Reversion of fruit-dependent inhibition of flowering in Citrus requires sprouting of buds with epigenetically silenced CcMADS19.

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.

Fruit-dependent epigenetic regulation of flowering in Citrus.

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.

Bud sprouting and floral induction and expression of FT in loquat [Eriobotrya japonica (Thunb.) Lindl.].

MAIN CONCLUSION: EjFT1 and EjFT2 genes were isolated and sequenced from leaves of loquat. EjFT1 is involved in bud sprouting and leaf development, and EjFT2 in floral bud induction. Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an evergreen species belonging to the family Rosaceae, such as apple and pear, whose reproductive development, in contrast with these species, is a continuous process that is not interrupted by winter dormancy. Thus, the study of the mechanism of flowering in loquat has the potential to uncover the environmental and genetic networks that trigger flowering more accurately, contributing for a better understanding of the Rosaceae floral process. As a first step toward understanding the molecular mechanisms controlling flowering, extensive defoliation and defruiting assays, together with molecular studies of the key FLOWERING LOCUS T (FT) gene, were carried out. FT exhibited two peaks of expression in leaves, the first one in early to mid-May, the second one in mid-June. Two FT genes, EjFT1 and EjFT2, were isolated and sequenced and studied their expression. Expression of EjFT1 and EjFT2 peaks in mid-May, at bud sprouting. EjFT2 expression peaks again in mid-June, coinciding with the floral bud inductive period. Thus, when all leaves of the tree were continuously removed from early to late May vegetative apex differentiated into panicle, but when defoliation was performed from early to late June apex did not differentiate. On the other hand, fruit removal advanced EjFT1 expression in old leaves and the sooner the fruit detached, the sooner the bud sprouted. Accordingly, results strongly suggest that EjFT1 might be related to bud sprouting and leaf development, while EjFT2 might be involved in floral bud induction. An integrative model for FT functions in loquat is discussed.

Self-pollination and parthenocarpic ability in developing ovaries of self-incompatible Clementine mandarins (Citrus clementina).

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 (GA1 and GA4 ), 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 9 days 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 GA1 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.

Warm temperature during floral bud transition turns off EjTFL1 gene expression and promotes flowering in Loquat (Eriobotrya japonica Lindl.).

The Rosaceae family includes several deciduous woody species whose flower development extends over two consecutive growing seasons with a winter dormant period in between. Loquat (Eriobotrya japonica Lindl.) belongs to this family, but it is an evergreen species whose flower bud initiation and flowering occur within the same growing year. Vegetative growth dominates from spring to late summer when terminal buds bloom as panicles. Thus, its floral buds do not undergo winter dormancy until flowering, but a summer heat period of dormancy is required for floral bud differentiation, and that is why we used loquat to study the mechanism by which this summer rest period contributes to floral differentiation of Rosaceae species. As for the deciduous species, the bud transition to the generative stage is initiated by the floral integrator genes. There is evidence that combinations of environmental signals and internal cues (plant hormones) control the expression of TFL1, but the mechanism by which this gene regulates its expression in loquat needs to be clarified for a better understanding of its floral initiation and seasonal growth cycles. Under high temperatures (>25ºC) after floral bud inductive period, EjTFL1 expression decreases during meristem transition to the reproductive stage, and the promoters of flowering (EjAP1 and EjLFY) increase, indicating that the floral bud differentiation is affected by high temperatures. Monitoring the apical meristem of loquat in June-August of two consecutive years under ambient and thermal controlled conditions showed that under lower temperatures (<25ºC) during the same period, shoot apex did not stop growing and a higher EjTFL1 expression was recorded, preventing the bud to flower. Likewise, temperature directly affects ABA content in the meristem paralleling EjTFL1 expression, suggesting signaling cascades could converge to refine the expression of EjTFL1 under specific conditions (Tª<25ºC) during the floral transition stage.

Relationship between soil temperature and fruit colour development of 'Clemenpons' Clementine mandarin (Citrus clementina Hort ex. Tan).

BACKGROUND: In Citrus, root temperature regulates rind colouration. However, few studies have investigated the range of temperatures and timing which determine rind colour break. The objective of this study was to determine the relationship between range of soil temperature (ST) and rind colour development in the precocious 'Clemenpons' Clementine mandarin. Reflective white plastic mulch was used to modify root temperature. RESULTS: Mulching increased reflected light and reduced daily maximum ST and temperature range, major differences being established 70-30 days before harvest. Rind colour-break correlated positively with 20 °C < ST < 23 °C; thus, 20-23 °C appears to be the ST threshold interval for fruit colouration. The sooner the soil reached it, the sooner the fruit changed rind colour. In our experiments, control trees accumulated 565 h at this ST interval before fruit changed colour, whereas in treated trees it occurred 2 weeks earlier. Hence, in treated trees the colour break was advanced by 2 weeks and this increased the percentage of fruit harvested at the first picking date by up to 2.5-fold. CONCLUSIONS: Fruit colour-break does not take place at a certain ST, but after several hours at a ST of 20-23 °C. In our experiments, reducing ST during the 2 months before harvest advances the first picking date in the 'Clemenpons' Clementine mandarin.

Timing of the inhibitory effect of fruit on return bloom of 'Valencia' sweet orange (Citrus sinensis (L.) Osbeck).

BACKGROUND: In Citrus the inhibitory effect of fruit on flower formation is the main cause of alternate bearing. Although there are some studies reporting the effect on flowering of the time of fruit removal in a well-defined stage of fruit development, few have investigated the effect throughout the entire fruit growth stage from early fruitlet growth to fruit maturity. The objective of this study was to determine the phenological fruit developmental stage at which the fruit begins its inhibitory effect on flowering in sweet orange by manual removal of fruits, and the role of carbohydrates and nitrogen in the process. RESULTS: Fruit exerted its inhibitory effect from the time it was close to reaching its maximum weight, namely 90% of its final size (November) in the present experiments, to bud sprouting (April). The reduction in flowering paralleled the reduction in bud sprouting. This reduction was due to a decrease in the number of generative sprouted buds, whereas mixed-typed shoots were largely independent of the time of fruit removal, and vegetative shoots increased in frequency. The number of leaves and/or flowers per sprouted shoot was not significantly modified by fruit load. CONCLUSION: In 'Valencia' sweet orange, fruit inhibits flowering from the time it completes its growth. Neither soluble sugar content nor starch accumulation in leaves due to fruit removal was related to flowering intensity, but some kind of imbalance in nitrogen metabolism was observed in trees tending to flower scarcely.

Gibberellin reactivates and maintains ovary-wall cell division causing fruit set in parthenocarpic Citrus species.

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 GA1 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 GA1 content with GA3 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.

Loquat fruit ripening is associated with root depletion. Nutritional and hormonal control.

In woody species, it is known that there is a competition for nutrients, water and carbohydrates between root and fruit-shoot systems, however the influence of root development on fruit quality has received little attention. This research aims to identify the network of mechanisms involved in loquat (Eriobotrya japonica Lindl.) fruit ripening in connection with root activity. The study includes root growth rate measurements paralleling the ongoing fruit developmental stages, photosynthate translocation to the root by using (13)CO2 tracing, and nitrogen fractions (N-NH4(+), N-NO3(-), and N-proteinaceous) as well as their upward translocation to the fruit. The role of hormones (IAA, zeatin and ABA) in regulating the responses is also addressed. The experiment was conducted during two consecutive years on adult and 3-year-old loquat trees from early fruit developmental stage (10% of final size, 701 BBCH scale) to fully developed fruit colour (809 BBCH scale). This approach revealed that root development depends on the growing fruit sink strength, which reduces carbohydrates translocation to the roots and prevents them for further elongation. A nitrate accumulation in roots during the active fruit growth period takes place, which also contributes to slowing elongation and paralleled reduced ammonium and proteinaceous nitrogen concentrations. Concomitantly, the concentration of IAA and zeatin were lowest while that of ABA was highest when root exhibited minimum elongation. The depletion in zeatin and nitrogen supply by the roots paralleling the high ABA transport to the fruit allowed for colour break. These results suggest that loquat fruit changes colour by reducing root growth, as fruit increases sugars and ABA concentrations and reduces nitrogen and zeatin concentrations.

Carbon utilization by fruit limits shoot growth in alternate-bearing citrus trees.

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. (13)C- and (15)N-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. (13)C-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 (15)N. 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.

Cell division interference in newly fertilized ovules induces stenospermocarpy in cross-pollinated citrus fruit.

Seedlessness is a highly desirable characteristic in fresh fruits. However, post-fertilization seed abortion of cross-pollinated citrus fruit is uncommon. The factors regulating stenospermocarpy in citrus are unknown. In this research, we induced stenospermocarpy interfering in newly fertilized ovule cell division. The research also elucidates the most sensitive stage for ovule/seed abortion in citrus. Experiments were conducted with 'Afourer' mandarin that cross-pollinates with several cultivars and species. Cross-pollinated fruitlets were treated with maleic hydrazide (MH), a systemic growth regulator that specifically interferes in cell division. MH reduced ovule growth rate, the number of cell layers in nucella and inhibited embryo sac expansion; moreover, the treatment increased callose accumulation in nucella and surrounding the embryo sac. Fruits developed an early-aborted seed type with an immature, soft and edible seed coat. Seed number (-80%) and seed weight (-46%) were reduced in mature fruits. MH also hampered cell division in ovary walls, mesocarp and endocarp, thus reducing daily fruitlet growth and increasing fruit abscission. Stenospermocarpy could only be induced for a short period of time in the progamic phase of fertilization, specifically, when ovules are ready to be fertilized (7 days after anthesis) to early stages of embryo sac development (14 days after anthesis).

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

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 DIGE 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.