the other hand, the expression of the homologues of TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS C (FLC) did not appear to be related to fruit load. Conclusions These results suggest for the first time that fruit inhibits flowering by repressing CiFT and SOC1 expression in leaves of alternate-bearing citrus. Fruit also reduces CsAP1 expression in leaves, and the significant increase in leaf CsLFY expression from off trees in late February was associated with the onset of floral differentiation.
Proyecto:


Use or abuse of bioinformatic tools: a response to Samach

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • González-Mas, María C.
  • Primo-Millo, Eduardo
  • Agusti, Manuel
  • Iglesias, Domingo J.
In a recent paper, we described for the first time the effects of fruit on the expression of putative homologues of genes involved in flowering pathways. It was our aim to provide insight into the molecular mechanisms underlying alternate bearing in citrus. However, a bioinformatics-based critique of our and other related papers has been given by Samach in the preceding Viewpoint article in this issue of Annals of Botany. The use of certain bioinformatic tools in a context of structural rather than functional genomics can cast doubts about the veracity of a large amount of data published in recent years. In this response, the contentions raised by Samach are analysed, and rebuttals of his criticisms are presented.
Proyecto:


Proteomic study of 'Moncada' mandarin buds from on- versus off-crop trees

  • Munoz-Fambuena, Natalia
  • Mesejo, Carlos
  • Reig, Carmina
  • Agusti, Manuel
  • Tarraga, Susana
  • Lison, Purificacion
  • Iglesias, Domingo J.
  • Primo-Millo, Eduardo
  • González-Mas, María C.
A proteomic analysis of buds from mandarin trees with contrasting fruit load (on- and off-crop trees) was carried out during the onset of low-temperature induction. The aim of the study was to find out more about the molecular mechanism relating to alternate bearing in Citrus and its relationship with flowering. The 'Moncada' variety (Clementine 'Oroval' x 'Kara' mandarin), displaying remarkable behaviour in alternate production, was used in this study. From 2D DICE gel, 192 spots were isolated: 97 showed increased expression in the off-crop buds as compared to the on-crop buds, while 95 exhibited enhanced expression in the on-crop buds versus the off-crop buds. These spots were identified by MALDI-MS or LC -MS-MS. The largest groups of proteins up-expressed in the off-crop buds were the proteins involved in carbohydrate and amino acid metabolism, and the proteins expressed in response to stimuli such as reactive oxygen species. The largest groups of proteins up-expressed in the on-crop buds were related to primary metabolism, oxidative stress and defence responses. Depending on their function, some of these proteins can stimulate the flowering, such as fructose-bisphosphate aldolase or leucine-rich repeat transmembrane protein kinase, while others can inhibit it, such as cytochrome c oxidase subunit II. Twenty-two other proteins with unknown functions were up-expressed in the on- or off-crop buds. (C) 2013 Elsevier Masson SAS. All rights reserved.
Proyecto:


Hydraulic and Chemical Responses of Citrus Seedlings to Drought and Osmotic Stress

  • Forner-Giner, María A.
  • Rodriguez-Gamir, Juan
  • Primo-Millo, Eduardo
  • Iglesias, Domingo J.
In this work we investigated the function of abscisic acid (ABA) as a long-distance chemical signal communicating water shortage from the root to the shoot in citrus plants. Experiments indicated that stomatal conductance, transpiration rates, and leaf water potential decline progressively with drought. ABA content in roots, leaves, and xylem sap was also increased by the drought stress treatment three- to sevenfold. The addition of norflurazon, an inhibitor of ABA biosynthesis, significantly decreased the intensity of the responses and reduced ABA content in roots and xylem fluid, but not in leaves. Polyethylene glycol (PEG)-induced osmotic stress caused similar effects and, in general, was counteracted only by norflurazon at the lowest concentration (10%). Partial defoliation was able to diminish only leaf ABA content (22.5%) at the highest PEG concentration (30%), probably through a reduction of the active sites of biosynthesis. At least under moderate drought (3-6 days without irrigation), mechanisms other than leaf ABA concentration were required to explain stomatal closure in response to limited soil water supply. Measurements of xylem sap pH revealed a progressive alkalinization through the drought condition (6.4 vs. 7.1), that was not counteracted with the addition of norflurazon. Moreover, in vitro treatment of detached leaves with buffers iso-osmotically adjusted at pH 7.1 significantly decreased stomatal conductance (more than 30%) as much as 70% when supplemented with ABA. Taken together, our results suggest that increased pH generated in drought-stressed roots is transmitted by the xylem sap to the leaves, triggering reductions in shoot water loss. The parallel rise in ABA concentration may act synergistically with pH alkalinization in xylem sap, with an initial response generated from the roots and further promotion by the stressed leaves.
Proyecto:


Hydrogel substrate amendment alleviates drought effects on young citrus plants

  • Arbona, Vicent
  • Iglesias, Domingo J.
  • Jaques, Josep A.
  • Primo-Millo, Eduardo
  • Talón, Manuel
  • Gomez-Cadenas, A.
Water deficits affect citrus physiology, yield, fruit size and quality. Citrus can respond to drought stress conditions through endogenous hormonal regulation of water status and leaf abscission. In this work, we assayed the efficiency of an amendment to soilless media in delaying the drought stress effect in young citrus seedlings and trees. Substrate amendment promoted plant survival of citrus seedlings subjected to several cycles of drought stress and rehydration. In budded trees, the amendment increased substrate water content, leaf water potential, leaf number, root biomass, CO2 assimilation and stomatal conductance over that of control plants growing in non-amended substrates. We conclude that the substrate amendment reduced the damaging effects of drought stress in citrus plants. The longer survival of seedlings in the amended treatment together with the reduction in leaf abscission and the improvement of physiological parameters, can account for a higher vigour of citrus grown under water stress conditions.
Proyecto:


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