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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.

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] 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.

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.