Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Improvement of shikimic acid production in <Emphasis Type="Italic">Escherichia coli</Emphasis> with growth phase-dependent regulation in the biosynthetic pathway from glycerol

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33 g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.     

Recombinant expression of glpK and glpD genes improves the accumulation of shikimic acid in E. coli grown on glycerol

Shikimic acid (SA) is an industrially important chiral compound used in diverse commercial applications, and the insufficient supply by isolation from plants and expensive chemical synthesis of SA has increased the importance of developing strategies for SA synthesis. In our previous studies, glycerol was observed to be an effective carbon source for SA accumulation in E. coli DHPYAAS-T7, where the PTS operon (ptsHIcrr) and aroL and aroK genes were inactivated, and the tktA, glk, aroE, aroF ^fbr , and aroB genes were overexpressed. For further investigation of the effects of glycerol aerobic fermentation on SA accumulation in E. coli BL21(DE3), the glpD, glpK genes and tktA, glk, aroE, aroF ^fbr , aroB genes were overexpressed simultaneously. The results indicated that SA production was increased 5.6-fold, while the yield was increased 5.3-fold over that of parental strain in shake flasks. It is demonstrated that the aerobic fermentation of glycerol associated with glpD and glpK gene overexpression increased glycerol flux, resulting in higher SA accumulation in E. coli BL21(DE3)-P-DK.     

The Response of Physiological Characteristics,Expression of OSC Genes,and Accumulation of Triterpenoids in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Sukto MeJA and SA Treatment

The pentacyclic triterpenoids from birch (Betula platyphylla suk) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we explored the effects of spraying 3-year-old white birch with different concentration of methyl jasmonate (MeJA) and salicylic acid (SA) on the expression of key genes in triterpenoid biosynthesis pathways and on the accumulation and physiological characteristics of triterpenoids in birch saplings. The results showed that spraying different concentration of MeJA and SA could obviously promote accumulation of total triterpenoids in 3-year-old white birch. The triterpenoid content in the stem bark was increased by 46.11 %, reaching 81.86 mg/g, after 1 day of treatment with 1 mmol·L^?1 MeJA (MJ2), and by 45.07 %, reaching 91.4 mg/g, after 14 days of treatment with 5 mmol·L^?1 SA (SA1). In addition, MeJA and SA treatment increased the contents of chlorophyll a and b, antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as photosynthetic performance, and affected the content of soluble sugar and soluble protein in birch leaf. Fluorescence quantitative polymerase chain reaction (qPCR) results showed that MeJA and SA treatment deferentially enhanced the key gene expression of cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and beta-amyrin synthase (BPY) in triterpenoid synthesis pathway in birch bark and leaves. The results showed that MeJA and SA induced triterpenoid synthesis of birch plant is closely related with not only the expression of key genes of triterpenoid synthesis pathway but also photosynthesis, anti-stress response and physiological indexes, suggesting that regulation of triterpenoid synthesis of birch by MeJA and SA may involve in more complex mechanisms at physiological and molecular levels.     

Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta)

The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300–2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum (‘F’ clade) and K. nitens of the ‘E2’ clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg^?1, Ψ?=??2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg^?1, Ψ?=??1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 μmol O₂ h^?1 mg^?1 chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ?=??5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 μmol O₂ h^?1 mg^?1 chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium.     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

Sensitive,selective gas chromatographic—mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity

One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[¹³C]sorbitol, ¹³C₆H₁₄O₆, 98.7%) as an internal standard. Gas chromatography—mass spectrometry (GC—MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[¹³C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1–8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r=0.999, r=0.997 and r=0.997, respectively. The precision of the GC—MS measurement of standard solution was C.V.=4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, K_m and V_max values of the reductase in erythrocytes were 115±19 mmol/l and 4.42±0.26 nmol/min/g of hemoglobin. In human liver, on the other hand, they were 75±132 mmol/l and 0.77±0.090 nmol/min/mg of protein, respectively. This difference of K_m values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.     

Salicylic acid-mediated reductions in yield in Nicotiana attenuata challenged by aphid herbivory

Aphid herbivory decreases primary production in natural ecosystems and reduces crop yields. The mechanism for how aphids reduce yield is poorly understood as some studies suggest aphid feeding directly impedes photosynthesis, whereas other studies suggest a change in allocation of resources from growth to defense compounds reduces yield. To determine the mechanisms underlying reduced plant growth by aphids, Nicotiana attenuata plants, native tobacco, were infested with Myzus persicae ssp. nicotianae, tobacco-adapted green peach aphids, at low and high densities, and plant performance including fitness was assessed. To test the direct defense capacity of salicylic acid (SA) on aphid performance, we fed aphids an artificial diet with varying levels of SA and measured their survivorship and fecundity. There was no detectable effect of aphid herbivory on net photosynthesis, yet herbivory reduced plant growth, final biomass (43 % at high aphid density), and seed set (18 % at high aphid density) at both low and high aphid infestation levels. High-density aphid attack during the rosette and flowering stage caused an increase in SA levels, but caused only a transient decrease in jasmonic acid concentration at low aphid density. SA concentrations similar to those found in infested flowering plants decreased aphid fecundity, suggesting that SA was an effective chemical defense response against aphids. These results suggest that as aphid densities increased the proximal cause of reduced growth and yield was not reduced photosynthesis, but instead resources may have been mobilized for defense via the SA pathway, decreasing the availability of resources for building plant biomass.     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.     

Modification of glycolysis and its effect on the production of l-threonine in Escherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and l-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an l-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more l-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less l-threonine than THRD, THRDΔpfkB produced levels of l-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high l-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of l-threonine.     

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-d-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.     

Assessment of synergistic combination potential of probiotic and bacteriophage against antibiotic-resistant Staphylococcus aureus exposed to simulated intestinal conditions

This study was designed to evaluate the combined effect of probiotic Lactobacillus rhamnosus and bacteriophage SA11 on the control of antibiotic-sensitive Staphylococcus aureus (ASSA) and antibiotic-resistant S. aureus (ARSA) under the simulated intestinal conditions. The survivability of ASSA and ARSA were determined in the simulated phosphate-buffered saline (PBS)-, trypticase soy broth (TSB)-, and milk-based gastric juices adjusted to pH 2.0, 3.0, and 5.0 at 37 °C for 30 min. The inhibitory effect of bacteriophage SA11 and probiotic on the growth of ASSA and ARSA was evaluated in the simulated intestinal juices at 37 °C for 20 h. The least reductions in the numbers of ASSA and ARSA were observed in the milk-based gastric juices at pH 2.0 (<1 log). No significant changes in the teichoic acid-mediated sliding motility were observed for ASSA and ARSA after 30-min exposure to the simulated gastric juices (pH 2.0, 3.0, and 5.0), responsible for the enhanced bacterial attachment to the epithelial cells. The bacteriophage SA11 was stable down to pH 5.0 and up to 0.06 % bile salts. The bacteriophage SA11 combined with probiotic effectively inhibited the growth of ASSA and ARSA in the simulated intestinal conditions, showing more than 4 log reduction. The relative expression levels of adhesion-related genes (clfA, eno, and fnbA) and efflux-related genes (mdeA, norB, and norC) were less decreased in ARSA than in ASSA after exposure to the simulated gastrointestinal conditions. These results might shed light on the application of bacteriophage to control the ingested antibiotic-resistant foodborne pathogens in the intestinal tract.     

Natronomonas gomsonensis sp. nov., isolated from a solar saltern

A halophilic archaeal strain, SA3^T, was isolated from sediment of a solar saltern in Gomso Bay, Republic of Korea. Cells of strain SA3^T were observed to be coccoid-shaped, to lyse in distilled water, Gram stain-negative and to form red-pigmented colonies. Strain SA3^T was found to require at least 18 % (w/v) NaCl for growth. Optimal growth was observed at 24 % (w/v) NaCl and 6 % (w/v) MgCl₂. The optimum pH and temperature for growth were determined to be pH 7.0 and 40 °C, respectively, while the strain was found to grow within pH and temperature ranges of 5.5–8.0 and 20–45 °C, respectively. The polar lipids were determined to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified phosphoglycolipids and unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SA3^T was most closely related to the members of the genus Natronomonas, Natronomonas moolapensis JCM 14361^T (95.2 %) and Natronomonas pharaonis JCM 8858^T (95.1 %). The genomic DNA G+C content (61.8 mol%) determined for strain SA3^T was slightly lower than those of N. moolapensis JCM 14361^T (63.4 mol%) and N. pharaonis JCM 8858^T (64.3 mol%). DNA–DNA hybridization values between N. moolapensis JCM 14361^T and N. pharaonis JCM 8858^T and strain SA3^T were <20 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe a new species of the genus Natronomonas, represented by strain SA3^T (=JCM 17867^T = KCTC 4088^T), for which we propose the name Natronomonas gomsonensis sp. nov.     

Low-cost culture medium for the production of proteases by <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products

The present study aims to maximize proteases production by Bacillus mojavensis SA strain and their use to produce bioactive protein hydrolysates from a meat by-product. The production of SA bacteria proteases was maximized using a culture medium based on wheat bran, which offer an advantage in minimizing the production cost and enhancing the enzyme activity by using agro-industrial wastes. The composition of media and cultural conditions for optimal proteases production by B. mojavensis SA strain were investigated. A successful and significant improvement of the alkaline proteases production (four folds) by the SA strain was achieved using the medium composed of (g/l): wheat bran, 50.0; KH₂PO₄, 0.5; K₂HPO₄, 0.5; CaCl₂, 2.0; pH 6.0, where the growth conditions were monitored at 37 °C with an agitation speed of 200 rpm. Interestingly, the enzyme preparation of B. mojavensis was applied for the preparation of protein hydrolysates from a meat by-product. Hydrolysis was carried out for 180 min at pH 12.0. The resulting hydrolysate displayed an important antioxidant activity as evaluated by the radical scavenging capacity, the reducing power, and the β-carotene bleaching inhibition. The present study showed the high proteases’ producing level by B. mojavensis SA strain in a low-cost fermentation medium (wheat bran) and their potential use in the production of bioactive protein hydrolysate from meat by-products.     

An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue

A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.     

Overexpression of GASA5 increases the sensitivity of Arabidopsis to heat stress

Basal thermotolerance is very important for plant growth and development when plants are subjected to heat stress. However, little is known about the functional mechanism of gibberellins (GAs) in the basal thermotolerance of plants. In the present work, we provide molecular evidence that a member of the gene family encoding the GA-stimulated Arabidopsis (GASA) peptides, namely GASA5, is involved in the regulation of seedling thermotolerance. The GASA5-overexpressing plants displayed a weak thermotolerance, with a faster cotyledon-yellowing rate, lower seedling-survival rate, and slower hypocotyl elongation, in comparison to the wild-type and GASA5 null-mutant (gasa5-1) plants, after heat-stress treatment. The short-hypocotyl phenotype of GASA5-overexpressing plants could be rescued by the exogenous application of salicylic acid (SA), the hormone found to protect plants from heat stress-induced damage. GASA5 expression was inhibited by heat stress but unaffected by the application of exogenous SA. However, expression of the gene encoding the noexpresser of PR genes 1 (NPR1), a key component of the SA-signaling pathway, was downregulated by GASA5 overexpression. Importantly, when different GASA5-genotype plants were treated with heat stress, several genes encoding heat-shock proteins, including HSP101, HSP70B, HSP90.1, HSP17.6-C1, and HSP60, were inhibited by GASA5 overexpression. Meanwhile, hydrogen peroxide was accumulated at high levels in heat stress-treated GASA5-overexpressing plants. These results suggest that the Arabidopsis GASA5 gene acts as a negative regulator in thermotolerance by regulating both SA signaling and heat shock-protein accumulation.