Multiple promoters driving the expression of astaxanthin biosynthesis genes can enhance free-form astaxanthin production.

Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.

Genome-wide analysis of GDSL-type esterases/lipases in Arabidopsis.

KEY MESSAGE: In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Identification of single nucleotide polymorphism markers associated with resistance to bruchids (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) and cultivated V. radiata through genotyping by sequencing and quantitative trait locus analysis.

BACKGROUND: Bruchid beetles are an important storage pest of grain legumes. Callosobruchus sp. infect mungbean (Vigna radiata) at low levels in the field, multiply during grain storage and can destroy seed stocks in a few months. Resistance against bruchid beetles has been found in wild mungbean V. radiata var. sublobata TC1966 and in cultivated mungbean line V2802. RESULTS: Bruchid resistance data were obtained from recombinant inbred line populations TC1966 (V. radiata var. sublobata) × NM92 (F12) and V2802 (V. radiata) × NM94 (F7). More than 6,000 single nucleotide polymorphic markers were generated through genotyping by sequencing (GBS) for each of these populations and were used to map bruchid resistance genes. One highly significant quantitative trait locus (QTL) associated with bruchid resistance was mapped to chromosome 5 on genetic maps of both populations, suggesting that TC1966 and V2802 contain the same resistance locus. Co-segregation of all markers associated with resistance indicated the presence of only one major resistance QTL on chromosome 5, while QTL analysis based on physical map positions of the markers suggested the presence of multiple QTLs on different chromosomes. The diagnostic capacity of the identified molecular markers located in the QTL to correctly predict resistance was up to 100 %. CONCLUSIONS: Molecular markers tightly linked to bruchid resistance loci of two different mungbean resistance sources were developed and validated. These markers are highly useful for developing resistant lines.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BACKGROUND: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean. RESULTS: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype. CONCLUSIONS: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a.

Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.

A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives.

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

BACKGROUND: Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. RESULTS: In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. CONCLUSIONS: We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Purification and immobilization of the recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as potential biocatalyst for the production of chlorophyllide and phytol.

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.

Arabidopsis SFAR4 is a novel GDSL-type esterase involved in fatty acid degradation and glucose tolerance.

BACKGROUND: SFARs (seed fatty acid reducers) belonging to the GDSL lipases/esterases family have been reported to reduce fatty acid storage and composition in mature Arabidopsis seeds. GDSL lipases/esterases are hydrolytic enzymes that possess multifunctional properties, such as broad substrate specificity, regiospecificity, and stereoselectivity. Studies on the physiological functions and biochemical characteristics of GDSL lipases/esterases in plants are limited, so it is important to elucidate the molecular functions of GDSL-type genes. RESULTS: We found that SFAR4 (At3g48460), a fatty acid reducer belonging to the Arabidopsis GDSL lipases/esterases family, was intensely expressed in embryo protrusion, early seedlings, and pollen. The characterization of recombinant SFAR4 protein indicated that it has short-length p-nitrophenyl esterase activity. In addition, SFAR4 enhanced the expression of genes involved in fatty acid metabolism during seed germination and seedling development. SFAR4 elevated the expression of COMATOSE, which transports fatty acids into peroxisomes, and of LACS6 and LACS7, which deliver long-chain acetyl-CoA for ß-oxidation. Furthermore, SFAR4 increased the transcription of PED1 and PNC1, which function in importing peroxisomal ATP required for fatty acid degradation. SFAR4 has another function on tolerance to high glucose concentrations but had no significant effects on the expression of the glucose sensor HXK1. CONCLUSIONS: The results demonstrated that SFAR4 is a GDSL-type esterase involved in fatty acid metabolism during post-germination and seedling development in Arabidopsis. We suggested that SFAR4 plays an important role in fatty acid degradation, thus reducing the fatty acid content.

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Identification of cucurbitacins and assembly of a draft genome for Aquilaria agallocha.

BACKGROUND: Agarwood is derived from Aquilaria trees, the trade of which has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. RESULTS: In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. DNA and RNA data are utilized to annotate many genes and protein functions in the draft genome. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. agallocha to biotic stress and a set of homologous genes in Arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qRT-PCR. CONCLUSIONS: This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria. The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants.

EMBRYONIC FLOWER1 and ULTRAPETALA1 Act Antagonistically on Arabidopsis Development and Stress Response.

Epigenetic regulation of gene expression is of fundamental importance for eukaryotic development. EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene that participates in Polycomb group-mediated transcriptional repression of target genes such as the flower MADS box genes AGAMOUS, APETALA3, and PISTILLATA. Here, we investigated the molecular mechanism underlying the curly leaf and early flowering phenotypes caused by reducing EMF1 activity in the leaf primordia of LFYasEMF1 transgenic plants and propose a combined effect of multiple flower MADS box gene activities on these phenotypes. ULTRAPETALA1 (ULT1) functions as a trithorax group factor that counteracts Polycomb group action in Arabidopsis (Arabidopsis thaliana). Removing ULT1 activity rescues both the abnormal developmental phenotypes and most of the misregulated gene expression of LFYasEMF1 plants. Reducing EMF1 activity increases salt tolerance, an effect that is diminished by introducing the ult1-3 mutation into the LFYasEMF1 background. EMF1 is required for trimethylating lysine-27 on histone 3 (H3K27me3), and ULT1 associates with ARABIDOPSIS TRITHORAX1 (ATX1) for trimethylating lysine-3 on histone 4 (H3K4me3) at flower MADS box gene loci. Reducing EMF1 activity decreases H3K27me3 marks and increases H3K4me3 marks on target gene loci. Removing ULT1 activity has the opposite effect on the two histone marks. Removing both gene activities restores the active and repressive marks to near wild-type levels. Thus, ULT1 acts as an antirepressor that counteracts EMF1 action through modulation of histone marks on target genes. Our analysis indicates that, instead of acting as off and on switches, EMF1 and ULT1 mediate histone mark deposition and modulate transcriptional activities of the target genes.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing.

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. BIOLOGICAL SIGNIFICANCE: Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Molecular and functional characterization of broccoli EMBRYONIC FLOWER 2 genes.

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.

Arabidopsis ENDO2: its catalytic role and requirement of N-glycosylation for function.

The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.

Paclobutrazol pre-treatment enhanced flooding tolerance of sweet potato.

The objective of this experiment was to study changes of antioxidants and antioxidative enzymes in the flooding-stressed sweet potato leaf, as affected by paclobutrazol (PBZ) treatment at 24 h prior to flooding. Sweet potato 'Taoyuan 2' were treated with 0 and 0.5 mg/plant of PBZ, afterwards subjected to non-flooding and flooding-stress conditions for 0, 1, 3, and 5 d, followed by a 2 d drainage period. The study was conducted as a factorial experiment in completely randomized blocks with three replications maintained within a screen house. Plants with various antioxidative systems responded differently to flooding stress according to the duration of the flooding period and subsequent drainage period. The increased levels of antioxidants and antioxidative enzymes observed on different days of flooding afforded the sweet potato leaf with improved flooding tolerance. Glutathione reductase activity in the leaf was significantly enhanced over 5 d continuous flooding followed by a drainage period, in comparison with non-flooding conditions. Under non-flooding conditions, antioxidative system of leaf was regulated and elevated by PBZ pre-treatment. PBZ treatment may enable sweet potato 'Taoyuan 2' to maintain the balance between the formation and the detoxification of activated oxygen species. Our results also show that under flooding-stress conditions, the level of 'Taoyuan 2' antioxidative system is linked to PBZ treatment. Pre-treating with PBZ may increase levels of various components of antioxidative systems after exposure to different durations of flooding and drainage, thus inducing flooding tolerance. PBZ exhibited the important function of enhancing the restoration of leaf oxidative damage under flooding stress after the pre-application of 0.5 mg/plant. These findings may have greater significance for farming in frequently flooded areas.