Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

First Clarification of the Involvement of Glycosyltransferase MdUGT73CG22 in the Detoxification Metabolism of Nicosulfuron in Apple.

Nicosulfuron, an acetolactate synthase (ALS) inhibitor herbicide, is a broad-spectrum and highly effective post-emergence herbicide. Glycosyltransferases (GTs) are widely found in organisms and transfer sugar molecules from donors to acceptors to form glycosides or sugar esters, thereby altering the physicochemical properties of the acceptor molecule, such as participating in detoxification. In this study, nine glycosyltransferases in group D of the apple glycosyltransferase family I were predicted to possibly be involved in the detoxification metabolism of ALS-inhibiting herbicides based on gene chip data published online. In order to confirm this, we analysed whether the expression of the nine glycosyltransferase genes in group D was induced by the previously reported ALS-inhibiting herbicides by real-time PCR (polymerase chain reaction). It was found that the ALS-inhibiting herbicide nicosulfuron significantly increased the expression of the MdUGT73CG22 gene in group D. Further investigation of the mechanism of action revealed that the apple glycosyltransferase MdUGT73CG22 glycosylated and modified nicosulfuron both in vivo and ex vivo to form nicosulfuron glycosides, which were involved in detoxification metabolism. In conclusion, a new glycosyltransferase, MdUGT73CG22, was identified for the first time in this study, which can glycosylate modifications of the ALS-inhibiting herbicide nicosulfuron and may be involved in the detoxification process in plants, which can help to further improve the knowledge of the non-targeted mechanism of herbicides.

Synthesis, Herbicidal Activity, and Molecular Mode of Action Evaluation of Novel Aryloxyphenoxypropionate/Amide Derivatives Containing a Quinazolinone Moiety.

To develop aryloxyphenoxypropionate herbicides with a novel structure and improved activity, a total of 39 aryloxyphenoxypropionate/amide derivatives containing quinazolinone moiety were synthesized and further bioevaluated. The bioassay results in the greenhouse showed that most of the target compounds had good herbicidal activity under postemergence conditions, of which, QPP-I-6 displayed excellent herbicidal activity against Echinochloa crusgalli, Digitaria sanguinalis, Spartina alterniflora, Eleusine indica, and Pennisetum alopecuroides with inhibition rates >90% at a dosage of 187.5 g ha-1. More importantly, QPP-I-6 displayed higher crop safety to Gossypium hirsutum, Glycine max, and Arachis hypogaea than the commercial herbicide quizalofop-p-ethyl. Studying the molecular mode of action by phenotypic observation, membrane permeability evaluation, transcriptomic analysis, and in vivo ACCase activity evaluation reveals that QPP-I-6 is a novel ACCase inhibitor. The present work demonstrates that QPP-I-6 can serve as a lead compound for further developing novel ACCase-inhibiting herbicides.

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70 g/L vs 2.56 g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6 g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

Highly drug/target-tolerant neutralizing antibody (NAb) assay development through target-based drug depletion and drug-based NAb extraction for an anti-EGFR therapeutic monoclonal antibody.

The reduction of immunogenicity is fundamental for the development of biobetter Erbitux, given that the development of an immune response reduces treatment efficacy and may lead to potential side effects. One of the requirements for the clinical research of a Erbitux biobetter candidate (CMAB009) is to develop a neutralizing antibody (NAb) assay, and sufficient drug and target tolerance for the assay is necessary. Here, we describe the development of a competitive ligand binding (CLB) assay for CMAB009 with high drug and target tolerance through target-based drug depletion and drug-based NAb extraction, the integrated experimental strategy was implemented to simultaneously mitigate drug interference and enhance target tolerance. Following troubleshooting and optimization, the NAb assay was validated for clinical sample analysis with the sensitivity of 92 ng/mL, drug tolerance of 70 µg/mL and target tolerance of 798 ng/mL. The innovative drug depletion and NAb extraction achieved though the combination of drug and target beads would enable the development of reliable NAb assays for many other therapeutics that overcome drug and its target interference for more precise and sensitive NAb assessment.

Diphenyl Ether Derivatives as Novel GPR120 Agonists for the Treatment of Type 2 Diabetes Mellitus.

Diabetes mellitus (DM) is a serious disease affecting human health. Numerous attempts have been made to develop safe and effective new antidiabetic drugs. Recently, a series of G protein-coupled receptors for free fatty acids (FFAs) have been described and characterized, and small molecule agonists and antagonists of these receptors show considerable promise for managing diabetes and related complications. FFA-activated GPR120 could stimulate the release of glucagon-like peptide-1(GLP-1), which can enhance the glucose-dependent secretion of insulin from pancreatic ß cells. GPR120 is a promising target for treating type 2 DM (T2DM). Herein we designed and synthesized a series of novel GPR120 agonists based on the structure of TUG-891, which was the first potent and selective GPR120 agonist. Among the designed compounds, 18 f showed excellent GPR120 activation activity and high selectivity for GPR40 in vitro. Compound 18 f dose-dependently improved glucose tolerance in normal mice, and no hypoglycemic side effects were observed at high dose. In addition, compound 18 f increased insulin release and displayed good antidiabetic effect in diet-induced obese mice. Molecular simulations illustrated that compound 18 f could enter the active site of GPR120 and interact with Arg99. Based on these observations, compound 18 f may be a promising lead compound for the design of novel GPR120 agonists to treat T2DM.

Design, synthesis, and biological evaluation of membrane-active honokiol derivatives as potent antibacterial agents.

Infections caused by drug-resistant bacteria have emerged to be one of the greatest threats to global public health, and new antimicrobial agents with novel mechanisms of action hence are in an urgent need to combat bacterial resistance. Herein, we reported the design, synthesis, and antibacterial evaluation of novel honokiol derivatives as mimics of antimicrobial peptides (AMPs). These mimics showed potent antimicrobial properties against Gram-positive bacteria. Among them, the most promising compound 13b exhibited excellent antibacterial activity, rapid bactericidal properties, avoidance of antibiotic resistance, and weak hemolytic and cytotoxic activities. In addition, compound 13b not only inhibited the biofilm formation but also destroy the preformed biofilm. Mechanism studies further revealed that compound 13b killed bacteria rapidly by interrupting the bacterial membrane. More intriguingly, compound 13b exhibited potent in vivo antibacterial efficacy in a mouse septicemia model induced by Staphylococcus aureus ATCC43300. These results highlight the potential of 13b to be used as therapeutic agents.

Genome-wide analysis of the apple family 1 glycosyltransferases identified a flavonoid-modifying UGT, MdUGT83L3, which is targeted by MdMYB88 and contributes to stress adaptation.

The plant family 1 UDP-glycosyltransferases (UGTs) are increasingly being investigated because of their contribution to plant secondary metabolism and other diverse biological roles. The apple (Malus domestica) is one of the most widely cultivated fruit trees with great economic importance. However, little is known regarding the apple UGTs. In this study, we identified 229 members of family 1 through a genome-wide analysis of the apple UGTs, which were clustered into 18 groups, from A to R. We also performed detailed analysis of 34 apple UGTs by quantitative RT-PCR, and discovered a number of stress-regulated UGTs. Among them, we characterized the role of MD09G1064900, also named MdUGT83L3, which was significantly induced by salt and cold. In vivo analysis showed that it has high activity towards cyanidin, and moderate activity towards quercetin and keampferol. Transgenic callus and regenerated apple plants overexpressing MdUGT83L3 showed enhanced tolerance to salt and cold treatments. Overexpression of MdUGT83L3 also increased anthocyanin accumulation in the callus tissues and enhanced ROS clearing upon exposure to salt and cold stresses. Furthermore, via yeast-one-hybrid assay, EMSA and CHIP analyses, we also found that MdUGT83L3 could be directly regulated by MdMYB88. Our study indicated that MdUGT83L3, under the regulation of MdMYB88, plays important roles in salt and cold stress adaptation via modulating flavonoid metabolism in apple.

Characterization of the complete chloroplast genome of Toddalia asiatica (L.) Lam.

Toddalia asiatica (L.) Lam. belongs to family Rutaceae and mainly distributes in dry areas of bushes in tropical Africa, Asia, and Swaziland. Sometimes it can be used as fodder for goats, but it has been used as herbs in traditional medical treatment for 1000 years. In this study, we sequenced the sample of T. asiatica and determined its complete chloroplast genome. The length of CP genome is 158,434 bp, includes two invert repeats (IR) regions of 27,008 bp, a large single-copy (LSC) region of 86,132 bp, and a short single-copy (SSC) region of 18,286 bp. There are 133 genes, which includes 88 protein-coding genes, 8 rRNA and 37 tRNA, and 38.5% overall GC content. Each of trnK-UUU, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC, and ndhA genes contains a intron, clpP and ycf3 contains 2 intron. The phylogenetic analysis result shows that T. asiatica has the closest relationship with Zanthoxylum armatum (MT990984) and Zanthoxylum nitidum (MN508801).

Characterization of the complete chloroplast genome of Syzygium nervosum.

Syzygium nervosum A.Cunn. ex DC (also named as Cleistocalyx operculatus) is a member of genus Syzygium mainly distributed in China, Vietnam and some other tropical countries and belongs the family of Myrtaceae. Syzygium nervosum is a popular medicinal plant, some species of genus Syzygium have been used in folk medicine. In this study, we sequenced the sample of Syzygium nervosum and determined its complete chloroplast genome. The complete chloroplast genome of Syzygium nervosum is 158,979 bp in length, and contained a large single copy (LSC) with 88,099 bp in length, a small single copy (SSC) with 18,756 bp in length and two inverted repeat (IR) regions of 26,062 bp each. It includes 85 protein coding genes, 8 rRNA and 37 tRNA, and 37% overall GC content. Each of trnK-UUU, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC and ndhA genes contain an intron, the gene clpP and ycf3 contain 2 introns. The phylogenetic position shows that Syzygium nervosum has the closest relationship with Syzygium aromaticum.

Identification and functional characterization of a new flavonoid synthase gene MdFLS1 from apple.

MAIN CONCLUSION: The flavonoid synthase gene MdFLS1 from apple, which possibly plays an important role in anthocyanin synthesis, accumulates in the purple-red branches of Malus 'Pink spire'. Flavonoid metabolism serves an important function in plant growth and development. In this study, we selected 20 varieties of apple lines, 10 green and ten red branches, from the plant nursery of Qingdao Agriculture Academy. Metabolite analysis revealed that large amounts of anthocyanins accumulated in the purple-red branches of M. 'Pink spire'. Real-time polymerase chain reaction showed that the expression of the flavonol synthase gene MdFLS1 was over 1500-fold higher in M. 'Pink spire' than in the other varieties. A single base A was inserted at the first three bases of the active binding site of MdFLS1 to prove that the purple-red colour of apple leaves and stems in M. 'Pink spire' may be caused by the inactivation of MdFLS1 protein. The results of in vitro enzymatic reaction revealed that the MdFLS1 protein lost its activity. MdFLS1 was expressed in Arabidopsis thaliana to explore further its functions. High-expression wild-type strains (OE1 and OE2) and high-expression strains of A-base insertion (A-OE1 and A-OE2) were obtained. Compared with the wild-type strains, the overexpression lines showed lighter tissue colour and less accumulation of anthocyanins. However, A-OE1 and A-OE2 showed no difference in colouration. In conclusion, we speculated that the MdFLS1 gene in M. 'Pink spire' cannot bind flavonoids, triggering the synthesis of anthocyanins in another branch of the flavonoid metabolic pathway and resulting in the purple-red colouration of apple leaves and stems. These results suggest that MdLS1 is a potential genetic target for breeding high-flavonoid apples in future cultivar development.

Characterization of the complete chloroplast genome of Sansevieria trifasciata var. Laurentii.

Sansevieria trifasciata var. laurentii (S. trifasciata) is a kind of popular in-door and out-door plant around world, it is not only known as the ornamental plant, but also as medical plant. It belongs to the Draceanaceae family, Draceanaceae includes more than 60 species distributed in tropical and subtropical dry climate regions. In this study, we sequenced the sample of S. trifasciata and determined its complete chloroplast genome. The length of CP genome is 155,179 bp, includes two invert repeats (IR) regions of 26,513 bp, a large single copy (LSC) region of 83,680 bp and a short single copy (SSC) region of 18,473 bp. There are 133 genes, which includes 87 protein coding genes, 8 rRNA and 38 tRNA, and 37.5% overall GC content. Each of trnK-UUU, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC and ndhA genes contains a intron, clpP and ycf3 contains 2 intron. The phylogenetic position shows that S. trifasciata has the closest relationship with Rohdea Chinensis (MH356725.1).

Phosphine-catalyzed [3 + 2] annulation of ß-sulfonamido-substituted enones with trans-α-cyano-α,ß-unsaturated ketones for the synthesis of highly substituted pyrrolidines.

To synthesize highly substituted pyrrolidines, we developed a phosphine-catalyzed [3 + 2] annulation of ß-sulfonamido-substituted enones with trans-α-cyano-α,ß-unsaturated ketones. We prepared a series of pyrrolidines under mild conditions with high yields and moderate-to-good diastereoselectivities. A catalytic mechanism for this reaction is suggested.

Cold-tolerant introgression line construction and low-temperature stress response analysis for bell pepper.

We aimed to screen cold-tolerant introgression lines (ILs) of bell pepper and investigate stress responses of these bell peppers under low temperature. Seedlings of cold-resistant wild-type bell pepper CA157, cultivated bell pepper CA52, and their ILs were evaluated for their tolerance to low temperature. Electrical conductivity measurement was performed on ILs and two parents. Then, contents of physiological and biochemical indexes including malondialdehyde (MDA), proline, and soluble sugar content were examined. Moreover, the superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) activities were further investigated. Finally, the chlorophyll fluorescence (PSII) parameters in all pant leaves were examined.A total of 22 IL plants showed better resistance than their recurrent parent CA52. CL122 was one of the most outstanding plants in ILs that had similar performance with wild bell pepper CA157. Cold resistance analysis based on physiological and biochemical indexes showed that factors such as electrical conductivity, MDA, and PSII were closely related to cold resistance among CA157, CA52, and CL122 under low-temperature stress. In conclusion, ILs constructed in the current study might be used for cold resistance gene introgression between wild and cultured species. Moreover, CL122 might be a novel bridge material for understanding low-temperature response in bell pepper. Furthermore, electrical conductivity, MDA, and PSII might be used to identify the low-temperature resistance of bell pepper plants in a period of obvious differentiation.

Design and Synthesis of Novel 4-Hydroxyl-3-(2-phenoxyacetyl)-pyran-2-one Derivatives for Use as Herbicides and Evaluation of Their Mode of Action.

In order to develop a novel herbicide containing the ß-triketone motif, a series of 4-hydroxyl-3-(2-phenoxyacetyl)-pyran-2-one derivatives were designed and synthesized. The bioassay results showed that compound II15 had good pre-emergent herbicidal activity even at a dosage of 187.5 g ha-1. Moreover, compound II15 showed a broader spectrum of weed control when compared with a commercial herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and displayed good crop safety to Triticum aestivum L. and Zea mays Linn. when applied at 375 g ha-1 under pre-emergence conditions, which indicated its great potential as a herbicide. More importantly, studying the molecular mode of action of compound II15 revealed that the novel triketone structure is a proherbicide of its corresponding phenoxyacetic acid auxin herbicide, which has a herbicidal mechanism similar to that of 2,4-D. The present work indicates that the 4-hydroxyl-3-(2-phenoxyacetyl)-pyran-2-one motif may be a potential lead structure for further development of novel auxin-type herbicides.

Identification and characterization of the first cytokinin glycosyltransferase from rice.

BACKGROUND: Cytokinins are one of the five major hormones families in plants and are important for their normal growth and environmental adaptability. In plants, cytokinins are mostly present as glycosides in plants, and their glycosylation modifications are catalyzed by family 1 glycosyltransferases. Current research on cytokinin glycosylation has focused on the biochemical identification of enzymes and the analysis of metabolites in Arabidopsis. There are few studies that examine how cytokinin glycosylation affects its synthesis and accumulation in plants. It is particularly important to understand these processes in food crops such as rice (Oryza sativa); however, to date, cytokinin glycosyltransferase genes in rice have not been reported. RESULTS: In this study, we identified eight rice genes that were functionally homologous to an Arabidopsis cytokinin glycosyltransferase gene. These genes were cloned and expressed in a prokaryotic system to obtain their purified proteins. Through enzymatic analysis and liquid chromatography-mass spectrometry, a single rice glycosyltransferase, Os6, was identified that glycosylated cytokinin in vitro. Os6 was overexpressed in Arabidopsis, and the extraction of cytokinin glycosides showed that Os6 is functionally active in planta. CONCLUSIONS: The identification and characterization of the first cytokinin glycosyltransferase from rice is important for future studies on the cytokinin metabolic pathway in rice. An improved understanding of rice cytokinin glycosyltransferases may facilitate genetic improvements in rice quality.

Constitutive expression of CaHSP22.5 enhances chilling tolerance in transgenic tobacco by promoting the activity of antioxidative enzymes.

Chilling stress limits the productivity and geographical distribution of many organisms throughout the world. In plants, the small heat shock proteins (sHSPs) belong to a group of proteins known as chaperones. The sweet pepper (Capsicum annuum L.) cDNA clone CaHSP22.5, which encodes an endoplasmic reticulum-located sHSP (ER-sHSP), was isolated and introduced into tobacco (Nicotiana tabacum L.) plants and Escherichia coli. The performance index and the maximal efficiency of PSII photochemistry (Fv/Fm) were higher and the accumulation of H2O2 and superoxide radicals (O2-) was lower in the transgenic lines than in the untransformed plants under chilling stress, which suggested that CaHSP22.5 accumulation enhanced photochemical activity and oxidation resistance. However, purified CaHSP22.5 could not directly reduce the contents of H2O2 and O2- in vitro. Additionally, heterologously expressed recombinant CaHSP22.5 enhanced E. coli viability under oxidative stress, helping to elucidate the cellular antioxidant function of CaHSP22.5 in vivo. At the same time, antioxidant enzyme activity was higher, which was consistent with the lower relative electrolyte conductivity and malondialdehyde contents of the transgenic lines compared with the wild-type. Furthermore, constitutive expression of CaHSP22.5 decreased the expression of other endoplasmic reticulum molecular chaperones, which indicated that the constitutive expression of ER-sHSP alleviated endoplasmic reticulum stress caused by chilling stress in plants. We hypothesise that CaHSP22.5 stabilises unfolded proteins as a chaperone and increases the activity of reactive oxygen species-scavenging enzymes to avoid oxidation damage under chilling stress, thereby suggesting that CaHSP22.5 could be useful for improving the tolerance of chilling-sensitive plant types.

Enzymatic single-step preparation and antioxidant activity of hetero-chitooligosaccharides using non-pretreated housefly larvae powder.

A novel chitinolytic enzyme-producing bacterium Chitiniphilus sp. LZ32 was isolated. Non-pretreated Housefly larvae powder (HLP) was used as an adsorbent to purify chitinolytic enzymes. The optimal conditions for hydrolysis of HLP by purifying enzymes chitinolytic enzymes were investigated. HPLC and MALDI-TOF analyses indicated that HLP enzymatic hydrolyzates mainly contain N-acetylglucosamine (GlcNAc) and hetero-chitooligosaccharides (COS) composed of GlcN and GlcNAc. The hetero-chitooligosaccharides (COS) had a degree of polymerization (DP) in the 2-6 range. The maximum production of COS was 158.3µg/mL after 72h of incubation. Maximum pentamer (51.2µg/mL) and hexamer concentrations (36.1µg/mL) were achieved at hydrolysis times of 72 and 84h, respectively. Antioxidant activities of purified COS products (PCOS) from different hydrolysis times were investigated in vitro. PCOS produced by hydrolysis times of 72h (PCOS-72) exhibited the strongest hydroxyl-scavenging ability and reducing power. These results indicate the potential of Chitiniphilus sp. LZ32 for COS production using HLP.

TM6, a novel nuclear matrix attachment region, enhances its flanking gene expression through influencing their chromatin structure.

Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.

The protective mechanisms of CaHSP26 in transgenic tobacco to alleviate photoinhibition of PSII during chilling stress.

A known sweet pepper cDNA clone, CaHSP26 encoding the chloroplast-localized small heat shock protein (CPsHSP), was isolated and introduced into tobacco plants. It has been reported that CaHSP26 is a member of the CPsHSP gene family related to extreme temperature tolerance in plants. In the present work, the transcripts were detected in the transgenic tobacco lines. The actual quantum yield of photosynthesis (ΦPSII), non-photochemical quenching, and stomatal conductance (gs) in the transgenic lines overexpressing CaHSP26 were higher than those in the wild-type plants under a range of photosynthetic photon flux density during chilling stress. Electron microscopic analysis showed that the transgenic line (L1) had larger size of stomata to lessen stomatal limitation. The activities of ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) were also higher in the transgenic lines than those in wild-type plants. Additionally, a significant increase in cis-unsaturated fatty acid contents was observed in transgenic lines due to lower temperatures. These results suggested that CaHSP26 protein plays an important role in protection of PSII by maintaining the antioxidative enzyme activities to avoid or mitigate photooxidation and increasing the fluidity of the thylakoid membrane during chilling stress under low irradiance. Key message CaHSP26 protein protects PSII by maintaining the antioxidative enzyme activities to avoid or mitigate photooxidation and increases the fluidity of the thylakoid membrane during chilling stress under low irradiance.