Impact of smoking on tooth loss in adults.

DesignCohort studyCohort selectionParticipants were recruited between 1994 and 1998 from the general population with the preferred ages of 35 to 65 years in women and 40 to 65 years in men.Exposure measurementSmoking was assessed using a questionnaire from which pack years of smoking were calculated. Educational attainment, body mass index, hypertension, diabetes, alcohol consumption and vitamin or mineral supplements were assessed from measurements and questionnaires. Tooth loss was also assessed by questionnaire returned between 2004 and 2006. With the exception of the tooth loss data analysis was based on data collected at baseline.Data analysisThe 24,373 participants who returned the tooth loss questionnaire were analysed. Two hundred and eighty-six (1.2%) were excluded, as they did not respond to either of the tooth loss questions, and an additional 106 (0.4%) were excluded because they gave inconsistent responses to the questions on tooth loss. Four hundred and thirteen (1.7%) participants with missing data on cigarette smoking and 192 (0.8%) participants with missing data in any of the covariates were also excluded. The association between smoking and number of teeth at baseline was assessed using negative binomial regression models to obtain relative risks and 95% confidence intervals (CIs).ResultsThe sample of 23,376 included 9,032 men and 14,344 women of which 4,394 (19%) were current cigarette smokers, and 7,268 (31%) were cigarette smokers. 1,566 (6.7%) were edentulous at baseline. Compared with never smokers, current smokers were more likely to be male, less educated, more likely to be hypertensive, and less likely to take vitamins/mineral supplements, and they had higher alcohol consumption. Cigarette smoking was associated with higher prevalence of tooth loss at baseline as well as higher incidence of tooth loss during follow-up. The association between cigarette smoking and incident tooth loss during follow-up for the fully adjusted model (adjusted for age, sex, education, diabetes, body mass index, waist-to-hip ratio, hormone replacement therapy, contraception, intake of vitamin and mineral supplements, physical activity, alcohol intake, hypertension, and cardiovascular disease) is shown in the table.ConclusionsThere is a strong dose-dependent association between cigarette smoking and the risk of tooth loss. The risk declines after cessation of cigarette smoking; however, the risk may remain elevated for up to 20 years compared with never smokers. Efforts to improve the oral health of the population should include the prevention of smoking as well the promotion of smoking cessation.

Extending dental nurses' duties: a national survey investigating skill-mix in Scotland's child oral health improvement programme (Childsmile).

BACKGROUND: Childsmile is Scotland's national child oral health improvement programme. To support the delivery of prevention in general dental practice in keeping with clinical guidelines, Childsmile sought accreditation for extended duty training for dental nurses to deliver clinical preventive care. This approach has allowed extended duty dental nurses (EDDNs) to take on roles traditionally undertaken by general dental practitioners (GDPs). While skill-mix approaches have been found to work well in general medicine, they have not been formally evaluated in dentistry. Understanding the factors which influence nurses' ability to fully deliver their extended roles is necessary to ensure nurses' potential is reached and that children receive preventive care in line with clinical guidance in a cost-effective way. This paper investigates the supplementation of GDPs' roles by EDDNs, in general dental practice across Scotland. METHODS: A cross-sectional postal survey aiming to reach all EDDNs practising in general dental practice in Scotland was undertaken. The survey measured nurses': role satisfaction, perceived utility of training, frequency, and potential behavioural mediators of, preventive delivery. Frequencies, correlations and multi-variable linear regression were used to analyse the data. RESULTS: Seventy-three percent of practices responded with 174 eligible nurses returning questionnaires. Respondents reported a very high level of role satisfaction and the majority found their training helpful in preparing them for their extended role. While a high level of preventive delivery was reported, fluoride vanish (FV) was delivered less frequently than dietary advice (DA), or oral hygiene advice (OHA). Delivering FV more frequently was associated with higher role satisfaction (p < 0.001). Those nurses who had been practising longer reported delivering FV less frequently than those more recently qualified (p < 0.001). Perceived difficulty of delivering preventive care (skills) and motivation to do so were most strongly associated with frequency of delivery (p < 0.001 for delivery of FV, DA and OHA). CONCLUSIONS: This study has provided insight into EDDNs' experiences and demonstrates that with appropriate training and support, EDDNs can supplement GDPs' roles in general dental practice in Scotland. However, some barriers to delivery were identified with delivery of FV showing scope for improvement.

A nurse-run walk-in clinic: cost-effective alternative to non-urgent emergency department use by the uninsured.

Non-urgent healthcare problems are responsible for more than 9 million visits to the emergency department (ED) in US hospitals each year, largely due to patients' lack of access to a primary care physician. To avoid costly and unnecessary ED usage for non-urgent health problems, a walk-in clinic run by nurses (CHEER Clinic) was developed as an extension of the services provided by an existing free clinic in a low-income neighborhood of Providence, RI, with the goal of providing uninsured patients with a convenient, no-cost means of accessing healthcare. An evaluation and cost-effectiveness analysis of the clinic's first 5 months of operation were performed. During this pilot period, 256 patients were seen. When incorporating the quality-adjusted-life-year value of preventive services rendered, an estimated $1.28 million in future healthcare costs was avoided. Dividing these cost-savings by the clinic's operational cost yielded a mean return on investment of $34 per $1 invested. Adding nurse-run walk-in hours at a free clinic significantly expanded access to healthcare for uninsured patients and was cost-effective for both the clinic and the patient. Ultimately, replication of this model in community clinics serving the uninsured could reduce ED burden by treating a substantial number of non-urgent medical concerns at a lower cost than would be incurred for treatment of the same problems in EDs.

Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: comparison with girdled fruit.

Distribution of viable Candidatus Liberibacter asiaticus (CaLas) in sweet orange fruit and leaves ('Hamlin' and 'Valencia') and transcriptomic changes associated with huanglongbing (HLB) infection in fruit tissues are reported. Viable CaLas was present in most fruit tissues tested in HLB trees, with the highest titre detected in vascular tissue near the calyx abscission zone. Transcriptomic changes associated with HLB infection were analysed in flavedo (FF), vascular tissue (VT), and juice vesicles (JV) from symptomatic (SY), asymptomatic (AS), and healthy (H) fruit. In SY 'Hamlin', HLB altered the expression of more genes in FF and VT than in JV, whereas in SY 'Valencia', the number of genes whose expression was changed by HLB was similar in these tissues. The expression of more genes was altered in SY 'Valencia' JV than in SY 'Hamlin' JV. More genes were also affected in AS 'Valencia' FF and VT than in AS 'Valencia' JV. Most genes whose expression was changed by HLB were classified as transporters or involved in carbohydrate metabolism. Physiological characteristics of HLB-infected and girdled fruit were compared to differentiate between HLB-specific and carbohydrate metabolism-related symptoms. SY and girdled fruit were smaller than H and ungirdled fruit, respectively, with poor juice quality. However, girdling did not cause misshapen fruit or differential peel coloration. Quantitative PCR analysis indicated that many selected genes changed their expression significantly in SY flavedo but not in girdled flavedo. Mechanisms regulating development of HLB symptoms may lie in the host disease response rather than being a direct consequence of carbohydrate starvation.

Improving participation and engagement with a COVID-19 surveillance programme in an outpatient setting.

BACKGROUND: On 3 August 2020, Public Health Scotland commenced a prospective surveillance study to monitor the prevalence of COVID-19 among asymptomatic outpatients attending dental clinics across 14 health boards in Scotland. OBJECTIVES: The primary aim of this quality improvement project was to increase the number of COVID-19 tests carried out in one of the participating sites, Glasgow Dental Hospital and School. The secondary aim was to identify barriers to patient participation and staff engagement when implementing a public health initiative in an outpatient setting. METHOD: A quality improvement working group met weekly to discuss hospital findings, identify drivers and change ideas. Details on reasons for patient non-participation were recorded and questionnaires on project barriers were distributed to staff. In response to findings, rapid interventions were implemented to fast-track increases in the numbers of tests being carried out. RESULTS: Over 16 weeks, 972 tests were carried out by Glasgow Dental Hospital and School Secondary Care Services. The number of tests per week increased from 19 (week 1) to 129 (week 16). This compares to a similar 'control' site, where the number of tests carried out remained unchanged; 38 (week 1) to 36 (week 16). The most frequent reason given for non-participation was fear that the swab would hurt. For staff, lack of time and forgetting to ask patients were identified as the most significant barriers. CONCLUSION: Public health surveillance programmes can be integrated rapidly into outpatient settings. This project has shown that a quality improvement approach can be successful in integrating such programmes. The key interventions used were staff engagement initiatives and front-line data collection. Implementation barriers were also identified using staff questionnaires.

Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

Response of sweet orange (Citrus sinensis) to 'Candidatus Liberibacter asiaticus' infection: microscopy and microarray analyses.

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

CsPLDalpha1 and CsPLDgamma1 are differentially induced during leaf and fruit abscission and diurnally regulated in Citrus sinensis.

Understanding leaf and fruit abscission is essential in order to develop strategies for controlling the process in fruit crops. Mechanisms involved in signalling leaf and fruit abscission upon induction by abscission agents were investigated in Citrus sinensis cv. 'Valencia'. Previous studies have suggested a role for phospholipid signalling; hence, two phospholipase D cDNA sequences, CsPLDalpha1 and CsPLDgamma1, were isolated and their role was examined. CsPLDalpha1 expression was reduced in leaves but unaltered in fruit peel tissue treated with an ethylene-releasing compound (ethephon), or a fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP). By contrast, CsPLDgamma1 expression was up-regulated within 6 h (leaves) and 24 h (fruit peel) after treatment with ethephon or CMNP, respectively. CsPLDalpha1 expression was diurnally regulated in leaf blade but not fruit peel. CsPLDgamma1 exhibited strong diurnal oscillation in expression in leaves and fruit peel with peak expression around midday. While diurnal fluctuation in CsPLDalpha1 expression appeared to be light-entrained in leaves, CsPLDgamma1 expression was regulated by light and the circadian clock. The diurnal expression of both genes was modulated by ethylene-signalling. The ethephon-induced leaf abscission and the ethephon- and CMNP-induced decrease in fruit detachment force were enhanced by application during rising diurnal expression of CsPLDgamma1. The results indicate differential regulation of CsPLDalpha1 and CsPLDgamma1 in leaves and fruit, and suggest possible roles for PLD-dependent signalling in regulating abscission responses in citrus.

Induction of phytohormones and differential gene expression in citrus flowers infected by the fungus Colletotrichum acutatum.

Colletotrichum acutatum infects citrus petals and induces premature fruit drop and the formation of persistent calyces. The accumulation of hormones and other growth regulators, and differential gene expression in affected flowers and young fruit, was examined following fungal infection. Ethylene evolution increased threefold and indole-3-acetic acid (IAA) accumulation was as much as 140 times. Abscisic acid (ABA) levels showed no significant response. After infection, both trans- and cis-12-oxo-phytodienoic acid increased 8- to 10-fold. No significant difference of transjasmonic acid (JA) was observed in citrus flower petals or pistils. However, a fivefold increase of cis-JA was detected. The amount of salicylic acid (SA) was elevated twofold in affected petals, but not in pistils. Northern blot analyses revealed that the genes encoding ACC oxidase or ACC synthase, and 12-oxo-phytodienoic acid (12-oxo-PDA) reductase, were highly expressed in affected flowers. The genes encoding auxin-related proteins also were upregulated. Application of 2-(4-chlorophenoxy)-2-methyl-propionic acid (clofibrate; a putative auxin inhibitor), 2,3,5-triiodobenzolic acid (an auxin transport inhibitor), or SA after inoculation significantly decreased the accumulation of the gene transcripts of auxin-responsive, GH3-like protein and 12-oxo-PDA reductase, but resulted in higher percentages of young fruit retention. The results indicate that imbalance of IAA, ethylene, and JA in C. acutatum-infected flowers may be involved in symptom development and young fruit drop.

Phenylalanine ammonia lyase gene expression during abscission in citrus.

Phenylalanine ammonia lyase (PAL) gene expression was examined in fruit and leaf abscission zones of Valencia orange for periods up to 72 h after induction of abscission with Ethrel(R) (CEPA) or the citrus mature fruit-specific abscission material 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-P). PAL gene expression was detected in mature fruit abscission zones beginning 6 and 24 h after CEPA and CMN-P application, respectively, and continued up to 72 h. PAL gene expression was detected in leaf abscission zones 6 h after CEPA application and continued throughout the remainder of the study. In contrast, PAL gene expression was not detected in leaf abscission zones treated with CMN-P. 2-aminoindan-2-phosphonic acid (AIP), a specific inhibitor of PAL enzyme activity, decreased CMN-P-induced PAL expression in fruit abscission zones, and this was accompanied by a lack of fruit drop. PAL transcripts were low in abscission zones of immature fruit; however, the transcript was induced by CMN-P but less by CEPA application. The data suggest that downstream products of PAL activity may be important not only for wound healing and defense reactions that occur at the abscission zone fracture plane, but also for regulation of PAL gene expression during abscission.

A pectinmethylesterase gene associated with a heat-stable extract from citrus.

A putative thermostable pectinmethylesterase (TSPME) protein of 36 kDa was isolated from heat-treated citrus finisher pulp. After purification and partial sequencing of the protein, a reverse genetic approach was used to obtain the complete genomic sequence of a new pectinmethylesterase (PME) gene, CsPME4, from Citrus sinensis (L.) Osb. cv. Valencia. The CsPME4 gene contained two exons of 1203 and 690 bp interrupted by a single positionally conserved intron of 1230 bp. A full-length CsPME4 cDNA clone amplified from Valencia orange juice vesicles shared 98% identity with the genomic clone. The encoded protein of the full-length CsPME4 cDNA shared 66 and 39% amino acid identity with the full-length encoded proteins of the citrus PME, CsPME1, and CsPME3, respectively, whereas the predicted mature protein of CsPME4 shared 80 and 61% identity with the predicted mature proteins of CsPME1 and CsPME3, respectively. Southern analysis demonstrated that CsPME4 was present in at least two copies in the Valencia orange genome. Northern analysis revealed that CsPME4 mRNA was accumulated mainly in young and developing tissues of Valencia orange. Several approaches to express recombinant CsPME4 in different systems failed to obtain active protein. Further research will be necessary to successfully express the putative TSPME gene CsPME4 for biochemical characterization.

Modification of carotenoid levels by abscission agents and expression of carotenoid biosynthetic genes in 'valencia' sweet orange.

The effect of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) and ethephon on peel color, flavedo carotenoid gene expression, and carotenoid accumulation was investigated in mature 'Valencia' orange ( Citrus sinensis L. Osbeck) fruit flavedo at three maturation stages. Abscission agent application altered peel color. CMNP was more effective than ethephon in promoting green-to-red (a) and blue-to-yellow (b) color at the middle and late maturation stages and total carotenoid changes at all maturation stages. Altered flow of carotenoid precursors during maturation due to abscission agents was suggested by changes in phytoene desaturase (Pds) and ζ-carotene desaturase (Zds) gene expression. However, each abscission agent affected downstream expression differentially. Ethephon application increased ß-carotene hydroxilase (ß-Chx) transcript accumulation 12-fold as maturation advanced from the early to middle and late stages. CMNP markedly increased ß- and ε-lycopene cyclase (Lcy) transcript accumulation 45- and 15-fold, respectively, at midmaturation. Patterns of carotenoid accumulation in flavedo were supported in part by gene expression changes. CMNP caused greater accumulation of total flavedo carotenoids at all maturation stages when compared with ethephon or controls. In general, CMNP treatment increased total red carotenoids more than ethephon or the control but decreased total yellow carotenoids at each maturation stage. In control fruit flavedo, total red carotenoids increased and yellow carotenoids decreased as maturation progressed. Trends in total red carotenoids during maturation were consistent with measured a values. Changes in carotenoid accumulation and expression patterns in flavedo suggest that regulation of carotenoid accumulation is under transcriptional, translational, and post-translational control.

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.

A citrus abscission agent induces anoxia- and senescence-related gene expression in Arabidopsis.

The mechanisms of negative effects of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP), a pyrazole-derived plant growth regulator used as a citrus abscission agent, were explored in Arabidopsis by integrating transcriptomic, physiological, and ultrastructural analyses. CMNP promoted starch degradation and senescence-related symptoms, such as chloroplast membrane disruption, electrolyte leakage, and decreased chlorophyll and protein content. Symptoms of plant decline were evident 12 h after CMNP treatment. Microarray analysis revealed that CMNP influenced genes associated with stress, including those related to anoxia, senescence, and detoxification. Sucrose treatment arrested CMNP-induced plant decline. The results demonstrate that the plant response to CMNP shares common elements with various stresses and senescence at physiological and molecular levels.

G-protein-coupled alpha2A-adrenoreceptor agonists differentially alter citrus leaf and fruit abscission by affecting expression of ACC synthase and ACC oxidase.

Temporal and spatial expression patterns of genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS1 and ACS2) and ACC oxidase (ACO), ACC concentration, and ethylene production in leaves and fruit of 'Valencia' orange (Citrus sinensis [L.] Osbeck) were examined in relation to differential abscission after treatment with 2-chloroethylphosphonic acid (ethephon) alone or in combination with guanfacine or clonidine, two G-protein-coupled alpha(2A)-adrenoreceptor selective agonists. Guanfacine and clonidine markedly reduced ethephon-enhanced leaf abscission, but had little effect on ethephon-enhanced fruit loosening. Ethephon-enhanced fruit and leaf ethylene production, and ACC concentration in fruit abscission zones, fruit peel, leaf abscission zones, and leaf blades were decreased by guanfacine. Guanfacine reduced ethephon-enhanced expression of ACS1 and ACO genes in leaf abscission zones and blades, but to a lesser extent in fruit abscission zones. The expression pattern of the ACS2 gene, however, was not associated with abscission. The results demonstrate that differential expression of ACS1 and ACO genes is associated with reduction of ethephon-enhanced leaf abscission by guanfacine, and suggest a link between G-protein-related signalling and abscission.

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene.

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.

Isolation and characterization of a cDNA encoding a lipid transfer protein expressed in 'Valencia' orange during abscission.

The genetics and expression of a lipid transfer protein (LTP) gene was examined during abscission of mature fruit of 'Valencia' orange. A cDNA encoding an LTP, CsLTP, was isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-pyrazole (CMN-pyrazole). A full-length cDNA clone of 652 nucleotides was isolated using 5' and 3' RACE followed by cDNA library screening and PCR amplification. The cDNA clone encoded a protein of 155 amino acid residues with a molecular mass and isoelectric point of 9.18 kDa and 9.12, respectively. A partial genomic clone of 505 nucleotides containing one intron of 101 base pairs was amplified from leaf genomic DNA. Southern blot hybridization demonstrated that at least two closely related CsLTP genes are present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones were examined by northern hybridization. Increased expression of CsLTP mRNA was detected in RNA of mature fruit abscission zones 6, 24, 48, and 72 h after application of a non-specific abscission agent, ethephon. Low expression of CsLTP transcripts was observed after treatment of CMN-pyrazole until 24 h after application. After this time, expression markedly increased. The results suggest that CsLTP has a role in the abscission process, possibly by assisting transport of cutin monomers to the fracture plane of the abscission zone or through its anti-microbial activity by reducing the potential of microbial attack.

A beta-galactosidase gene is expressed during mature fruit abscission of 'Valencia' orange (Citrus sinensis).

beta-galactosidases have been detected in a wide range of plants and are characterized by their ability to hydrolyse terminal non-reducing beta-D-galactosyl residues from beta-D-galactosides. These enzymes have been detected in a wide range of plant organs and tissues. In a search for differentially expressed genes during the abscission process in citrus, sequences encoding beta-galactosidase were identified. Three cDNA fragments of a beta-galactosidase gene were isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after the application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-pyrazole). Based on sequence information derived from these fragments, a full-length cDNA of 2847 nucleotides (GenBank accession number AY029198) encoding beta-galactosidase was isolated from mature fruit abscission zones by 5'- and 3'-RACE approaches. The beta-galactosidase cDNA encoded a protein of 737 amino acid residues with a calculated molecular weight of 82 kDa. The deduced protein was highly homologous to plant beta-galactosidases expressed in fruit ripening. Southern blot analysis demonstrated that at least two closely related beta-galactosidase genes were present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones indicated beta-galactosidase mRNA was detected 48 h after treatment of CMN-pyrazole and ethephon in mature fruit abscission zones. beta-galactosidase transcripts were detected in leaf abscission zones only after ethephon application. The citrus beta-galactosidase was expressed in stamens and petals of fully opened flowers and young fruitlets. The results suggest that this beta-galactosidase may play a role during abscission as well as early growth and development processes in flowers and fruitlets.