Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones.

Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.

Low-temperature storage and cryopreservation of grapefruit (Citrus paradisi Macfad.) seeds.

BACKGROUND: Grapefruit is an economically important fruit worldwide, but our knowledge of its seed biology is rather poor. OBJECTIVE: The present study aimed to develop techniques for banking and cryopreservation of grapefruit seeds. MATERIALS AND METHODS: Grapefruit seeds with the exotesta removed were used. Seeds were desiccated to three moisture levels between 5-9 % and stored at 15 degree C, 4 degree C and -20 degree C for up to 24, months to investigate seed lifespan in conventional seed bank. Meanwhile seeds desiccated by silica gel or saturated salt solution and embryonic axes by flash drying were employed to develop cryopreservation protocols. RESULTS: It was confirmed that grapefruit seeds have some intermediate properties, being able to withstand removal of type II water up to 7 % MC, but sensitive to -20 degree C storage. For cryopreservation, the excised embryonic axes had a wider moisture window between 5 % and 15%, with a maximum past-thaw emergence of 95 %, while seeds survived only with a maximum past-thaw emergence of 50 % or 70 % from a much narrow moisture window. CONCLUSION: In contrast to previous reports on another type II seed, coffee, we found that citrus seeds desiccated by silica gel had better post-thaw viability than those subjected to equilibrium desiccation with saturated salt solutions. Further investigation is required to elucidate the mechanisms that contribute to variable cryopreservation tolerance.

Global changes in gene expression of grapefruit peel tissue in response to the yeast biocontrol agent Metschnikowia fructicola.

To gain a better understanding of the molecular changes taking place in citrus fruit tissue following the application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. Using a cut-off of P < 0.05 and a 1.5-fold change difference as biologically significant, the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. Microarray results of selected genes were validated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The data indicated that yeast application induced the expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. Moreover, suppression was correlated with significantly higher levels of hydrogen peroxide, superoxide anion and hydroxyl radical production in yeast-treated surface wounds. Interestingly, large amounts of hydrogen peroxide were detected inside yeast cells recovered from wounded fruit tissue, indicating the ability of the yeast to activate reactive oxygen species when it is in contact with plant tissue. This study provides the first global picture of gene expression changes in grapefruit in response to the yeast antagonist M. fructicola.

Grapefruit juice and licorice increase cortisol availability in patients with Addison's disease.

OBJECTIVE: Failure to mirror the diurnal cortisol profile could contribute to the impaired subjective health status in Addison's disease (AD). Some patients report benefit from the use of various nutritional compounds. The objective of this study was to investigate the impact of licorice and grapefruit juice (GFJ) on the absorption and metabolism of cortisone acetate (CA). DESIGN: Patients (n=17) with AD on stable CA replacement therapy were recruited from the outpatient clinic at Haukeland University Hospital, Norway. They were assessed on their ordinary CA medication and following two 3-day periods of co-administration of licorice or GFJ. METHODS: Time series of glucocorticoids (GCs) in serum and saliva were obtained, and GCs in 24 h urine samples were determined. The main outcome measure was the area under the curve (AUC) for serum cortisol in the first 2.6 h after orally administered CA. RESULTS: Compared with the ordinary treatment, the median AUC for serum cortisol increased with licorice (53 783 vs 50 882, P<0.05) and GFJ (60 661 vs 50 882, P<0.05). Median cortisol levels in serum were also elevated 2.6 h after tablet ingestion (licorice 223 vs 186 nmol/l, P<0.05; GFJ 337 vs 186 nmol/l, P<0.01). Licorice increased the median urinary cortisol/cortisone ratio (0.43 vs 0.21, P<0.00001), whereas GFJ increased the (allo-tetrahydrocortisol+tetrahydrocortisol)/tetrahydrocortisone ratio (0.55 vs 0.43, P<0.05). CONCLUSION: Licorice and in particular GFJ increased cortisol available to tissues in the hours following oral CA administration. Both patients and physicians should be aware of these interactions.

Population ecology and phenology of Diaphorina citri (Hemiptera: Psyllidae) in two Florida citrus groves.

Studies were conducted to assess population densities and phenology of the psyllid Diaphorina citri Kuwayama at two citrus groves in east-central Florida. One grove contained young, irrigated grapefruit trees and the other contained mature, nonirrigated orange trees. The two groves were sampled weekly for eggs, nymphs, and adults on flush shoots; for adults on mature leaves; and for adults captured on yellow sticky card traps. Because infestations of immature D. citri develop strictly on young flush, the abundance of flush was assessed weekly. Overall means of 26.5, 16.8, and 0.27 eggs, nymphs, and adults per flush shoot, respectively, were observed in the young grapefruit trees. In the grove of mature orange trees, overall means of 16.0, 12.7, and 0.31 eggs, nymphs, and adults per flush shoot were observed, respectively. Flush abundance was an inconsistent indicator of the mean density of D. citri per flush shoot. Mean density per shoot by itself was an inconsistent indicator of overall population levels of D. citri at each study site because few shoots were sometimes present when mean densities per shoot were high. May, June, and July were periods of time when immature D. citri were consistently present and most abundant at each study site, but the study indicated large infestations could occur at any time of the year depending on environmental factors and flush availability. Yellow sticky traps were effective for both male and female D. citri and useful for gauging adult population trends.

Proximity to forest edge does not affect crop production despite pollen limitation.

A decline in pollination function has been linked to agriculture expansion and intensification. In northwest Argentina, pollinator visits to grapefruit, a self-compatible but pollinator-dependent crop, decline by approximately 50% at 1km from forest edges. We evaluated whether this decrease in visitation also reduces the pollination service in this crop. We analysed the quantity and quality of pollen deposited on stigmas, and associated limitation of fruit production at increasing distances (edge: 10, 100, 500 and 1000m) from the remnants of Yungas forest. We also examined the quantitative and qualitative efficiency of honeybees as pollen vectors. Pollen receipt and pollen tubes in styles decreased with increasing distance from forest edge; however, this decline did not affect fruit production. Supplementation of natural pollen with self- and cross-pollen revealed that both pollen quantity and quality limited fruit production. Despite pollen limitation, honeybees cannot raise fruit production because they often do not deposit sufficient high-quality pollen per visit to elicit fruit development. However, declines in visitation frequency well below seven visits during a flower's lifespan could decrease production beyond current yields. In this context, the preservation of forest remnants, which act as pollinator sources, could contribute to resilience in crop production. Like wild plants, pollen limitation of the yield among animal-pollinated crops may be common and indicative not only of pollinator scarcity, but also of poor pollination quality, whereby pollinator efficiency, rather than just abundance, can play a broader role than previously appreciated.

Postharvest heat and conditioning treatments activate different molecular responses and reduce chilling injuries in grapefruit.

A combination of hot water (a rinse at 62 degrees C for 20 s) and conditioning (pre-storage at 16 degrees C for 7 d) treatments synergistically reduced chilling injury development in grapefruit (Citrus paradisi, cv. "Star Ruby") during cold storage at 2 degrees C, suggesting that the treatments may activate different chilling tolerance responses. To study the molecular mechanisms involved, chilling- and conditioning-responsive genes were isolated by polymerase chain reaction (PCR) cDNA subtraction, cDNA libraries were constructed from hot water- and conditioning-treated fruit, and cDNA sequencing was used to identify putative stress-responsive and chilling tolerance genes. PCR cDNA subtraction revealed the identification of 17 chilling-responsive and heat- and conditioning-induced genes, and the expression patterns of 11 additional stress-related genes, antioxidant defensive genes, and genes encoding enzymes involved in membrane lipid modifications were characterized. It was found that hot water and conditioning treatments had little effect on gene expression by themselves, but rather had a priming effect, and enabled the fruit to activate their defence responses after subsequent exposure to chilling. RNA gel blot hybridizations revealed that the expression patterns of eight genes, including HSP19-I, HSP19-II, dehydrin, universal stress protein (USP), EIN2, 1,3;4-beta-D-glucanase, and superoxide dismutase (SOD), were specifically regulated by the heat treatment, and four genes, including fatty acid desaturase2 (FAD2) and lipid transfer protein (LTP), were specifically regulated by the conditioning treatment. Furthermore, four more genes were identified, including a translation initiation factor (SUI1), a chaperonin, and alcohol dehydrogenase (ADH), that were commonly regulated by both heat and conditioning treatments. According to these data, it is suggested that pre-storage heat and conditioning treatments may enhance fruit chilling tolerance by activating different molecular mechanisms. The hot water treatment activates mainly the expression of various stress-related genes, whereas the conditioning treatment activates mainly the expression of lipid membrane modification enzymes.

Effect of grapefruit juice volume on the reduction of fexofenadine bioavailability: possible role of organic anion transporting polypeptides.

OBJECTIVE: The purpose of this study was to elucidate the potential clinical relevance and mechanism(s) of action of 2 different volumes of grapefruit juice on the reduction of bioavailability of fexofenadine, a substrate of organic anion transporting polypeptides. METHODS: Grapefruit juice or water at normal (300 mL) or high (1200 mL) volume was ingested concomitantly with 120 mg fexofenadine by 12 healthy volunteers in a randomized 4-way crossover study, and fexofenadine pharmacokinetics were determined over a period of 8 hours. RESULTS: The 300-mL volume of grapefruit juice decreased the mean area under the plasma drug concentration-time curve (AUC) and the peak plasma drug concentration of fexofenadine to 58% (P < .001) and 53% (P < .001), respectively, of those with the corresponding volume of water, and 1200 mL grapefruit juice reduced these parameters to 36% ( P < .001) and 33% ( P < .001), respectively, of those with the corresponding volume of water. The 300-mL volume of grapefruit juice diminished the AUC of fexofenadine variably among individuals. This decline correlated with baseline AUC of fexofenadine with water at equivalent volume (r(2) = 0.97, P < .0001). The 1200-mL volume of grapefruit juice decreased the AUC of fexofenadine more than the 300-mL volume of grapefruit juice compared with the corresponding volume of water in each subject by a constant amount. Grapefruit juice, 300 mL and 1200 mL, reduced the coefficient of variation of the AUC of fexofenadine by 2-fold compared with that with a matching volume of water. CONCLUSIONS: Grapefruit juice at a commonly consumed volume diminished the oral bioavailability of fexofenadine sufficiently to be pertinent clinically, likely by direct inhibition of uptake by intestinal organic anion transporting polypeptide A (OATP-A; new nomenclature, OATP1A2). A much higher volume caused an additional modest effect, possibly from reduced intestinal concentration and transit time of fexofenadine. This food-drug interaction appears to be novel and may be relevant to other fruit juices and drugs.

Moderate shade can increase net gas exchange and reduce photoinhibition in citrus leaves.

Daily variations in net gas exchange, chlorophyll a fluorescence and water relations of mature, sun-acclimated grapefruit (Citrus paradisi Macfady.) and orange (Citrus sinensis L. Osbeck) leaves were determined in tree canopies either shaded with 50% shade screens or left unshaded (sunlit). Mean daily maximum photosynthetic photon flux density (PPFD) under shade varied from 500 to 700 micromol m-2 s-1 and was sufficient to achieve maximum net CO2 assimilation rates (A CO2). Responses of grapefruit and orange leaves to shading were remarkably similar. At midday, on bright clear days, the temperatures of sunlit leaves were 2-6 degrees C above air temperature and 1-4 degrees C above the temperatures of shaded leaves. Although midday depressions of stomatal conductance (gs) and A CO2 were observed in both sunlit and shaded leaves, shaded leaves had lower leaf-to-air vapor pressure differences (D) along with higher gs, A CO2 and leaf water-use efficiency than sunlit leaves. Estimated stomatal limitation to A CO2 was generally less than 25% and did not differ between shaded and sunlit leaves. Leaf intercellular CO2 partial pressure was not altered by shade treatment and did not change substantially with increasing D. Radiation and high temperature stress-induced non-stomatal limitation to A CO2 in sunlit leaves was greater than 40%. Reversible photoinhibition of photosystem II efficiency was more pronounced in sunlit than in shaded leaves. Thus, non-stomatal factors play a major role in regulating A CO2 of citrus leaves during radiation and high temperature stress.