Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Long-term <Emphasis Type="Italic">ex vivo</Emphasis> monitoring of <Emphasis Type="Italic">in vivo</Emphasis> microRNA activity in liver using a secreted luciferase sensor

Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology. However, in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs. Sensors for miRNA were developed by adding miRNA target sequences to the 3′-untranslated region of Gaussia princeps luciferase (Gluc) mRNA. These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood. Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells, but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing. Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities. We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122, miR142 and miR-34a activities, as well as for exogenous miR-34a activity. Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.     

Chemoorganoheterotrophic Growth of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrosomonas eutropha</Emphasis>

The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, α-ketoglutarate, or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein (mmol pyruvate)⁻¹ and the maximum growth rates of N. europaea and N. eutropha are 0.094 d⁻¹ and 0.175 d⁻¹, respectively. In the presence of pyruvate and CO₂ about 80% of the incorporated carbon derives from pyruvate and about 20% from CO₂. Pyruvate is used as energy and only carbon source in the absence of CO₂ (chemoorganoheterotrophic growth). CO₂ stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase is down-regulated at increasing CO₂ concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced.     

RETRACTED ARTICLE: Archaeal <Emphasis Type="Italic">amoA</Emphasis> Genes Outnumber Bacterial <Emphasis Type="Italic">amoA</Emphasis> Genes in Municipal Wastewater Treatment Plants in Bangkok

The contribution of ammonia-oxidizing archaea (AOA) to nitrogen removal in wastewater treatment plants (WWTPs) remains unknown. This study investigated the abundance of archaeal (AOA) and bacterial (ammonia-oxidizing bacteria (AOB)) amoA genes in eight of Bangkok’s municipal WWTPs. AOA amoA genes (3.28 × 10⁷ ± 1.74 × 10⁷–2.23 × 10¹¹ ± 1.92 × 10¹¹ copies l⁻¹ sludge) outnumbered AOB amoA genes in most of the WWTPs even though the plants’ treatment processes, influent and effluent characteristics, removal efficiencies, and operation varied. An estimation of the ammonia-oxidizing activity of AOA and AOB suggests that AOA involved in autotrophic ammonia oxidation in the WWTPs. Statistical analysis shows that the numbers of AOA amoA genes correlated negatively to the ammonium levels in effluent wastewater, while no correlation was found between the AOA amoA gene numbers and the oxygen concentrations in aeration tanks. An analysis of the AOB sequences shows that AOB found in the WWTPs limited to only two AOB clusters which exhibit high or moderate affinity to ammonia. In contrast to AOB, AOA sequences of various clusters were retrieved, and they were previously recovered from a variety of environments, such as thermal and marine environments.     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Alginate production and <Emphasis Type="Italic">alg8</Emphasis> gene expression by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> in continuous cultures

Alginates are polysaccharides that are used as thickening agents, stabilizers, and emulsifiers in various industries. These biopolymers are produced by fermentation with a limited understanding of the processes occurring at the cellular level. The objective of this study was to evaluate the effects of agitation rate and inlet sucrose concentrations (ISC) on alginate production and the expression of the genes encoding for alginate-lyases (algL) and the catalytic subunit of the alginate polymerase complex (alg8) in chemostat cultures of Azotobacter vinelandii ATCC 9046. Increased alginate production (2.4 g l⁻¹) and a higher specific alginate production rate (0.1 g g⁻¹ h⁻¹) were obtained at an ISC of 15 g l⁻¹. Carbon recovery of about 100% was obtained at an ISC of 10 g l⁻¹, whereas it was close to 50% at higher ISCs, suggesting that cells growing at lower sucrose feed rates utilize the carbon source more efficiently. In each of the steady states evaluated, an increase in algL gene expression was not related to a decrease in alginate molecular weight, whereas an increase in the molecular weight of alginate was linked to higher alg8 gene expression, demonstrating a relationship between the alg8 gene and alginate polymerization in A. vinelandii for the first time. The results obtained provide a possible explanation for changes observed in the molecular weight of alginate synthesized and this knowledge can be used to build a recombinant strain able to overexpress alg8 in order to produce alginates with higher molecular weights.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

Mediated catalysis of <Emphasis Type="Italic">Paracoccus pantotrophus</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase by <Emphasis Type="Italic">P. pantotrophus</Emphasis> pseudoazurin: kinetics of intermolecular electron transfer

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E ⁰′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 10⁵ M⁻¹ s⁻¹ was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Quantification of Sulfate-reducing Bacteria in Industrial Wastewater,by Real-time Polymerase Chain Reaction (PCR) Using <Emphasis Type="Italic">dsrA</Emphasis> and <Emphasis Type="Italic">apsA</Emphasis> Genes

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5′-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 × 10² and 9.5 × 10⁶ copies/μL and between 1.2 × 10³ and 1.2 × 10⁷ copies/μL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10⁷–10⁸ targets mL⁻¹) and lower during the winter (about 10⁴–10⁵ targets mL⁻¹). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Effect of high temperature on photosynthesis and transpiration of sweet corn (<Emphasis Type="Italic">Zea mays</Emphasis> L. var. <Emphasis Type="Italic">rugosa</Emphasis>)

Four temperature treatments were studied in the climate controlled growth chambers of the Georgia Envirotron: 25/20, 30/25, 35/30, and 40/35 °C during 14/10 h light/dark cycle. For the first growth stage (V3-5), the highest net photosynthetic rate (P _N) of sweet corn was found for the lowest temperature of 28–34 μmol m⁻² s⁻¹ while the P _N for the highest temperature treatment was 50–60 % lower. We detected a gradual decline of about 1 P _N unit per 1 °C increase in temperature. Maximum transpiration rate (E) fluctuated between 0.36 and 0.54 mm h⁻¹ (≈5.0–6.5 mm d⁻¹) for the high temperature treatment and the minimum E fluctuated between 0.25 and 0.36 mm h⁻¹ (≈3.5–5.0 mm d⁻¹) for the low temperature treatment. Cumulative CO₂ fixation of the 40/35 °C treatment was 33.7 g m⁻² d⁻¹ and it increased by about 50 % as temperature declined. The corresponding water use efficiency (WUE) decreased from 14 to 5 g(CO₂) kg⁻¹(H₂O) for the lowest and highest temperature treatments, respectively. Three main factors affected WUE, P _N, and E of Zea: the high temperature which reduced P _N, vapor pressure deficit (VPD) that was directly related to E but did not affect P _N, and quasi stem conductance (QC) that was directly related to P _N but did not affect E. As a result, WUE of the 25/20 °C temperature treatment was almost three times larger than that of 40/35 °C temperature treatment.