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[EN] In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

[EN] In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

[EN] In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

[EN] In Citrus, certain species and/or cultivars are prone to alternate bearing, i.e., a year of heavy crop (ON year) is followed by one of low flowering and light crop (OFF year), and vice-versa. To counteract the fruit effect, we propose a guided mechanical pruning initiated in the spring of an ON year and carried out annually. Our hypothesis is that this gives rise to abundant shoots with well-developed leaves that reach the floral bud inductive period ready to receive the inductive signal and, thus, to induce the bud to bloom the following spring. The experiment was carried out during four consecutive years (2013¿16) using a commercial plantation of `Nadorcott¿ mandarin in southwest Spain. Trees were mechanically pruned: hedging (H) or topping by completely removing the upper shoots of the canopy (T) or by cutting ¿ of their length (T¿). Unpruned trees were used as controls. The experiment began in the spring of an ON-year. T and T¿ carried out in the spring restart the meristem and give rise new shoots that develop in summer and autumn. At the inductive period, the leaves on these new shoots showed a CiFT2 gene expression 4- and 3.7-fold higher than their respective old leaves. In the case of the T¿ their buds avoided the inhibitory effect of the fruit and bloomed the following spring even with the fruit in the cut-back shoot. For the two periods of ON-OFF years of our experiment, cumulative yield of the T treated trees barely increased, whereas T¿ significantly increased with respect to the unpruned trees. We conclude that cutting flowering shoots annually in half-length by means of mechanical pruning (T¿), attenuates alternate bearing behaviour in 'Nadorcott' mandarin. In this experiment, cumulative yield increased by 25% with regard to unpruned trees during the four years of the trial.

[EN] In Citrus, certain species and/or cultivars are prone to alternate bearing, i.e., a year of heavy crop (ON year) is followed by one of low flowering and light crop (OFF year), and vice-versa. To counteract the fruit effect, we propose a guided mechanical pruning initiated in the spring of an ON year and carried out annually. Our hypothesis is that this gives rise to abundant shoots with well-developed leaves that reach the floral bud inductive period ready to receive the inductive signal and, thus, to induce the bud to bloom the following spring. The experiment was carried out during four consecutive years (2013¿16) using a commercial plantation of `Nadorcott¿ mandarin in southwest Spain. Trees were mechanically pruned: hedging (H) or topping by completely removing the upper shoots of the canopy (T) or by cutting ¿ of their length (T¿). Unpruned trees were used as controls. The experiment began in the spring of an ON-year. T and T¿ carried out in the spring restart the meristem and give rise new shoots that develop in summer and autumn. At the inductive period, the leaves on these new shoots showed a CiFT2 gene expression 4- and 3.7-fold higher than their respective old leaves. In the case of the T¿ their buds avoided the inhibitory effect of the fruit and bloomed the following spring even with the fruit in the cut-back shoot. For the two periods of ON-OFF years of our experiment, cumulative yield of the T treated trees barely increased, whereas T¿ significantly increased with respect to the unpruned trees. We conclude that cutting flowering shoots annually in half-length by means of mechanical pruning (T¿), attenuates alternate bearing behaviour in 'Nadorcott' mandarin. In this experiment, cumulative yield increased by 25% with regard to unpruned trees during the four years of the trial.

[EN] In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

[EN] In Citrus, certain species and/or cultivars are prone to alternate bearing, i.e., a year of heavy crop (ON year) is followed by one of low flowering and light crop (OFF year), and vice-versa. To counteract the fruit effect, we propose a guided mechanical pruning initiated in the spring of an ON year and carried out annually. Our hypothesis is that this gives rise to abundant shoots with well-developed leaves that reach the floral bud inductive period ready to receive the inductive signal and, thus, to induce the bud to bloom the following spring. The experiment was carried out during four consecutive years (2013¿16) using a commercial plantation of `Nadorcott¿ mandarin in southwest Spain. Trees were mechanically pruned: hedging (H) or topping by completely removing the upper shoots of the canopy (T) or by cutting ¿ of their length (T¿). Unpruned trees were used as controls. The experiment began in the spring of an ON-year. T and T¿ carried out in the spring restart the meristem and give rise new shoots that develop in summer and autumn. At the inductive period, the leaves on these new shoots showed a CiFT2 gene expression 4- and 3.7-fold higher than their respective old leaves. In the case of the T¿ their buds avoided the inhibitory effect of the fruit and bloomed the following spring even with the fruit in the cut-back shoot. For the two periods of ON-OFF years of our experiment, cumulative yield of the T treated trees barely increased, whereas T¿ significantly increased with respect to the unpruned trees. We conclude that cutting flowering shoots annually in half-length by means of mechanical pruning (T¿), attenuates alternate bearing behaviour in 'Nadorcott' mandarin. In this experiment, cumulative yield increased by 25% with regard to unpruned trees during the four years of the trial.

[EN] Background and Aims In woody species, the juvenile period maintains the axillary meristems in a vegetative stage, unable to flower, for several years. However, in adult trees, some 1-year-old meristems flower whereas others remain vegetative to ensure a polycarpic growth habit. Both types of trees, therefore, have non-flowering meristems, and we hypothesize that the molecular mechanism regulating flower inhibition in juvenile trees is different from that in adult trees. Methods In adult Citrus trees, the main endogenous factor inhibiting flower induction is the growing fruit. Thus, we studied the expression of the main flowering time, identity and patterning genes of trees with heavy fruit load (not-flowering adult trees) compared to that of 6-month-old trees (not-flowering juvenile trees). Adult trees without fruits (flowering trees) were used as a control. Second, we studied the expression of the same genes in the meristems of 6-month, and 1-, 3-, 5-and 7-year-old juvenile trees compared to 10-year-old flowering trees. Key Results The axillary meristems of juvenile trees are unable to transcribe flowering time and patterning genes during the period of induction, although they are able to transcribe the FLOWERING LOCUS T citrus orthologue (CiFT2) in leaves. By contrast, meristems of not-flowering adult trees are able to transcribe the flowering network genes but fail to achieve the transcription threshold required to flower, due to CiFT2 repression by the fruit. Juvenile meristems progressively achieve gene expression, with age-dependent differences from 6 months to 7 years, FD-like and CsLFY being the last genes to be expressed. Conclusions During the juvenile period the mechanism inhibiting flowering is determined in the immature bud, so that it progressively acquires flowering ability at the gene expression level of the flowering time programme, whereas in the adult tree it is determined in the leaf, where repression of CiFT2 gene expression occurs.

[EN] Background and Aims In woody species, the juvenile period maintains the axillary meristems in a vegetative stage, unable to flower, for several years. However, in adult trees, some 1-year-old meristems flower whereas others remain vegetative to ensure a polycarpic growth habit. Both types of trees, therefore, have non-flowering meristems, and we hypothesize that the molecular mechanism regulating flower inhibition in juvenile trees is different from that in adult trees. Methods In adult Citrus trees, the main endogenous factor inhibiting flower induction is the growing fruit. Thus, we studied the expression of the main flowering time, identity and patterning genes of trees with heavy fruit load (not-flowering adult trees) compared to that of 6-month-old trees (not-flowering juvenile trees). Adult trees without fruits (flowering trees) were used as a control. Second, we studied the expression of the same genes in the meristems of 6-month, and 1-, 3-, 5-and 7-year-old juvenile trees compared to 10-year-old flowering trees. Key Results The axillary meristems of juvenile trees are unable to transcribe flowering time and patterning genes during the period of induction, although they are able to transcribe the FLOWERING LOCUS T citrus orthologue (CiFT2) in leaves. By contrast, meristems of not-flowering adult trees are able to transcribe the flowering network genes but fail to achieve the transcription threshold required to flower, due to CiFT2 repression by the fruit. Juvenile meristems progressively achieve gene expression, with age-dependent differences from 6 months to 7 years, FD-like and CsLFY being the last genes to be expressed. Conclusions During the juvenile period the mechanism inhibiting flowering is determined in the immature bud, so that it progressively acquires flowering ability at the gene expression level of the flowering time programme, whereas in the adult tree it is determined in the leaf, where repression of CiFT2 gene expression occurs.

[EN] Gibberellins (GA) and auxin (indole-3-acetic acid, IAA) are considered the main compounds involved in the induction of fruit set. Citrus trees flower profusely but exhibit dramatically low fruit set rates and, in particular, seeded orange cultivars also require pollination for fruit to adequately set. Consequently, they represent an excellent model to investigate the interactions between both hormones and their effect on fruit set and development. Unpollinated ovaries from 'Pineapple' sweet orange trees were treated with IAA and pollinated ones with TIBA (2,3,5-triiodobenzoic acid, inhibitor of auxin transport), and changes in ovaries were registered shortly after the treatments. The highest IAA levels were found in unpollinated ovaries treated with auxin (twofold increase compared to pollinated ones), and the lowest corresponded to pollinated ones supplemented with TIBA (30% reduction). GA content also differed substantially among samples. In general, expression of the GA-biosynthetic gene GA20ox2 in the ovule and pericarp paralleled the changes in GA(20) content in both tissues, and also expression of GA3ox1 and GA(1) content but only in the ovule. The levels of these GA in unpollinated ovaries were promoted in response to exogenous IAA, whereas expression of the GA-inactivation gene GA2ox1 and the concentration of the GA-catabolite GA(8) were reduced by this treatment. Significantly, treatments with GA(3) or IAA to unpollinated ovaries recovered fruit set to the level reached by free pollinated ones. Our study demonstrates that IAA alters GA metabolism in citrus leading to marked changes in the active GA(1) levels in ovules and pericarp, mainly through the regulation of GA-biosynthetic genes and the inhibition of the catabolic pathway.

[EN] Gibberellins (GA) and auxin (indole-3-acetic acid, IAA) are considered the main compounds involved in the induction of fruit set. Citrus trees flower profusely but exhibit dramatically low fruit set rates and, in particular, seeded orange cultivars also require pollination for fruit to adequately set. Consequently, they represent an excellent model to investigate the interactions between both hormones and their effect on fruit set and development. Unpollinated ovaries from 'Pineapple' sweet orange trees were treated with IAA and pollinated ones with TIBA (2,3,5-triiodobenzoic acid, inhibitor of auxin transport), and changes in ovaries were registered shortly after the treatments. The highest IAA levels were found in unpollinated ovaries treated with auxin (twofold increase compared to pollinated ones), and the lowest corresponded to pollinated ones supplemented with TIBA (30% reduction). GA content also differed substantially among samples. In general, expression of the GA-biosynthetic gene GA20ox2 in the ovule and pericarp paralleled the changes in GA(20) content in both tissues, and also expression of GA3ox1 and GA(1) content but only in the ovule. The levels of these GA in unpollinated ovaries were promoted in response to exogenous IAA, whereas expression of the GA-inactivation gene GA2ox1 and the concentration of the GA-catabolite GA(8) were reduced by this treatment. Significantly, treatments with GA(3) or IAA to unpollinated ovaries recovered fruit set to the level reached by free pollinated ones. Our study demonstrates that IAA alters GA metabolism in citrus leading to marked changes in the active GA(1) levels in ovules and pericarp, mainly through the regulation of GA-biosynthetic genes and the inhibition of the catabolic pathway.

Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks—Citrus macrophylla (CM) and Carrizo citrange (CC)—were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and Citrus tristeza virus (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloroplastic genome. Thus, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis such as CM, and have a higher tolerance to salinity than the sensitive CC. These hybrids have potential as improved rootstocks to grow citrus in areas with calcareous and saline soils where CTV is present, such as the Mediterranean region. The provided knowledge on the effects of somatic hybridization on the genome composition, anatomy, and physiology of citrus rootstocks will be key for breeding programs that aim to address current and future needs of the citrus industry.

Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks—Citrus macrophylla (CM) and Carrizo citrange (CC)—were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and Citrus tristeza virus (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloroplastic genome. Thus, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis such as CM, and have a higher tolerance to salinity than the sensitive CC. These hybrids have potential as improved rootstocks to grow citrus in areas with calcareous and saline soils where CTV is present, such as the Mediterranean region. The provided knowledge on the effects of somatic hybridization on the genome composition, anatomy, and physiology of citrus rootstocks will be key for breeding programs that aim to address current and future needs of the citrus industry.

Tesis por compendio, [EN] Polyploidy is very common in higher plants and is one of the main forces driving evolution, as it leads to changes that may favor plant adaptation. In citrus, polyploid breeding has provided great advantage, enabling the development of new tetraploid (4x) rootstocks and triploid seedless varieties. Rootstock breeding programs aim to combine the characters of interest in a single genotype to provide the required tolerances to biotic and abiotic constraints. In Spain, the presence of citrus tristeza virus (CTV) and the abundance of calcareous and saline soils limit the number of suitable citrus rootstocks. Citrus breeding has been traditionally carried out by sexual hybridization but this method has limitations which are mainly imposed by their complex reproductive biology and their high heterozygosity. To overcome these restrictions, some breeding programs working at tetraploid level have been initiated to facilitate the development of new citrus rootstock ideotypes. The spontaneous appearance of autotetraploid plants in seedlings of apomictic citrus genotypes brings a relatively simple breeding method, offering the opportunity of evaluating their properties and selecting them as new rootstocks. The impact of genome duplication on citrus is variable between species and remains poorly described. To assess the usefulness of 4x citrus rootstocks it is required to evaluate the effect of genome duplication on stress physiology, tree vigor and agronomical behavior. Citrus protoplast fusion allows the somatic hybridization between sexually incompatible species, leading to combine the whole genomes of parents in a single genotype and adding complementary dominant characters, regardless of parental heterozygosity. The purpose of this thesis was to assess the potential interest of the 4x versions of the main rootstocks used in Spanish citrus industry, which are citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) and C. macrophylla W. (CM) and, [ES] La poliploidía es una característica muy común en plantas superiores y actúa como motor evolutivo promoviendo su adaptación. En cítricos, la mejora genética a nivel poliploide agiliza el desarrollo de portainjertos tetraploides (4x) y permite obtener variedades sin semilla triploides. El portainjerto es un elemento fundamental en la citricultura para proporcionar al cultivo la tolerancia a estreses bióticos y abióticos. En España, el alto contenido en caliza y sales de la mayoría de los suelos citrícolas y el virus de la tristeza de los cítricos (CTV) limitan en el número de portainjertos adecuados para el cultivo de cítricos. El desarrollo de nuevos portainjertos tiene como objetivo reunir en un genotipo las características de interés. La hibridación sexual ha sido el método tradicionalmente empleado, aunque en cítricos plantea inconvenientes debido a su compleja biología reproductiva y a su alta heterocigosidad. La autotetraploidía, que aparece espontáneamente en los cítricos apomícticos, posibilita la selección y evaluación de las variantes 4x de cada portainjerto. El efecto de la tetraploidía es variable entre las diferentes especies de cítricos y apenas ha sido descrito. Conocer su influencia sobre la fisiología y morfología de los cítricos permitiría valorar su utilidad agronómica. La alotetraploidía, obtenida mediante fusión de protoplastos, permite hibridar especies sexualmente incompatibles y reunir los genomas completos de los parentales, acumulando sus caracteres dominantes complementarios con independencia de la heterozigosidad. El propósito de ésta tesis ha sido evaluar las variantes 4x de los portainjertos más utilizados en España, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) y C. macrophylla (CM) para valorar su utilidad agronómica y describir sus propiedades. Además se han caracterizado dos híbridos alotetraploides obtenidos mediante fusión de protoplastos de CM y CC para valorar su posible utilización como p, [CA] La poliploïdia és molt comuna en plantes superiors i es un dels principals motors evolutius per promoure canvis que afavoreixen la adaptació. En cítrics, la millora genètica a nivell poliploide ha contribuït agilitzant el desenvolupament de nous portaempelts tetraploides (4x) i de varietats sense llavors triploides. En Espanya, el virus de la tristesa dels cítrics (CTV) i l'alt contingut salí i calcari de la majoria dels sols limiten el numere de portaempelts adequats per al cultiu. La millora de portaempelts de cítrics té l'objectiu de reunir el major numere de característiques d'interés en un únic cultivar. La millora genètica tradicional de cítrics, duta a terme mitjançant hibridació sexual, planteja inconvenients deguts a la seua complexa biologia reproductiva i a l'alta heterocigositat. Per aixó s'han iniciat programes de millora de portaempelts a nivell 4x, amb l'objectiu d'agilitzar el seu desenvolupament. La autotetraploïdia, que apareix de manera espontània en especies apomíctiques de cítrics, permet seleccionar les variants 4x dels portaempelts, com un mètode de millora relativament senzill. L'efecte de la tetraploïdia és variable entre les espècies de cítrics i la seua incidència a penes havia sigut descrita. Conéixer la seua repercussió en la tolerància a estressos i el vigor de l'arbre permetria avaluar la utilitat de les versions 4x de cada portaempelt. D'altra banda, l'al¿lotetraploïdia, obtinguda mitjançant la fusió de protoplastos, possibilita l'obtenció d'híbrids d'espècies sexualment incompatibles i reunir en una sola hibridació els genomes complets de les parentals d'interés, permetent l'addició de caràcters dominants complementaris amb independència de l'heterozigositat dels parentals. La finalitat d'esta tesi ha sigut avaluar les propietats d'interés agronòmic de les variants 4x dels portaempelts més utilitzats en la citricultura española, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) i C. macrophylla (C

Tesis por compendio, [EN] Polyploidy is very common in higher plants and is one of the main forces driving evolution, as it leads to changes that may favor plant adaptation. In citrus, polyploid breeding has provided great advantage, enabling the development of new tetraploid (4x) rootstocks and triploid seedless varieties. Rootstock breeding programs aim to combine the characters of interest in a single genotype to provide the required tolerances to biotic and abiotic constraints. In Spain, the presence of citrus tristeza virus (CTV) and the abundance of calcareous and saline soils limit the number of suitable citrus rootstocks. Citrus breeding has been traditionally carried out by sexual hybridization but this method has limitations which are mainly imposed by their complex reproductive biology and their high heterozygosity. To overcome these restrictions, some breeding programs working at tetraploid level have been initiated to facilitate the development of new citrus rootstock ideotypes. The spontaneous appearance of autotetraploid plants in seedlings of apomictic citrus genotypes brings a relatively simple breeding method, offering the opportunity of evaluating their properties and selecting them as new rootstocks. The impact of genome duplication on citrus is variable between species and remains poorly described. To assess the usefulness of 4x citrus rootstocks it is required to evaluate the effect of genome duplication on stress physiology, tree vigor and agronomical behavior. Citrus protoplast fusion allows the somatic hybridization between sexually incompatible species, leading to combine the whole genomes of parents in a single genotype and adding complementary dominant characters, regardless of parental heterozygosity. The purpose of this thesis was to assess the potential interest of the 4x versions of the main rootstocks used in Spanish citrus industry, which are citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) and C. macrophylla W. (CM) and, [ES] La poliploidía es una característica muy común en plantas superiores y actúa como motor evolutivo promoviendo su adaptación. En cítricos, la mejora genética a nivel poliploide agiliza el desarrollo de portainjertos tetraploides (4x) y permite obtener variedades sin semilla triploides. El portainjerto es un elemento fundamental en la citricultura para proporcionar al cultivo la tolerancia a estreses bióticos y abióticos. En España, el alto contenido en caliza y sales de la mayoría de los suelos citrícolas y el virus de la tristeza de los cítricos (CTV) limitan en el número de portainjertos adecuados para el cultivo de cítricos. El desarrollo de nuevos portainjertos tiene como objetivo reunir en un genotipo las características de interés. La hibridación sexual ha sido el método tradicionalmente empleado, aunque en cítricos plantea inconvenientes debido a su compleja biología reproductiva y a su alta heterocigosidad. La autotetraploidía, que aparece espontáneamente en los cítricos apomícticos, posibilita la selección y evaluación de las variantes 4x de cada portainjerto. El efecto de la tetraploidía es variable entre las diferentes especies de cítricos y apenas ha sido descrito. Conocer su influencia sobre la fisiología y morfología de los cítricos permitiría valorar su utilidad agronómica. La alotetraploidía, obtenida mediante fusión de protoplastos, permite hibridar especies sexualmente incompatibles y reunir los genomas completos de los parentales, acumulando sus caracteres dominantes complementarios con independencia de la heterozigosidad. El propósito de ésta tesis ha sido evaluar las variantes 4x de los portainjertos más utilizados en España, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) y C. macrophylla (CM) para valorar su utilidad agronómica y describir sus propiedades. Además se han caracterizado dos híbridos alotetraploides obtenidos mediante fusión de protoplastos de CM y CC para valorar su posible utilización como p, [CA] La poliploïdia és molt comuna en plantes superiors i es un dels principals motors evolutius per promoure canvis que afavoreixen la adaptació. En cítrics, la millora genètica a nivell poliploide ha contribuït agilitzant el desenvolupament de nous portaempelts tetraploides (4x) i de varietats sense llavors triploides. En Espanya, el virus de la tristesa dels cítrics (CTV) i l'alt contingut salí i calcari de la majoria dels sols limiten el numere de portaempelts adequats per al cultiu. La millora de portaempelts de cítrics té l'objectiu de reunir el major numere de característiques d'interés en un únic cultivar. La millora genètica tradicional de cítrics, duta a terme mitjançant hibridació sexual, planteja inconvenients deguts a la seua complexa biologia reproductiva i a l'alta heterocigositat. Per aixó s'han iniciat programes de millora de portaempelts a nivell 4x, amb l'objectiu d'agilitzar el seu desenvolupament. La autotetraploïdia, que apareix de manera espontània en especies apomíctiques de cítrics, permet seleccionar les variants 4x dels portaempelts, com un mètode de millora relativament senzill. L'efecte de la tetraploïdia és variable entre les espècies de cítrics i la seua incidència a penes havia sigut descrita. Conéixer la seua repercussió en la tolerància a estressos i el vigor de l'arbre permetria avaluar la utilitat de les versions 4x de cada portaempelt. D'altra banda, l'al¿lotetraploïdia, obtinguda mitjançant la fusió de protoplastos, possibilita l'obtenció d'híbrids d'espècies sexualment incompatibles i reunir en una sola hibridació els genomes complets de les parentals d'interés, permetent l'addició de caràcters dominants complementaris amb independència de l'heterozigositat dels parentals. La finalitat d'esta tesi ha sigut avaluar les propietats d'interés agronòmic de les variants 4x dels portaempelts més utilitzats en la citricultura española, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) i C. macrophylla (C

[EN] Citrus is a wide genus in which most of the cultivated species and cultivars are natural parthenocarpic mutants or hybrids (i.e. orange, mandarin, tangerine, grapefruit). The autonomous increase in GA(1) ovary concentration during anthesis was suggested as being the stimulus responsible for parthenocarpy in Citrus regardless of the species. To determine the exact GA-role in parthenocarpic fruit set, the following hypothesis was tested: GA triggers and maintains cell division in ovary walls causing fruit set. Obligate and facultative parthenocarpic Citrus species were used as a model system because obligate parthenocarpic Citrus sp (i.e. Citrus unshiu) have higher GA levels and better natural parthenocarpic fruit set compared to other facultative parthenocarpic Citrus (i.e. Citrus clementina). The autonomous activation of GA synthesis in C. unshiu ovary preceded cell division and CYCA1.1 up regulation (a G2-stage cell cycle regulator) at anthesis setting a high proportion of fruits, whereas C. clementina lacked this GA-biosynthesis and CYCA1.1 up-regulation failing in fruit set. In situ hybridization experiments revealed a tissue-specific expression of GA20ox2 only in the dividing tissues of the pericarp. Furthermore, CYCA1.1 expression correlated endogenous GA(1) content with GA(3) treatment, which stimulated cell division and ovary growth, mostly in C clementina. Instead, paclobutrazol (GA biosynthesis inhibitor) negated cell division and reduced fruit set. Results suggest that in parthenocarpic citrus the specific GA synthesis in the ovary walls at anthesis triggers cell division and, thus, the necessary ovary growth rate to set fruit. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

[EN] In this work, we study the capacity to biosynthesize gibberellins (GA) of ovules (either fertilised or unfertilised), developing seeds and pericarp from fruitlets and their relation with fruit set capacity. Experiments were performed in adult, 12-year-old trees of seeded (Pineapple) and seedless parthenocarpic (Washington navel) sweet orange [Citrus sinensis L. Osbeck] cultivars. The activity of GA20-, GA3- and GA2-oxidases and gibberellin levels were measured in the ovules and pericarp of fruitlets in different development states. The results indicate that ovules are the main sites of gibberellin synthesis in fruitlets during the post-anthesis period. The most intense GA(1) synthesis-coincident with the highest expression of GA20ox2, GA3ox1 and GA2ox1-was detected in the ovules of the seeded cultivar, probably induced by fecundation and associated with low early fruitlet abscission rates. By contrast, the low activity detected in the sterile cultivar appears to be rather developmentally or constitutively regulated. As a fruitlet develops, the GA(1) concentration is augmented in the pericarp in comparison to ovules or developing seeds, and levels therein did not exhibit noticeable differences between varieties. Furthermore, developing seeds from pineapple had higher GA(1) content than the unfertilised abortive ovules from Washington navel. Taken together, data suggest a main role for this hormone in the control of fruitlet abscission, and also demonstrate a function in seed development.

[EN] In this work, we study the capacity to biosynthesize gibberellins (GA) of ovules (either fertilised or unfertilised), developing seeds and pericarp from fruitlets and their relation with fruit set capacity. Experiments were performed in adult, 12-year-old trees of seeded (Pineapple) and seedless parthenocarpic (Washington navel) sweet orange [Citrus sinensis L. Osbeck] cultivars. The activity of GA20-, GA3- and GA2-oxidases and gibberellin levels were measured in the ovules and pericarp of fruitlets in different development states. The results indicate that ovules are the main sites of gibberellin synthesis in fruitlets during the post-anthesis period. The most intense GA(1) synthesis-coincident with the highest expression of GA20ox2, GA3ox1 and GA2ox1-was detected in the ovules of the seeded cultivar, probably induced by fecundation and associated with low early fruitlet abscission rates. By contrast, the low activity detected in the sterile cultivar appears to be rather developmentally or constitutively regulated. As a fruitlet develops, the GA(1) concentration is augmented in the pericarp in comparison to ovules or developing seeds, and levels therein did not exhibit noticeable differences between varieties. Furthermore, developing seeds from pineapple had higher GA(1) content than the unfertilised abortive ovules from Washington navel. Taken together, data suggest a main role for this hormone in the control of fruitlet abscission, and also demonstrate a function in seed development.

[EN] Citrus is a wide genus in which most of the cultivated species and cultivars are natural parthenocarpic mutants or hybrids (i.e. orange, mandarin, tangerine, grapefruit). The autonomous increase in GA(1) ovary concentration during anthesis was suggested as being the stimulus responsible for parthenocarpy in Citrus regardless of the species. To determine the exact GA-role in parthenocarpic fruit set, the following hypothesis was tested: GA triggers and maintains cell division in ovary walls causing fruit set. Obligate and facultative parthenocarpic Citrus species were used as a model system because obligate parthenocarpic Citrus sp (i.e. Citrus unshiu) have higher GA levels and better natural parthenocarpic fruit set compared to other facultative parthenocarpic Citrus (i.e. Citrus clementina). The autonomous activation of GA synthesis in C. unshiu ovary preceded cell division and CYCA1.1 up regulation (a G2-stage cell cycle regulator) at anthesis setting a high proportion of fruits, whereas C. clementina lacked this GA-biosynthesis and CYCA1.1 up-regulation failing in fruit set. In situ hybridization experiments revealed a tissue-specific expression of GA20ox2 only in the dividing tissues of the pericarp. Furthermore, CYCA1.1 expression correlated endogenous GA(1) content with GA(3) treatment, which stimulated cell division and ovary growth, mostly in C clementina. Instead, paclobutrazol (GA biosynthesis inhibitor) negated cell division and reduced fruit set. Results suggest that in parthenocarpic citrus the specific GA synthesis in the ovary walls at anthesis triggers cell division and, thus, the necessary ovary growth rate to set fruit. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport.

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport.

The endogenous levels of 13 gibberellins (GAs), three cytokinins (CKs), abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonic acid (JA) were analyzed in naturally pollinated ovaries of three mandarin cultivars selected for their different capacity to produce seeds and their differing parthenocarpic ability. The varieties compared were Murcott (pollen self-compatible, highly seeded), Moncada (self-incompatible, moderately seeded), and Moncalina (pollen sterile, seedless), obtained from Moncada by bud gamma-irradiation. As expected, the 13-hydroxylation pathway was predominant in ovaries and our results further indicate that cultivar differences exist in GA metabolism. The active gibberellin GA(1) levels in ovaries seems to be related with presence of fertilized ovules and, therefore, with the ability to produce the seeds of a variety. Sterility gamma irradiation arrested the biosynthesis of GA(1) and its precursor GA(19) in Moncalina ovaries if compared to Moncada. The productive efficiency of the studied cultivars also indicated that fruit set depends strongly on the GA(1) level achieved by ovaries, which is also closely related with carbohydrate content. The study of the expression of gibberellin-oxidase genes showed that the pollination/fecundation process enhances GA20ox2 and GA3ox1 activities in naturally pollinated Murcott and Moncada ovaries compared with unpollinated Murcott and Moncalina, respectively. GA2ox1 expression was lower in the ovaries of the highly seeded cultivar Murcott than in those of Moncada or Moncalina. Unpollinated Murcott ovaries contained much lower levels of GA(1) and IAA than the naturally pollinated ovaries of this cultivar. Conversely, unpollinated ovaries, which exhibited 100 % abscission, had more ABA and JA contents. Cytokinin activity seemed constitutive and independent of pollination/fecundation. However, trans-zeatin (t-Z) and 2-isopentenyl adenine (2-IP) concentrations were higher in Murcott ovaries than in Moncada/Monca

[EN] A proteomic study was used to know how the primary metabolism in Citrus leaves is affected by the fruit load. To this end, we researched the differential expression of proteins related to these processes between on-crop and off-crop 'Moncada' mandarin leaves. Samples were collected in November and proteins were extracted. From 2D DIGE gel of these extracts, 33 spots related to primary metabolism were isolated: 26 spots being associated with photosynthesis and carbohydrate metabolism, 4 spots related to Krebs cycle, 1 spot related to pentose phosphate pathway, and 2 spots related to nutrient reservoir activity. These spots were identified by MALDI-MS or LC-MS-MS. Between the proteins related to photosynthesis and carbohydrate metabolism, 16 were up-expressed in off-crop leaves, such as NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, RuBisCO large subunit, ADP-glucose pyrophosphorylase, cinnamoyl-CoA reductase, and carbonic anhydrase; while 10 proteins were up-expressed in on-crop leaves such as NADP-dependent malic enzyme, and 3,4-dihydroxy-2-butanone kinase. The isolated proteins related to pentose phosphate pathway (6-phosphogluconate dehydrogenase) and to nutrient reservoir activity (Granule-bound starch synthase Ib precursor) were up-expressed in off-crop leaves. Regarding the proteins related to Krebs cycle, only malate dehydrogenase was up-expressed in off-crop leaves, while the other proteins were up-expressed in on-crop leaves (citrate synthase and NADP-isocitrate dehydrogenase). According to these results, the primary metabolism seems to be more active in off-crop leaves, suggesting that fruit load inhibits the primary metabolism in 'Moncada' mandarins.

[EN] Fruit load in alternate-bearing citrus trees is reported to alter shoot number and growth during spring, summer, and autumn flushes, and the source-sink balance, which affects the storage and mobilization of reserve nutrients. The aim of this work was to assess the extent of shoot growth inhibition resulting from the presence of fruits in ` Moncada' mandarin trees loaded with fruit (ON) or with very light fruit load (OFF), and to identify the role of carbohydrates and nitrogenous compounds in the competition between fruits and shoots. Growth of reproductive and vegetative organs was measured on a monthly basis. C-13-and N-15-labeled compounds were supplied to trace the allocation of reserve nutrients and subsequent translocation from source to sink. At the end of the year, OFF trees produced more abundant flushes (2.4-and 4.9-fold higher in number and biomass, respectively) than ON trees. Fruits from ON trees accumulated higher C amounts at the expense of developing flushes, whereas OFF trees exhibited the opposite pattern. An inverse relationship was identified between the amount of C utilized by fruits and vegetative flush growth. C-13-labeling revealed an important role for mature leaves of fruit-bearing branches in supporting shoot/fruit growth, and the elevated sink strength of growing fruits on shoots. N availability for vegetative shoots was not affected by the presence or absence of fruits, which accumulated important amounts of N-15. In conclusion, our results show that shoot growth is resource-limited as a consequence of fruit development, and vegetative-growth inhibition is caused by photoassimilate limitation. The competence for N is not a decisive factor in limiting vegetative growth under the experimental conditions of this study. (C) 2014 Elsevier GmbH. All rights reserved.

[EN] A proteomic study was used to know how the primary metabolism in Citrus leaves is affected by the fruit load. To this end, we researched the differential expression of proteins related to these processes between on-crop and off-crop 'Moncada' mandarin leaves. Samples were collected in November and proteins were extracted. From 2D DIGE gel of these extracts, 33 spots related to primary metabolism were isolated: 26 spots being associated with photosynthesis and carbohydrate metabolism, 4 spots related to Krebs cycle, 1 spot related to pentose phosphate pathway, and 2 spots related to nutrient reservoir activity. These spots were identified by MALDI-MS or LC-MS-MS. Between the proteins related to photosynthesis and carbohydrate metabolism, 16 were up-expressed in off-crop leaves, such as NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, RuBisCO large subunit, ADP-glucose pyrophosphorylase, cinnamoyl-CoA reductase, and carbonic anhydrase; while 10 proteins were up-expressed in on-crop leaves such as NADP-dependent malic enzyme, and 3,4-dihydroxy-2-butanone kinase. The isolated proteins related to pentose phosphate pathway (6-phosphogluconate dehydrogenase) and to nutrient reservoir activity (Granule-bound starch synthase Ib precursor) were up-expressed in off-crop leaves. Regarding the proteins related to Krebs cycle, only malate dehydrogenase was up-expressed in off-crop leaves, while the other proteins were up-expressed in on-crop leaves (citrate synthase and NADP-isocitrate dehydrogenase). According to these results, the primary metabolism seems to be more active in off-crop leaves, suggesting that fruit load inhibits the primary metabolism in 'Moncada' mandarins.

The endogenous levels of 13 gibberellins (GAs), three cytokinins (CKs), abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonic acid (JA) were analyzed in naturally pollinated ovaries of three mandarin cultivars selected for their different capacity to produce seeds and their differing parthenocarpic ability. The varieties compared were Murcott (pollen self-compatible, highly seeded), Moncada (self-incompatible, moderately seeded), and Moncalina (pollen sterile, seedless), obtained from Moncada by bud gamma-irradiation. As expected, the 13-hydroxylation pathway was predominant in ovaries and our results further indicate that cultivar differences exist in GA metabolism. The active gibberellin GA(1) levels in ovaries seems to be related with presence of fertilized ovules and, therefore, with the ability to produce the seeds of a variety. Sterility gamma irradiation arrested the biosynthesis of GA(1) and its precursor GA(19) in Moncalina ovaries if compared to Moncada. The productive efficiency of the studied cultivars also indicated that fruit set depends strongly on the GA(1) level achieved by ovaries, which is also closely related with carbohydrate content. The study of the expression of gibberellin-oxidase genes showed that the pollination/fecundation process enhances GA20ox2 and GA3ox1 activities in naturally pollinated Murcott and Moncada ovaries compared with unpollinated Murcott and Moncalina, respectively. GA2ox1 expression was lower in the ovaries of the highly seeded cultivar Murcott than in those of Moncada or Moncalina. Unpollinated Murcott ovaries contained much lower levels of GA(1) and IAA than the naturally pollinated ovaries of this cultivar. Conversely, unpollinated ovaries, which exhibited 100 % abscission, had more ABA and JA contents. Cytokinin activity seemed constitutive and independent of pollination/fecundation. However, trans-zeatin (t-Z) and 2-isopentenyl adenine (2-IP) concentrations were higher in Murcott ovaries than in Moncada/Monca

La agricultura del s. XXI debe estar basada en el conocimiento científico, y no solo en el empirismo y la experiencia, por muy amplia que ésta sea. Solo de este modo el agricultor será capaz de comprender e interpretar la adaptación de las especies al medio y favorecer la producción de frutos. A todo ello contribuye el conocimiento en Anatomía, Bioquímica, Biología, Botánica, Climatología, Edafología, Entomología, Fisiología, Genética, Patología. El primer paso para el avance en el cultivo de las plantas es conocer cómo son sus órganos y sus tejidos. Anatomía de los Cítricos describe en profundidad los órganos de estas plantas (raíz, tallo, hoja, flor, semilla y fruto) explicando cómo están organizados y cómo son sus tejidos. El texto se apoya en una selección de 161 fotografías de microscopía óptica y electrónica obtenidas en trabajos de investigación durante los últimos 35 años.

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.

This study aimed to determine if self-pollination is needed to trigger facultative parthenocarpy in self-incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). 'Marisol' and 'Clemenules' mandarins were selected, and self-pollinated and un-pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self-pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time-course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA 1 and GA 4), auxin (IAA) and abscisic acid (ABA) content in the ovary. 'Clemenules' showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9days after pollination in both cultivars. Self-pollination did not stimulate parthenocarpy, whereas both un-pollinated and self-pollinated ovaries set fruit regardless of the cultivar. On the other hand, 'Marisol' un-pollinated flowers showed greater parthenocarpic ovary growth than 'Clemenules' un-pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of 'Marisol' paralleled higher levels of GA 1 in the ovary (+34% at anthesis). 'Marisol' ovary also showed higher hexoses and starch mobilization, but lower ABA levels (-64% at anthesis). Self-pollination did not modify carbohydrates or GA content in the ovary compared to un-pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination-independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self-incompatible mandarins is constitutively regulated. © Physiologia Plantarum 2012.

Euphorbia characias has drawn much attention as a potential bioenergy crop given its considerable amount of latex, rich in hydrocarbon-like compounds, and its ability to grow in large areas of semiarid lands. Compositions of major constituents with an energy value have been determined for the three phenological stages of this plant (preflowering, flowering, and postflowering) and different irrigation treatments. Metabolites from both nonpolar and polar extracts have been identified and quantified by GC-MS, GC-FID, HPLC-ELSD, and UPLC-PDA-MS. The results highlight that the end of the flowering period is the optimal harvesting time to maximize the yields of E. characias as a potential energy crop. The total water requirements to obtain the maximum yields of hexane- and methanol-extractables were determined for its annual development cycle.

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.

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.

The floral bud inductive period, PBZ promotes flowering in Citrus; however, our results indicate that this effect is fruit-load dependant. In 'Salustiana' and 'Navelina' sweet oranges, 'Hernandina' Clementine mandarin, and 'Afourer' and 'Moncada' hybrids, flowering intensity significantly increased the following spring for medium-to-low fruit-load trees treated with either 1-10g PBZ tree(-1) applied to the soil or 15 g tree(-1) sprayed on the canopy. PBZ significantly increased the percentage of sprouted buds and leafless floral shoots (both single-flowered shoots and inflorescences) and reduced the number of vegetative shoots. By contrast, heavy fruit load trees receiving the same amount of PBZ in the same season or at floral bud differentiation period scarcely flowered. Fruit nullified the effect of PBZ irrespective to treatment date (inductive period or bud burst) as well as the dose applied (1, 10 or 15 g tree(-1)) or the treatment method (soil application or canopy spraying). In conclusion, the effectiveness of PBZ in promoting flowering in Citrus depends on the fruit load since the tree showed a cultivar-dependant threshold value above which PBZ is unable to promote flowering. (C) 2012 Elsevier B.V. All rights reserved.

The floral bud inductive period, PBZ promotes flowering in Citrus; however, our results indicate that this effect is fruit-load dependant. In 'Salustiana' and 'Navelina' sweet oranges, 'Hernandina' Clementine mandarin, and 'Afourer' and 'Moncada' hybrids, flowering intensity significantly increased the following spring for medium-to-low fruit-load trees treated with either 1-10g PBZ tree(-1) applied to the soil or 15 g tree(-1) sprayed on the canopy. PBZ significantly increased the percentage of sprouted buds and leafless floral shoots (both single-flowered shoots and inflorescences) and reduced the number of vegetative shoots. By contrast, heavy fruit load trees receiving the same amount of PBZ in the same season or at floral bud differentiation period scarcely flowered. Fruit nullified the effect of PBZ irrespective to treatment date (inductive period or bud burst) as well as the dose applied (1, 10 or 15 g tree(-1)) or the treatment method (soil application or canopy spraying). In conclusion, the effectiveness of PBZ in promoting flowering in Citrus depends on the fruit load since the tree showed a cultivar-dependant threshold value above which PBZ is unable to promote flowering. (C) 2012 Elsevier B.V. All rights reserved.

Euphorbia characias has drawn much attention as a potential bioenergy crop given its considerable amount of latex, rich in hydrocarbon-like compounds, and its ability to grow in large areas of semiarid lands. Compositions of major constituents with an energy value have been determined for the three phenological stages of this plant (preflowering, flowering, and postflowering) and different irrigation treatments. Metabolites from both nonpolar and polar extracts have been identified and quantified by GC-MS, GC-FID, HPLC-ELSD, and UPLC-PDA-MS. The results highlight that the end of the flowering period is the optimal harvesting time to maximize the yields of E. characias as a potential energy crop. The total water requirements to obtain the maximum yields of hexane- and methanol-extractables were determined for its annual development cycle.

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.

This study aimed to determine if self-pollination is needed to trigger facultative parthenocarpy in self-incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). 'Marisol' and 'Clemenules' mandarins were selected, and self-pollinated and un-pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self-pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time-course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA 1 and GA 4), auxin (IAA) and abscisic acid (ABA) content in the ovary. 'Clemenules' showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9days after pollination in both cultivars. Self-pollination did not stimulate parthenocarpy, whereas both un-pollinated and self-pollinated ovaries set fruit regardless of the cultivar. On the other hand, 'Marisol' un-pollinated flowers showed greater parthenocarpic ovary growth than 'Clemenules' un-pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of 'Marisol' paralleled higher levels of GA 1 in the ovary (+34% at anthesis). 'Marisol' ovary also showed higher hexoses and starch mobilization, but lower ABA levels (-64% at anthesis). Self-pollination did not modify carbohydrates or GA content in the ovary compared to un-pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination-independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self-incompatible mandarins is constitutively regulated. © Physiologia Plantarum 2012.

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.

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.

The effects of iron (Fe) deficiency on the low-molecular-weight organic acid (LMWOA) metabolism have been investigated in Carrizo citrange (CC) [Citrus sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.] roots. Major LMWOAs found in roots, xylem sap and root exudates were citrate and malate and their concentrations increased with Fe deficiency. The activities of several enzymes involved in the LMWOA metabolism were also assessed in roots. In the cytosolic fraction, the activities of malate dehydrogenase (cMDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes were 132 and 100% higher in Fe-deficient conditions, whereas the activity of pyruvate kinase was 31% lower and the activity of malic enzyme (ME) did not change. In the mitochondrial fraction, the activities of fumarase, MDH and citrate synthase enzymes were 158, 117 and 53% higher, respectively, in Fe-deficient extracts when compared with Fe-sufficient controls, whereas no significant differences between treatments were found for aconitase (ACO) activity. The expression of their corresponding genes in roots of Fe-deficient plants was higher than that measured in Fe-sufficient controls, except for ACO and ME. Also, dicarboxylate-tricarboxylate carrier (DTC) expression was significantly increased in Fe-deficient roots. In conclusion, Fe deficiency in CC seedlings causes a reprogramming of the carbon metabolism that involves an increase of anaplerotic fixation of carbon via PEPC and MDH activities in the cytosol and a shift of the Krebs cycle in the mitochondria towards a non-cyclic mode, as previously described in herbaceous species. In this scheme, DTC could play an important role shuttling both malate and reducing equivalents between the cytosol and the mitochondria. As a result of this metabolic switch malate and citrate concentrations in roots, xylem sap and root exudates increase.

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.

La clorosis férrica es uno de los estreses abióticos más importantes para las plantas, y como tal, resulta un problema agrícola muy extendido especialmente para aquellos cultivos que se desarrollan en terrenos calizos, como es el caso de los cítricos en la comunidad Valenciana. El objetivo der este trabajo fue caracterizar el sistema de absorción de hierro en los cítricos y su respuesta ante distintas situaciones clorosantes que comprometen la disponibilidad del elemtento, provocanto la deficiencia de Fe en la planta. Los componentes del sistema de absorción del Fe -acidificación del medio, reducción del Fe(III), transporte a través de la membrana y alteración de la síntesis de ácidos orgánicos-, se analizaron a nivel fisiológico, bioquímico y molecular, en condiciones de ausencia de Fe externo, presencia de bicarbonato (HCO3) y de otros micronutrientes como zinc (Zn) y manganeso (Mn). Para la consecución de estos objetivos se utilizaron diversos genotipos de cítricos con diferente tolerancia a la clorosis férrica y se simularon diferentes condiciones clorosantes en el medio de cultivo, ya fuera eliminando el Fe o adicionando iones HCO3-, Zn2+ o Mn2+. En los distintos experimentos se evaluó, según cada caso, el crecimiento vegetativo de las plantas, el contenido de Fe total en los distintos órganos y en el apoplasto de la raíz, así como el estado del aparato fotosintético de las hojas tras la inducción de la clorosis. Así mismo, se determinó la capacidad de reducción y acidificación de las raíces, mediante el análisis de las actividades de los principales enzimas involucrados, como son la Quelato Férrico Reductasa (FC-R), la Protón-ATPasa (H+-ATPasa), la Fosfoenol Piruvato Carboxilasa (PEPCasa) y otros enzimas que intervienen en la biosíntesis de ácidos orgánicos, como la malato deshidrogenasa (MDH), la piruvato kinasa (PK), el enzima málico (ME), la citrato sintasa (CS), la aconitasa (ACO) y la fumarasa (FUM). Adicionalmente, se identificaron en el genoma de los cí

La superficie cultivada de cítricos en la comunitat Valenciana se mantiene prácticamente constante en el periodo objeto de este trabajo. En 1992 la superficie citrícola en la Comunitat Valenciana era de 180.110 ha. En 2008 la superficie citrícola era de 181.048 ha, lo que significa que en el periodo de 17 años, la superficie citrícola en la Comunitat Valenciana, se ha incrementado un 0,52%. La superficie cultivada de mandarinos se incrementa un 32,11% en el periodo estudiado, los mandarinos pasan de representar el 37,52% de la superficie citrícola al 50,6%. La superficie de mandarinos pasa de 67.585 ha en la campaña 1992/93 a 91.611 ha en la campaña 2008/09. La superficie cultivada de naranjo dulce se reduce un 18,6% en el periodo estudiado. Este grupo de cítricos pasa de representar el 53, 19% de la superficie citrícola, a representar el 43,19%, pasando de 16.230 ha en la campaña 1992/93 a 78.189 ha en la campaña 2008/09. La superficie cultivada de limonero disminuye un 34,3%. La superficie cultivada de limonero pasa de representar el 9,01% de la superficie citrícola al 5,89%, pasando de 16.230 ha a 10.636 ha en la última campaña. La producción de cítricos en la comunitat Valenciana se mantiene prácticamente constante a lo largo del período de las campañas estudiadas ya que desciende un 0,25% al pasar de 3.936.500 toneladas en la campaña 1992/93 a 3.953.46 toneladas en la última campaña estudiada. La producción de mandarinas se incrementa, al pasar del 35,6% al 45% de la producción citrícola total en el periodo estudiado. La producción del resto de cítricos desciende respecto de la producción citrícola total. La naranja dulce pasa del 56,5% al 49,65% de la producción citrícola total. La producción de limón pasa del 7,6% al 5,2% de la producción citrícola valenciana. La representación del grupo de pomelos a pesar de no ser significativa respecto del total también desciende al pasar del 0,31% al 0,14%.

La superficie cultivada de cítricos en la comunitat Valenciana se mantiene prácticamente constante en el periodo objeto de este trabajo. En 1992 la superficie citrícola en la Comunitat Valenciana era de 180.110 ha. En 2008 la superficie citrícola era de 181.048 ha, lo que significa que en el periodo de 17 años, la superficie citrícola en la Comunitat Valenciana, se ha incrementado un 0,52%. La superficie cultivada de mandarinos se incrementa un 32,11% en el periodo estudiado, los mandarinos pasan de representar el 37,52% de la superficie citrícola al 50,6%. La superficie de mandarinos pasa de 67.585 ha en la campaña 1992/93 a 91.611 ha en la campaña 2008/09. La superficie cultivada de naranjo dulce se reduce un 18,6% en el periodo estudiado. Este grupo de cítricos pasa de representar el 53, 19% de la superficie citrícola, a representar el 43,19%, pasando de 16.230 ha en la campaña 1992/93 a 78.189 ha en la campaña 2008/09. La superficie cultivada de limonero disminuye un 34,3%. La superficie cultivada de limonero pasa de representar el 9,01% de la superficie citrícola al 5,89%, pasando de 16.230 ha a 10.636 ha en la última campaña. La producción de cítricos en la comunitat Valenciana se mantiene prácticamente constante a lo largo del período de las campañas estudiadas ya que desciende un 0,25% al pasar de 3.936.500 toneladas en la campaña 1992/93 a 3.953.46 toneladas en la última campaña estudiada. La producción de mandarinas se incrementa, al pasar del 35,6% al 45% de la producción citrícola total en el periodo estudiado. La producción del resto de cítricos desciende respecto de la producción citrícola total. La naranja dulce pasa del 56,5% al 49,65% de la producción citrícola total. La producción de limón pasa del 7,6% al 5,2% de la producción citrícola valenciana. La representación del grupo de pomelos a pesar de no ser significativa respecto del total también desciende al pasar del 0,31% al 0,14%.

Background and Aims 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. Methods First defruiting experiments were performed to manipulate blossoming intensity in `Moncada¿ 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. Key Results 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. Conclusions 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 occu

Background and Aims 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. Methods First defruiting experiments were performed to manipulate blossoming intensity in `Moncada¿ 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. Key Results 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. Conclusions 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 occu

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 f

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 f