Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa

We have previously reported that the oligosaccharides transferred in vivo from dolichol-P-P derivatives in protein N-glycosylation in trypanosomatids are devoid of glucose residues and contain 2 N-acetylglucosamine and 6, 7, or 9 mannose units depending on the species. In this respect trypanosomatids differ from wild type mammalian, plant, insect, and fungal cells in which Glc3Man9GlcNAc2 is transferred. We are now reporting that incubation of Glc1-3Man9GlcNAc2-P-P-dolichol and Man7-9GlcNAc2-P-P-dolichol with membranes of Trypanosoma cruzi, Leptomonas samueli, Crithidia fasciculata, and Blastocrithidia culicis and an acceptor hexapeptide leads to the transfer of the six above mentioned lipid-linked oligosaccharides at the same rate. Control experiments performed under similar conditions but with rat liver and Saccharomyces cerevisiae membranes showed that, as already known, Glc3Man9GlcNAc2 is preferentially transferred in the latter systems. We have also previously reported that, once transferred to protein, the oligosaccharides become transiently glucosylated in trypanosomatids. Depending on the species, protein-linked Glc1Man5-9GlcNAc2 have been transiently detected in cells incubated with [14C] glucose. We are now reporting that glucosidase activities degrading both Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 were detected in T. cruzi, L. samueli, and C. fasciculata. The enzymatic activities were associated with a membrane fraction; they had a neutral optimum pH value, and similarly to mammalian glucosidase II, the enzyme acting on the monoglucosylated substrate showed a decreased affinity when the latter contained fewer mannose residues. No glucosidase I-like enzyme acting on Glc3Man9GlcNAc2 was detected in any of the three above-mentioned protozoan species. This result is consistent with the fact that no oligosaccharides containing 3 glucose units occur in trypanosomatids.     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn² and Asn¹⁵. The predominant glycan (60% of total) had thestructure GlcNAcß1–2Man1–3(Man1–6)Manß1–4GlcNAcß1–4GlcNAc-(Asn),with the remaining structures containing 1–3 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn² site. FAB-MS analysis of oligosaccharides releasedfrom the Asn² site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc₁Hex₆HexNAc₃(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin     

Evidence for Predominant Participation of Photosystem II{beta}-Units in Nitrogen Formation by Hydroxylamine-Treated Tobacco Chloroplasts

Relative optical cross sections for flash-induced O₂- and N₂-productionwith water and 1 mM NH₂OH as electron donors to PSII, respectively,as well as that for PSI-mediated O²-uptake have been measuredin tobacco chloroplasts by mass spectrometry. In the wild typetobacco these three reactions are driven by three populationsof photosynthetic units different with respect to the antennasize. The antenna size of O₂-evolving units is twice as largecompared to the N₂-evolving one, but both of these have thesame spectral characteristics in the far red region. In contrastto the wild type, the antenna sizes of O₂- and N₂-evolving unitsin the chlorophyll b-deficient tobacco mutant Su/su var. Aureaare the same as the sizes of the N₂-evolying units in the wildtype chloroplasts. Taking into account the data of Thielen andVan Gorkom [Biochim. Biophys. Acta (1981) 635:111] on a strongdifference in the relative amounts of PSII_ß and PSII_ßin the mutant compared with the wild type (the ratio about 1: 3 and 3 : 1 , respectively) it is concluded that mainly thestroma-exposed PSII_ß units are competent to N₂-evolution. (Received November 11, 1993; Accepted April 25, 1994)     

S-Locus-Specific Glycoproteins Associated with Self-Incompatibility in Brassica campestris

Specific S-glycoproteins were isolated from three Brassica campestriscultivars homozygous with respect to the S-alleles S₈, S₉ andS₁₂. Amino acid sequences of various peptide fragments of theS-proteins were determined using a gas-phase protein sequencer,and a nearly complete amino acid sequence of the S₈-glycoproteinwas determined on the basis of the revised cDNA sequence ofthe B. oleracea S-specific glycoprotein. The lysyl endopeptidasefragments of S₉ and S₁₂-glycoproteins were aligned in comparisonwith the sequence of the S₈-glycoprotein. Although extensivesequence homology was evident among the three S-glycoproteins,the sequences of the middle part were relatively different fromeach other. The numbers and positions of N-glycosylation alsodiffered among the S-glycoproteins of Brassica species. (Received April 20, 1987; Accepted July 29, 1987)     

Homonojirimycin and N-methyl-homonojirimycin inhibit N-Iinked oligosaccharide processing

Homonojirimycin (HNJ) and N-methylhomonojirimycin (MHNJ) weretested as inhibitors of the purified glycopro-tein processingenzymes, glucosidase I and glucosidase II. MHNJ was a reasonablygood inhibitor of glucosidase I (K₁ = 1 x 10^–6 M) andwas about three times as effective on this enzyme as was HNJ.On the other hand, HNJ inhibited glucosidase II with a K₁ ofabout 1 x 10^x6 M, whereas MHNJ was three times less effective(K₁ = 3 x 10^–5 M). However, the butyl derivative of HNJhad very low activity toward these two processing glucosidases.HNJ and its methyl derivative were also tested in vivo usinginfluenza virus-infected MDCK cells, and measuring the inhibitionof N-linked oligosaccharide processing of the viral envelopeglycoproteins. With 100 µg/ml of MHNJ in the medium, essentiallyall of the N-linked oligosaccharide chains of the virus wereof the "high-mannose" type with the major structure being characterizedas Glc₃Man₉(GlcNAc)₂. Similar results were obtained with HNJalthough this compound was less effective in vivo as well asin vitro. These results are in keeping with these inhibitorsbeing effective at the glucosidase I step. Both inhibitors werealso tested in MDCK cell cultures to determine whether theyaffected the in vivo synthesis of proteins, or of lipid-linkedsaccharides. In contrast to deoxynojirimycin, which has beenreported to inhibit the formation of lipid-linked saccharides,no effects were seen on either the incorporation of mannoseinto lipid-linked saccharides or the incorporation of leucineinto protein. glucosidase lipid influenza virus oligosaccharide     

Glutamine Synthetase Isoenzymes of Striga hermonthica and other Angiosperm Root Parasites

The occurrence of multiple forms of glutamine synthetase inStriga hermonthica and other angiosperm root parasites was investigated.The facultative chlorophyllous parasite Melampyrum arvense exhibitedtwo isoenzymes in leaf tissue, the cytosolic component (GS₁)comprised less than 30% of total glutamine synthetase. In contrastGS₁ was the major component (<70%) in photosynthetic tissueof Striga hermonthica and S. gesnerioides. Only a single isoenzyme(GS₁) was detectable in the achlorophyllous root parasites Orobancheand Lathraea and in non-photosynthetic tissue of S. gesnerioides.The kinetic and physical properties of GS₁ and GS₂ of theseangiosperm parasites were similar to those of the isoenzymesin other non-parasitic angiosperms. Key words: Glutamine synthetase, Angiosperms, Root parasites     

Reduced Phosphorus Requirement of a Mutant Azolla-Anabaena Symbiotic N2-fixing Complex

The use of the photo-autotrophic nitrogen-fixing water fernAzolla as an effective source of organic nitrogen in tropicalpaddy fields has been limited by a high phosphorus requirement.Azolla species with a minimum of 1.5 to 2.0 mM phosphate (P)requirement, under controlled conditions, are known. A local Azolla species requiring at least 1.5 mM sodium phosphatefor a normal rate of multiplication and N₂ fixation was exposedto N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The resultingmutant population had a significantly lower P requirement, butwas auxotrophic for glutamine with an extremely reduced glutaminesynthetase (GS) activity. An L-methionine-DL-sulphoximine (MSX)-resistant(MSX^r) Azolla population, having an approximately 1.5 timeshigher GS activity than that of the wild type (WT) parent organism,was cultured and subjected to MNNG-induced mutation for lowP requirement while putting MSX as a control in the mutant selectionmedium. The resulting population of mutant Azolla was a normalprototroph with a P requirement as low as 0.75 mM for its ‘WTparent-like’ usual growth and N₂ fixation. Key words: Azolla, phosphorus requirement, mutation     

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human {beta}1,2-N-acetylglucosaminyltransferase I

Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan₃GlcNAc₂. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells     

Novel Asn-linked oligosaccharides terminating in GaINAc{beta}(1->4)[Fuca(1->3)]GlcNAcB(1->{bullet}) are present in recombinant human Protein C expressed in human kidney 293 cells

Recombinant human Protein C (rHPC), expressed in human kidney293 cells, has a higher anticoagulant activity than plasma HPC,while its in vivo circulatory half-life is essentially unalteredcompared to that of the natural protein. In seeking to elucidatethe molecular basis for the improved efficacy of the recombinantantithrombotic drug, we focused on the carbohydrate moiety ofrHPC. Protein C is a heavily post-translationally modified serineprotease with four N-glycosylation sites. Glycosyl compositionanalysis of rHPC revealed a 5-fold higher fucose content anda 2-fold lower sialic acid content compared to plasma HPC. Inaddition, we found that rHPC contains N-acetylgaiac-tosamine(2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides,while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharidesof rHPC were released by N-glycanase and separated into 25 fractionsby high-pH anion-exchange chromatography. The most abundantoligosaccharides were structurally characterized by glycosylcomposition and linkage analysis, in conjunction with ¹H-NMRspectroscopy at 600 MHz. The structure of the major neutraloligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPCare: and Thus, many of the Asn-linked oligosaccharides in rHPC were foundto terminate in GaINAcß(1     

Growth rates of dominant planktonic ciliates in two freshwater bodies of different trophic degree

The in situ growth rates of dominant ciliate species were studiedduring and shortly after phytoplankton peaks in two water bodies:the eutrophic Rímov Reservoir (South Bohemia, Czech Republic)and the oligo-mesotrophic Piburger See (Tyrol, Austria). Growthrate estimates based on changes in ciliate abundances in incubatedpre-screened samples (E_N) were compared with those derived fromthe ciliate cell volume and ambient temperatures (E_T). The valuesof E_N were always rather lower than those of E_T. During thestudies, the food supply limited the ciliate growth dependingon the ciliate feeding mode. An ecological grouping into filterfeeding versus raptorial feeding (‘hunting’) species,on the one hand, and attached/crawling (browsing) versus freeswimming species, on the other hand, clearly affected experimentalestimation. Both fine filter feeders (namely attached) and browsersexhibited a calculated E_N closer to the theoretical (maximum)E_T than did hunters and coarse filter feeders. It was apparent,for example, comparing E_N and E_T (day^–1) of the followingspecies: filter feeders Halteria grandinella (E_N = 0.42; E_T>1.4), Strobilidium hexakinetum (0.34;>1.9), Pelagohalteriaviridis (0.27;>0.9), Vorticella aquadulcis complex (0.75;>1.0);raptorial Balanion planctonicum (0.65;>1.5), Urotricha furcata(in Rímov Reservoir 0.65;>2.1; in Piburger See 0.20;>1.5),Rhabdoaskenasia minima (0.22;>1.0), Askenasia acrostomia(0.12;>0.6); opportunistic Cyrtolophosis mucicola (0.42;>1.6)and Cinetochilum margaritaceum (0.86;>1.4). Predation byrotifers apparently affected measurements in several samplescontaining {small tilde}400 rotifers l^–1 however, it seemedto be of little importance in the water column.     

Recognition of N-Glycoforms in Human Chorionic Gonadotropin by Monoclonal Antibodies and Their Interaction Motifs

The glycosylation of human chorionic gonadotropin (hCG) plays an important role in reproductive tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related cancers. However, developing an immunoassay directly against the N-linked oligosaccharides is unlikely because of the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG was purified from the urine of invasive mole patients, and the structure of its N-linked oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG antibodies MCA329 and MCA1024 against hCG and hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. Hydrogen/deuterium exchange-MS reveals that the peptide β65–83 of the hCG β subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas others are not affected by N-glycosylation. It is promising to improve glycoprotein biomarker-based cancer diagnostics by developing combined immunoassays that can determine the level of protein and measure the degree of N-glycosylation simultaneously.     

Slow Leaf Development of Evergreen Broad-leaved Tree Species in Japanese Warm Temperate Forests

Rates of light-saturated net photosynthesis (P_Nmax) and darkrespiration (Rd) on a leaf area basis, leaf dry mass per area(LMA), leaf nitrogen content on a leaf area basis (LNa) andinstantaneous nitrogen use efficiency (NUE=P_Nmax/LNa) were followedduring leaf development in six evergreen broad-leaved tree speciestypical of warm-temperate forests in Japan. These species wereCastanopsissieboldii, Quercus myrsinaefolia, Quercus glauca, Machilus thunbergii,Cinnamomum japonicumandNeolitsea sericea.When expansion of leafarea was complete, P_Nmax was about one third of its peak valueand increased for another 15 to 44 d. Rd at full leaf expansionwas about 1.5 to 3.5-times greater than steady-state rates.These facts suggest that leaf development was still underwayat the time of full leaf area expansion. Low P_Nmax at full leafexpansion was caused both by low leaf nitrogen content and lowNUE. P_Nmax increased with the increase in LMA during leaf developmentin all six species; data from the literature for other specieswith different life forms also indicated a similar tendency.The steady-state LMA varied markedly among species. Becauseleaves with larger steady-state LMAs need more resources fortheir construction, they will also need longer periods for maturation.We hypothesized that the period required for the attainmentof peak P_Nmax, the ‘leaf maturation period’, dependson the steady-state LMA. Plotting data from the present studytogether with those from literature for other plants acrossseveral life forms showed a strong positive relationship betweenleaf maturation period and steady-state LMA, supporting thehypothesis.Copyright 1998 Annals of Botany Company. Castanopsis sieboldii, Cinnamomum japonicum,delayed period, expansion period, full leaf expansion,Machilus thunbergii,maturation period,Neolitsea sericea, Quercus glauca, Quercus myrsinaefolia,steady-state LMA.     

四种天敌对麦双尾蚜的功能反应

一种蚜小蜂 Aphelinus sp. 对麦双尾蚜Diuraphis noxia (Mordvilko) 的功能反应为Holling I型,直线方程为N_a=0.6060N-3.4700。七星瓢虫 Coccinella septempunctata L. 成虫对麦双尾蚜功能反应也为I型,直线方程为N_a=0.6020N+5.9000;多异瓢虫 Hippodamia variegata Goeze成虫和斑腹蝇 Leucopis annulipes Zett. 3龄幼虫对麦双尾蚜的功能反应均为II型,关系式分别为1/N_a=1.2550/N+0.0046和1/N_a=1.3280/N+0.0071。     

Free oligosaccharide regulation during mammalian protein N-glycosylation

During protein N-glycosylation in mammalian cells, free oligosaccharides(fOS) are generated from lipid-linked oligosaccharides by apyrophosphatase activity and oligosaccharyltransferase and frommisfolded glycoproteins by peptide:N-glycanase in both the ERand cytoplasm. Trafficking machinery comprising oligosaccharide-specificER and lysosomal transporters, an endo-β-N-acetyl-glucosaminidase,and the cytosolic M2C1 mannosidase drives a flux of fOS fromthe ER to cytoplasm and from the cytoplasm into lysosomes wherefOS are degraded. Transport of fOS out of the ER is normallyefficient and if inhibited causes fOS to be secreted via theGolgi apparatus. By contrast, fOS clearance from the cytosolinto lysosomes is less efficient resulting in low micromolarconcentrations of fOS in the cytoplasm. Structural analysisof cytosolic fOS reveals oligosaccharide families whose relativeabundance highlights the importance of different ER-associateddegradation (ERAD) pathways for misfolded glycoproteins andsuggests that in liver cells substantial amounts of glycoproteinsdestined for ERAD may transit early compartments of the Golgiapparatus. Glycoprotein quality control and ERAD are controlledby N-glycan/lectin interactions and the fOS trafficking pathwaywould seem to ensure that fOS do not interfere with these processeswhich occur in both the ER and cytoplasm. Although Saccharomycescerevisiae strains harbouring mutations in genes of the yeastfOS metabolic pathway do not display obvious phenotypes, mammalianfOS are quantitatively more important and the processes leadingto their regulation are more complex, raising the possibilitythat distinct phenotypes will be seen in mammalian cells oranimals in which fOS metabolism is modified.     

Differential Effects of Day and Night Temperature on Development to Flowering in Rice

There are conflicting reports with regard to difference in effectsof day temperature (T_D) and night temperatures (T_N) on plantdevelopment. The objective of this study is to determine whetherthere are different effects ofT_DandT_Non development from sowingto flowering in rice (Oryza sativaL.). Plants of 24 rice cultivars were grown in naturally-lightedgrowth chambers at five diurnally constant (22, 24, 26, 28 and32 °C) and four diurnally fluctuating temperatures (26 /22,30 /22, 22 /26 and 22 /30 °C forT_D/T_Nwith 12hd^-1each) witha constant photoperiod of 12hd^-1. The treatments were selectedto enable the separation of effects ofT_DandT_Non developmentrate (DR). The response of DR to constant temperatures was typically nonlinear.This nonlinearity could not explain the difference in floweringdates between fluctuating temperatures with the same mean dailyvalue but oppositeT_D/T_Ndifferences. Differential effects ofT_DandT_NonDR to flowering were detected in all but one cultivar. In mostcases,T_Dexerted a greater influence thanT_N, in contrast withmany previous reports based on the assumption of a linearitybetween DR and temperature. The data were further analysed bya nonlinear model which separated effects ofT_DandT_N. The estimatedvalue for the optimumT_Nwas generally 25 –29 °C, about2 –4 °C lower than the estimated optimumT_Din mostcultivars. The effects ofT_DandT_Non DR were found to be interactivein some cultivars. These results form a new basis for modellingflowering dates in rice. Oryza sativa; rice; flowering; development; day and night temperature; thermoperiodicity