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Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions.

Citrus fruits are sensitive to low temperatures and this often results in the development of chilling injuries during postharvest storage. In order to gain more insight into the molecular mechanisms involved in the acquisition of fruit chilling tolerance, we initiated a grapefruit (Citrus paradisi, cv. Marsh Seedless) flavedo cDNA sequencing project and used it to identify a cDNA similar to other Poncirus trifoliata and Citrus unshiu dehydrin genes reported to be responsive to low temperatures. The grapefruit dehydrin cDNA, designated cor15, encodes a predicted polypeptide of 15.1 kDa, that is almost completely identical with other reported citrus dehydrin proteins, except that it contains two large amino acid repeats, whereas P. trifoliata COR11 has only one such repeat and P. trifoliata COR19 and C. unshiu COR19 have three repeats. Together, the various grapefruit, P. trifoliata and C. unshiu dehydrins form a closely related and unique dehydrin gene family that differs from most other plant dehydrins in having an unusual K-segment similar to that of gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus. The grapefruit cor15 gene is consistently expressed in the fruit peel tissue at harvest, but its message levels dramatically decrease during storage at 2 degrees C. However, a pre-storage hot water treatment, which enhances fruit chilling tolerance, elicited retention of the constant level of cor15 gene expression during cold storage and eliminated its decline. The hot water treatment had no inductive effect on cor15 gene expression when the fruit were held at non-chilling temperatures. The effects of other stresses, such as exposure to ethylene, UV irradiation and wounding, on cor15 gene expression, were temporary and persisted for 1-2 days after the treatments.

Journal Article↗

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees.

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO3- runoff in "Navelina" orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis x Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K15NO3 and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of 15N/14N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75%) than with flooding system (64%). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 %), whereas the N retained as NO3- was 1% of the N applied under drip and 10% under flooding. In the last case, most of NO3- remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.

Citrus↗

Identification of sources of plant resistance to Diaprepes abbreviatus (Coleoptera: Curculionidae) by three bioassays.

Host plant resistance to the root weevil Diaprepes abbreviatus (L.) was assessed for 3 citrus rootstock cultivars, 5 promising hybrid rootstocks, and 3 citroid fruit trees using 3 bioassay methods: a pot bioassay with 1-yr seedlings; a new, 21-cm plastic cell bioassay with 5-mo seedlings; and a diet incorporation bioassay. The plastic cell bioassay is a more rapid screening method and is capable of evaluating a larger number of entries in a shorter period compared with current methods. The 3 bioassays yielded similar results. Larval growth was inhibited by 2 of the remote citroid fruit trees, Murraya koenigii (L.) Sprengel and Glycosmis pentaphylla (Retzius) Correa, compared with growth on commercial rootstock cultivars. Specifically, larvae allowed to feed on roots of M. koenigii or G. pentaphylla gained less weight compared with larvae fed on the commercial rootstock cultivar 'Swingle' [Citrus paradisi Macfayden x Poncirus trifoliata (L.) Rafinesque-Schmaltz]. The resistance of G. pentaphylla confirms previous reports. M. koenigii is a new source of resistance to D. abbreviatus.

Animals↗

Chloride absorption in salt-sensitive Carrizo citrange and salt-tolerant Cleopatra mandarin citrus rootstocks is linked to water use.

In this work, seedlings of two citrus rootstocks, the salt-tolerant Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and the salt-sensitive Carrizo citrange (Citrus sinensis [L.] Osb. x Poncirus trifoliata [L.] Raf.) were used to study the relationship between chloride and water uptake. The results indicated that net chloride uptake rates in both genotypes were alike and decreased linearly with the time of salinity exposure, although they were more rapidly reduced in the tolerant genotype. In each rootstock, chloride uptake rates paralleled the decreases in transpiration rates. When transpiration was modified, concomitant changes in leaf Cl(-) concentrations were observed. There was a high positive correlation between total chloride content per plant and total water absorbed. In addition, the data indicate that the tolerant genotype "excluded" more chloride, i.e. it absorbed lower amounts of chloride per volume of water. Cleopatra also possessed a less efficient root system for water uptake and a higher shoot-to-root ratio. The results show that, overall, chloride absorption is linked to water use and that further tolerance in Cleopatra is mostly conferred by superior root resistance to Cl(-) uptake. Therefore, it is proposed that chloride absorption and, hence, salt tolerance in citrus depends to a great extent upon water use.

Adaptation, Physiological↗

Offsetting effects of reduced root hydraulic conductivity and osmotic adjustment following drought.

Root hydraulic conductivity (L(p)) and leaf osmotic potential at full turgor (Psi(pi,o)) were measured in young, drought-stressed and nonstressed peach (Prunus persica (L.) Batsch), olive (Olea europaea L.), citrumelo (Poncirus trifoliata Raf. x Citrus paradisi Macf.) and pistachio (Pistachia integerrima L.). Drought stress caused a 2.5- to 4.2-fold reduction in L(p), depending on species, but Psi(pi,o) was reduced only in citrumelo and olive leaves by 0.34 and 1.4 MPa, respectively. No differences existed in L(p) among species for nonstressed plants. A simple model linking L(p) to osmotic adjustment through leaf water potential (Psi) quantified the offsetting effects of reduced L(p) and osmotic adjustment on the hypothetical turgor pressure difference between drought-stressed and nonstressed plants (DeltaPsi(p)). For olive, the 2.5-fold reduction in L(p) caused a linear decrease in DeltaPsi(p) such that the effect of osmotic adjustment was totally negated at Psi = -3.2 MPa. Thus, no stomatal closure would be required to maintain higher turgor in drought-stressed olive plants than in nonstressed plants over their typical diurnal range of Psi (-0.6 to -2.0 MPa). For citrumelo, osmotic adjustment was offset by reduced L(p) at Psi approximately -0.9 MPa. Unlike olive, stomatal closure would be necessary to maintain higher turgor in drought-stressed citrumelo plants than in nonstressed plants over their typical diurnal range of Psi (0 to -1.5 MPa). Regardless of species or the magnitude of osmotic adjustment, my analysis suggests that a drought-induced reduction in L(p) reduces or eliminates turgor maintenance through osmotic adjustment.

Journal Article↗

The dwarfing mechanism of citrus rootstocks F&A 418 and #23 is related to competition between vegetative and reproductive growth.

The annual development of Navelina (Citrus sinensis (L.) Osbeck) trees budded on three hybrid citrus rootstocks was studied. Two rootstocks, named #23 and #24, were obtained from the cross of Troyer citrange (C. sinensis x Poncirus trifoliata (L.) Raf.) x Cleopatra mandarin (C. reshni Hort. ex Tan.). The third rootstock, named F&A 418, came from a cross of Troyer citrange x common mandarin (C. deliciosa Ten.). Rootstocks #23 and F&A 418 are dwarfing rootstocks and reduce the size of the scion by about 75%. Rootstock #24 yields a standard size scion. Major growth differences that influenced tree size were apparent during the first summer after grafting and appeared to be related to fruit productivity, because defruiting the dwarfed scions caused a significant increase in vegetative shoot development, including summer sprouting. The reduced growth of the dwarfed scions was not restored by hormone application, indicating that a hormonal deficiency is unlikely to be the primary reason for scion dwarfing, although differences in gibberellin concentrations were found in actively growing shoots. Leaf photosynthesis was similar in scions on all three rootstocks, but the carbohydrate accumulation in fruits and fibrous roots during the summer sprouting period was significantly greater in the dwarfed trees than in the standard trees. Our results suggest that the dwarfing mechanism induced by the F&A 418 and #23 rootstocks is mediated by enhanced reproductive development and fruit growth, resulting in reduced vegetative development in the summer. Thus, a change in the pattern of assimilate distribution appears to be one of the main components of the dwarfing mechanism.

Citrus sinensis↗

Nitrate improves growth in salt-stressed citrus seedlings through effects on photosynthetic activity and chloride accumulation.

We analyzed the effects of nitrate availability on growth of Navelina (Citrus sinensis (L.) Osbeck) scions grafted on three citrus rootstocks differing in salt tolerance: Carrizo citrange (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.), Citrus macrophylla Wester and Cleopatra mandarin (Citrus reshni Hort. ex Tanaka). Salt stress reduced total plant biomass by 27-38%, whereas potassium nitrate supplementation partially counteracted this effect by increasing dry matter and new leaf area. Salinized Carrizo citrange had the greatest response to nitrate supplementation, whereas the effects on salinized Cleopatra mandarin and C. macrophylla were less apparent. Nitrogen and chlorophyll contents and photosynthetic activity also increased in leaves of the nitrate-supplemented salinized plants. In salinized plants, nitrate supplementation reduced leaf abscission, stimulated photosynthetic activity and increased growth of new leaves. The nitrate treatment did not modify chloride concentration in leaves, but it reduced chloride concentrations in Carrizo and Macrophylla roots. Therefore, in both rootstocks, chloride content was similar in mature leaves, higher in immature leaves and lower in roots of the nitrate-supplemented salinized plants compared with salinized plants unsupplemented with nitrate. We suggest that the nitrate-induced stimulation of growth reduced chloride concentration in roots through the reallocation of chloride to new leaves.

Chlorides↗

The persistence of engineered Agrobacterium tumefaciens in agroinfected plants.

Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C. sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd). Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria. These observations suggest the need for more stringent quarantine measures when handling A. tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids.

Agrobacterium tumefaciens↗

Host-directed processing of Citrus exocortis viroid.

Prolonged infection of tomato hybrid (Lycopersicon esculentum x Lycopersicon peruvianum) by Citrus exocortis viroid (CEVd) resulted in viroid-like enlarged structures, detected by gel electrophoresis. This population included two new enlarged variants or D-variants, D-87 and D-76, and three transient species or D-forms, D-38, D-40 and D-43. Sequence analyses exposed a locus near the terminal repeat region where major changes appeared consistently. In transmission tests to CEVd hosts, a variety of progeny populations were recovered, including progeny enlargements of and reversions to CEVd, as well as sequence fidelity to the inoculum. Transmission tests to citrus hosts of the genera Citrus, Poncirus or Fortunella were unsuccessful. The importance of host specificity to the recovery and processing of the various CEVd-related structures, as well as the temporal variability of progeny populations, was demonstrated.

Base Sequence↗

Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems.

Photosynthate partitioning was examined in seedings of sour orange (Citrus aurantium L.) and Carrizo citrange (Poncirus trifoliata [L.] Raf. x C. sinensis [L.] Osbeck) grown with split root systems inoculated on one side with vesicular-arbuscular mycorrhizal fungus (Glomus intraradices Schenck and Smith). Source-sink relations were studied without mitigating differences in mineral content or physiological age that can occur in separate plant comparisons, because phosphorus was evenly distributed between leaves on opposite sides of the seedlings. Above-ground portions of each plant were exposed to (14)CO(2) for 8.5 minutes and ambient air for 2 hours, followed by extraction and identification of labeled assimilates. Mycorrhizal halves of root systems accumulated 66 and 68% of the (14)C-labeled photosynthates translocated to roots of sour orange and ;Carrizo' citrange, respectively, as well as an average of 77% greater disintegrations per minute per gram fresh weight. Distribution of (14)C-labeled assimilates was independent of phosphorus effects on photosynthate partitioning in leaves and did not reflect fresh or dry weights of roots or degree of mycorrhizal dependency of the species. Differences in radioactivity between mycorrhizal and nonmycorrhizal root halves after 2 hours indicated at least 3 to 5% of the whole plant (14)C-labeled photosynthates were allocated to mycorrhizae-related events on one side and that twice this amount, or 6 to 10%, might be expected if the entire root system was infected.

Journal Article↗

Carbon Cost of the Fungal Symbiont Relative to Net Leaf P Accumulation in a Split-Root VA Mycorrhizal Symbiosis.

Translocation of (14)C-photosynthates to mycorrhizal (+ +), half mycorrhizal (0+), and nonmycorrhizal (00) split-root systems was compared to P accumulation in leaves of the host plant. Carrizo citrange seedlings (Poncirus trifoliata [L.] Raf. x Citrus sinensis [L.] Osbeck) were inoculated with the vesicular-arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith. Plants were exposed to (14) CO(2) for 10 minutes and ambient air for 2 hours. Three to 4% of recently labeled photosynthate was allocated to metabolism of the mycorrhiza in each inoculated root half independent of shoot P concentration, growth response, and whether one or both root halves were colonized. Nonmycorrhizal roots respired more of the label translocated to them than did mycorrhizal roots. Label recovered in the potting medium due to exudation or transport into extraradical hyphae was 5 to 6 times greater for (+ +) versus (00) plants. In low nutrient media, roots of (0+) and (+ +) plants transported more P to leaves per root weight than roots of (00) plants. However, when C translocated to roots utilized for respiration, exudation, etc., as well as growth is considered, (00) plant roots were at least as efficient at P uptake (benefit) per C utilized (cost) as (0+) and (+ +) plants. Root systems of (+ +) plants did not supply more P to leaves than (0+) plants in higher nutrient media, yet they still allocated twice the (14)C-photosynthate to the mycorrhiza as did (0+) root systems. This indicates there is an optimal level of mycorrhizal colonization above which the plant receives no enhanced P uptake yet continues to partition photosynthates to metabolism of the mycorrhiza.

Journal Article↗

Fatty Acid methyl ester profiles for characterization of glomalean fungi and their endomycorrhizae.

Arbuscule-forming fungi in the order Glomales form obligate endomycorrhizal associations with plants that make them difficult to quantify, and taxonomy of the group is only beginning to be objectively understood. Fatty acid methyl ester (FAME) profiles were analyzed to assess the diversity and quantity of fatty acids in 53 isolates of 24 glomalean species. Spores and endomycorrhizal roots of sudan grass (Sorghum sudanense) and the citrus rootstock Carrizo citrange (Poncirus trifoliata x Citrus sinensis) were examined. Spores yielded reproducible FAME profiles from replicate spore collections extracted from soil pot cultures despite being grown in association with a host plant and with contaminating microorganisms present. Unweighted pair group analysis revealed relatively tight clusters of groups at the intraspecific, specific, and generic levels; however, lipid profiles at the family level were convergent. Thus, FAME profile comparisons provided a robust measure of similarity below the family level. FAME profiles in sudan grass roots containing vesicles and/or spores of Glomus intraradices were more similar to spore profiles than to profiles from nonmycorrhizal roots. The FAME profiles for Gigaspora species, which do not form vesicles or spores in roots, were less distinct from nonmycorrhizal roots. G. intraradices and G. rosea produced fatty acids in roots that were distinguishable from each other as well as from the host root. Production in citrus roots of the fatty acid 16:1(inf(omega)5) cis by two Glomus species was correlated with the development of mycorrhizal colonization as measured by clearing and staining procedures and by estimates of total incidence and vesicle intensity. FAME analysis of roots not only provided a measure of colonization development but also served as an index of carbon allocated to intraradical fungal growth and lipid storage.

Journal Article↗

An evaluation of sequence tagged microsatellite site markers for genetic analysis within Citrus and related species.

Microsatellites, also called sequence tagged microsatellite sites (STMSs), have become important markers for genome analysis but are currently little studied in plants. To assess the value of STMSs for analysis within the Citrus plant species, two example STMSs were isolated from an intergeneric cross between rangpur lime (Citrus x limonia Osbeck) and trifoliate orange (Poncirus trifoliata (L.) Raf.). Unique flanking primers were constructed for polymerase chain reaction amplification both within the test cross and across a broad range of citrus and related species. Both loci showed length variation between test cross parents with alleles segregating in a Mendelian fashion to progeny. Amplification across species showed the STMS flanking primers to be conserved in every genome tested. The traits of polymorphism, inheritance, and conservation across species mean that STMS markers are ideal for genome mapping within Citrus, which contains high levels of genetic variability.

Alleles↗

Detection of cytosine methylation and mapping of a gene influencing cytosine methylation in the genome of Citrus.

A new method was developed to detect DNA methylation in the Citrus genome using random amplification coupled with restriction enzyme digestion. Genomic DNA from Citrus grandis (L.) Osb., Poncirus trifoliata (L.) Raf., and their F1 hybrid was amplified using 7 individual 10-mer random primers. Prior to amplification the DNA templates were digested with 2 pairs of restriction endonucleases (HpaII-MspI and (or) Sau3AI-NdeII) with different sensitivities to cytosine methylation and after PCR amplification their amplified products were further digested with the same enzymes. Using this method, it was possible to detect 28 methylation events involving 23 amplified bands with the 7 random primers and 2 pairs of enzymes. A methylation polymorphism was found at a Sau3AI site in a 1.2-kb band amplified with one primer. One locus influencing cytosine methylation at this restriction site was identified through genetic analysis of a BC1 population between C. grandis and P. trifoliata and was mapped to linkage group IV using an already developed core map. This technique for detecting methylation and methylation polymorphisms is simple and should be applicable to any eukaryotic species and to many situations where it is desirable to determine whether a sequence is methylated.

Base Sequence↗

The in vitro inhibitory effects of crude extracts of traditional Chinese herbs on 3-hydroxy-3-methylglutaryl-coenzyme A reductase on Vero cells.

Cardiovascular disease is still the leading cause of death in Western countries. Epidemiological studies have shown that hypercholesterolemia is a major risk factor for coronary artery disease. Clinical trials of lipid lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitor have been shown to decrease coronary events and mortality. Flavonoids are polyphenolic natural antioxidants occurring in natural products such as traditional Chinese herbs, fruits and beverages such as tea and wine. The aim of this study was to evaluate the effects of crude extracts from traditional Chinese herbs on HMG Co-A reductase. The methods for analysis of specific inhibitors of mevalonate biosynthesis have been well-established by using Vero cells, a cell line obtained from kidneys of African green monkeys. Crude extracts from different traditional Chinese herbs were dissolved in 1% Dulbecco's modified Eagle's medium and incubated with Vero cells with or without the addition of 1 mM mevalonate or 5 mM sodium acetate for 24 hours in order to observe cell growth. Pravastatin, a specific HMG Co-A reductase inhibitor, was used as a positive control which inhibits Vero cells growth effectively and cell growth inhibition was reversible after 1 mM mevalonate. Among 100 traditional Chinese herbs used for the study, only two herbs: Curcuma zedoaria Roscoe and Poncirus trifoliata Raf. showed significant growth inhibition of Vero cells. This study shows that some crude extracts isolated from traditional medicinal herbs were effective HMG Co-A reductase inhibitors which might be developed into new hypocholesterolemic agents.

Acetates↗

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu.

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.

Amino Acid Sequence↗

Flavonoid composition of fruit tissues of citrus species.

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.

Citrus↗

Interaction between warfarin and the herbal product quilinggao.

OBJECTIVE: To describe a patient with loss of anticoagulation control and bleeding after consumption of the combination herbal product quilinggao. CASE SUMMARY: A 61-year-old man who was stable on warfarin therapy regularly consumed a jelly-like herbal product called quilinggao ("essence of tortoise shell"). Five days after the daily consumption of a second brand of quilinggao, he developed easy gum bleeding, epistaxis, and skin bruising with an international normalized ratio (INR) >6.0. Warfarin therapy was temporarily withdrawn until the INR decreased to 1.9. On the day of hospital discharge, he took a third brand of quilinggao against medical advice. Three days later, his INR was 5.2. Warfarin therapy was again temporarily withheld and the patient counseled about an apparent herb-warfarin interaction. He could not remember the name of the third brand of quilinggao. DISCUSSION: Quilinggao is a very popular Chinese herbal product. There are many different brands, and the composition of herbal products varies between manufacturers. Chuanbeimu (Fritillaria cirrhosa) in the first brand and beimu (Fritillaria spp.), chishao (Paeoniae rubra, Chinese peony), jinyinhua (Lonicera japonica), and jishi (Poncirus trifoliata) in the second brand of quilinggao have antiplatelet and/or antithrombotic effects. Loss of anticoagulation control occurred after consumption of the second and third brands of quilinggao, possibly due to the presence of a greater number of interacting herbs. An objective causality assessment revealed that the observed reaction was highly probable to be related to the ingestion of the second and third brands of quilinggao. CONCLUSIONS: Quilinggao contains herbal ingredients that can interact with warfarin. Patients on warfarin therapy should be discouraged from taking herbal medicines, especially preparations that are already known to have antiplatelet and antithrombotic effects.

Blood Coagulation↗