Genetic inhibition of flowering differs between juvenile and adult Citrus trees.

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.

Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings.

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl- exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl- build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30-40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl- toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.

Euphorbia characias as bioenergy crop: a study of variations in energy value components according to phenology and water status.

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.

Characterization and regulation of ammonium transport systems in Citrus plants.

We have investigated both the kinetics and regulation of 15NH4+ influx in roots of 3-month-old hydroponically grown Citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata Blanco) seedlings. The 15NH4+ influx is saturable below an external ammonium concentration of 1 mM, indicating the action of a high-affinity transport system (HATS). The HATS is under feedback repression by the N status of the plant, being down-regulated in plants adequately supplied with N during growth, and up-regulated by N-starvation. When assayed between 1 and 50 mM [15NH4+]0, the 15NH4+ influx showed a linear response typical of a low-affinity transport system (LATS). The activity of the LATS increased in plants supplied with NH4+ as compared with plants grown on an N-free medium. Transfer of the plants to N-free solution resulted in a marked decrease in the LATS-mediated 15NH4+ influx. Accordingly, resupply of NH4+ after N-starvation triggered a dramatic stimulation of the activity of the LATS. These data provide evidence that in Citrus plants, the LATS or at least one of its components is inducible by NH4+. Even when up-regulated, both the HATS and the LATS displayed a limited capacity, as compared with that usually found in herbaceous species. The use of various metabolic uncouplers or inhibitors indicated that 15NH4+ influx mediated by the HATS is strongly dependent on energy metabolism and H+ transmembrane electrochemical gradient. By contrast, the LATS is not affected by protonophores or inhibitors of the H(+)-ATPase, suggesting that its activity is mostly driven by the NH4+/NH3 transmembrane gradient. In agreement with these hypotheses, the HATS-mediated 15NH4+ influx was strongly inhibited when the solution pH was raised from 4 to 7, whereas influx mediated by the LATS was slightly stimulated.

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus.

The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A1 (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions.

Pollination Increases Gibberellin Levels in Developing Ovaries of Seeded Varieties of Citrus.

Reproductive and vegetative tissues of the seeded Pineapple cultivars of sweet orange (Citrus sinensis L.) contained the following C-13 hydroxylated gibberellins (GAs): GA53, GA17, GA19, GA20, GA1, GA29, and GA8, as well as GA97, 3-epi-GA1, and several uncharacterized GAs. The inclusion of 3-epi-GA1 as an endogenous substance was based on measurements of the isomerization rates of previously added [2H2]GA1. Pollination enhanced amounts of GA19, GA20, GA29, and GA8 in developing ovaries. Levels of GA1 increased from 5.0 to 9.5 ng/g dry weight during anthesis and were reduced thereafter. The amount of GA in mature pollen was very low. Emasculation reduced GA levels and caused a rapid 100% ovary abscission. This effect was partially counteracted by either pollination or application of GA3. In pollinated ovaries, repeated paclobutrazol applications decreased the amount of GA and increased ovary abscission, although the pattern of continuous decline was different from the sudden abscission induced by emasculation. The above results indicate that, in citrus, pollination increases GA levels and reduces ovary abscission and that the presence of exogenous GA3 in unpollinated ovaries also suppresses abscission. Evidence is also presented that pollination and GAs do not, as is generally assumed, suppress ovary abscission through the reactivation of cell division.

Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots.

The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

Selection of a NaCl-tolerant Citrus plant.

The objective of the work was to select Citrus plants more tolerant to elevated NaCl concentrations in the irrigation water. For this purpose, unfertilized Troyer citrange ovules treated with a chemical mutagenic agent (ethyl methane sulphonate) were cultured in vitro. Whole plants were regenerated from embryos developed in the nucellar tissue of the ovule. The screening for salt tolerance was achieved by irrigating these plants with a nutrient solution containing 45 mM NaCl.Plants obtained from vegetative propagation of the selected plant show faster growth, less leaf damage and lower concentrations of Cl(-) and Na(+) in leaves than the original clone with increasing NaCl levels in the nutrient solution. In addition, the selected plant accumulated more Na(+) in shoots and roots and this was accompanied by a significant reduction in K(+)concentration. It can be concluded that the selected plant restricts the accumulation of Na(+) in leaves by concentrating Na(+) in shoots and roots.

1-Aminocyclopropane-1-Carboxylic Acid Transported from Roots to Shoots Promotes Leaf Abscission in Cleopatra Mandarin (Citrus reshni Hort. ex Tan.) Seedlings Rehydrated after Water Stress.

The effect of water stress and subsequent rehydration on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity, ethylene production, and leaf abscission was studied in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings. Leaf abscission occurred when drought-stressed plants were allowed to rehydrate, whereas no abscission was observed in plants under water stress conditions. In roots of water-stressed plants, a high ACC accumulation and an increase in ACC synthase activity were observed. Neither increase in ACC content nor significant ethylene production were detected in leaves of water-stressed plants. After rehydration, a sharp rise in ACC content and ethylene production was observed in leaves of water-stressed plants. Content of ACC in xylem fluid was 10-fold higher in plants rehydrated for 2 h after water stress than in nonstressed plants. Leaf abscission induced by rehydration after drought stress was inhibited when roots or shoots were treated before water stress with aminooxyacetic acid (AOA, inhibitor of ACC synthase) or cobalt ion (inhibitor of ethylene-forming enzyme), respectively. However, AOA treatments to shoots did not suppress leaf abscission. The data indicate that water stress promotes ACC synthesis in roots of Cleopatra mandarin seedlings. Rehydration of plants results in ACC transport to the shoots, where it is oxidized to ethylene. Subsequently, this ethylene induces leaf abscission.

Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins.

Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross-pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA(53), GA(44), GA(17), GA(19), GA(20), GA(29), GA(1), 3-epi-GA(1), GA(8), GA(24), GA(9), and GA(4) have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [(2)H(2)]- and [(13)C]-labeled internal standards, the levels of GA(53), GA(44), GA(19), GA(20), GA(1), GA(8), GA(4), and GA(9) were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13-hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA(53) (10.4x), GA(44) (13.9x), GA(19) (3.0x), GA(20) (11.2x), and GA(1) (2.0x). By contrast, levels of GA(8) were always higher in Clementine, whereas levels of GA(4) did not differ greatly. Levels of GA(9) were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA(1), respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA(3). GA(3) alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits.

Selection of Nicotiana tabacum Haploids of High Photosynthetic Efficiency.

Anthers of Nicotiana tabacum cultivar Wisconsin 38 were treated with the mutagen ethyl methane sulfonate and cultured to obtain populations of haploid plants of high genetic variability. The selection of these populations by their photosynthetic efficiency was carried out in a hydroponic culture chamber with a CO(2) atmosphere concentration close to the compensation point. Plants that survived 45 days of treatment were grown in pots in a greenhouse and their performance was compared to a population of unselected haploids. The growth characteristics, net photosynthesis, and chlorophyll content were measured and the haploid character verified. Selected plants were propagated by in vitro culture of buds and then diploidized to obtain seeds. Growth and other characteristics of the plants obtained were compared with those of the parental variety (Wisconsin 38) in a field assay. Growth, dry weight, leaf area, and net photosynthesis of selected plants were higher than in control plants.

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.).

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.