Genome-wide identification of SAMS gene family in Cucurbitaceae and the role of ClSAMS1 in watermelon tolerance to abiotic stress.

S-Adenosyl-L-methionine (SAM) is widely involved in plant growth, development, and abiotic stress response. SAM synthetase (SAMS) is the key enzyme that catalyzes the synthesis of SAM from methionine and ATP. However, the SAMS gene family has not been identified and their functions have not been characterized in most Cucurbitaceae plants. Here, a total of 30 SAMS genes were identified in nine Cucurbitaceae species and they were categorized into 3 subfamilies. Physicochemical properties and gene structure analysis showed that the SAMS protein members are tightly conserved. Further analysis of the cis-regulatory elements (CREs) of SAMS genes' promoter implied their potential roles in stress tolerance. To further understand the molecular functions of SAMS genes, watermelon SAMSs (ClSAMSs) were chosen to analyze the expression patterns in different tissues and under various abiotic stress and hormone responses. Among the investigated genes, ClSAMS1 expression was observed in all tissues and found to be up-regulated by abiotic stresses including salt, cold and drought treatments as well as exogenous hormone treatments including ETH, SA, MeJA and ABA. Furthermore, knockdown of ClSAMS1 via virus-induced gene silencing (VIGS) decreased SAM contents in watermelon seedings. The pTRSV2-ClSAMS1 plants showed reduced susceptibility to drought, cold and NaCl stress, indicating a positive role of ClSAMS1 in abiotic stresses tolerance. Those results provided candidate SAMS genes to regulate plant resistance against abiotic stresses in Cucurbitaceae plants.

Critical enzymes for biosynthesis of cucurbitacin derivatives in watermelon and their biological significance.

Various cucurbitacins have been isolated, and their structures have been elucidated. Owing to their economic potential and importance as active pharmacological compounds, their cytotoxicity in various cancer cells has been assessed. Here, we mined several candidate genes with potential involvement in cucurbitacin biosynthesis in watermelon (Citrullus lanatus) and performed in vitro enzymatic assays and instrumental analyses using various substrates to identify cucurbitacin functions and products. Enzymatic activities of two acetyltransferases (ACTs) and one UDP-glucosyltransferase (UGT) against cucurbitacins were confirmed, resulting in the synthesis of novel cucurbitacins in vivo and/or in vitro to our knowledge. As ACTs and UGT are involved in the dynamic conversion of cucurbitacins by catalyzing acetylation and glucosylation at moieties in the cucurbitacins skeleton, these findings improve our knowledge on how these genes contribute to the diversity of cucurbitacins.

Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.

Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. RESULTS: In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. CONCLUSION: We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.

Potential involvement of N-terminal acetylation in the quantitative regulation of the ε subunit of chloroplast ATP synthase under drought stress.

In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development.

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953-2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application.

BACKGROUND: Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system. RESULTS: A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml(-1) cell cultures) which can oxygenate linoleic acid resulting in high yields (18 µmol ml(-1) cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 µmol mg(-1) crude protein). The yield of C9-aldehydes from linoleic acid was 64%. CONCLUSION: The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.

Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system.

13-Lipoxygenase (13-LOX) and 13-hydroperoxide lyases (13-HPL) are the key enzymes for the production of the 'green note' compounds hexanal, (3Z)- and (2E)-hexenal in plant tissues. To produce high levels of 13-LOX and 13-HPL enzymatic activities for a biocatalytic process to generate C(6)-aldehydes on a large scale, soya bean 13-LOX (GmVLXC) and watermelon 13-HPL (ClHPL) genes were expressed in Nicotiana benthamiana using a viral vector system mediated by agroinfiltration. The N. benthamiana leaves produced high activity of watermelon HPL, but not GmVLXC 13-LOX. In addition, all leaves treated with bacterial suspension displayed a high activity of 9-LOX, indicating that the internal tobacco 9-LOX gene was highly induced through agroinfiltration because of wounding. GmVLXC and ClHPL transcripts could be detected in the corresponding transformed tobacco leaves by real-time RT-PCR analysis but the expression level of ClHPL was 24-fold higher than that of GmVLXC. Western blot analysis showed that LOX was present in all tobacco leaves which were treated with bacterial suspensions, but not in the untreated wild-type control. This result confirms that internal 9-LOX was highly induced by agroinfiltration. The highest levels of ClHPL activity under optimal infiltration conditions were 80 times the HPL activity of wild-type plants or plants transformed with control vector. A large amount of hexanal was formed when linoleic acid was incubated with extracts from N. benthamiana leaves over-expressing ClHPL in combination with GmVLXC-expressing yeast extracts. One gram of ClHPL-expressing N. benthamiana leaves (fresh weight) could produce 17 +/- 0.4 mg hexanal from 50 mg linoleic acid after 30 min.

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.).

A 1.8 kb 5'-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from -986 to -959 and from -472 to -424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative beta-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were approximately 10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves.

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.

A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources.

Six-carbon (C(6)) aldehydes and alcohols are important components of the aroma and flavor of fruits and vegetables. Soybean lipoxygenase (LOX) isozyme LOX 3 was reported not only to produce less 13-hydroperoxides, precursors of C(6) aldehydes, but also to convert them to ketodiene products. Here, we examined the effects of LOX 3 on hexenal formation from linolenic acid homogenized with watermelon 13-hydroperoxide lyase (HL)-overexpressing Nicotiana tabacum leaves and soybean acetone powder. Compared to the wild type, which contains LOXs 1, 2, and 3, the elimination of LOX 3 in LOX 1 + 2 facilitates greater production of hexenals. The use of LOX 2 alone yielded the highest hexenal production, while a two-step conversion was required for LOX 1 to produce hexenals at high levels due to different pH optima of the enzymes involved. These results clearly demonstrate that the soybeans lacking LOX 3 in combination with watermelon HL-overexpressing leaf tissues greatly enhance hexenal formation.

Co-expression of cytochrome b561 and ascorbate oxidase in leaves of wild watermelon under drought and high light conditions.

Despite carrying out C3 photosynthesis, wild watermelon (Citrullus lanatus sp.) exhibits exceedingly good tolerance to severe drought at high light intensities. However, the mechanism(s) by which this plant protects itself from photodamage has yet to be elucidated. In this study, we characterized wild watermelon cytochrome b561 (cyt b561), which potentially mediates regeneration of apoplastic ascorbate by transferring electrons from cytosolic ascorbate across the plasma membrane. Two cDNA species for wild watermelon cyt b561, designated CLb561A and CLb561B, were isolated. Levels of both CLb561A mRNA and protein were significantly elevated in the leaves during drought at a light intensity of 700 micromol photons m(-2) s(-1). The transcript of CLb561B was detected to a much lesser extent, but no CLb561B protein was produced under any condition used in this study. A transient expression assay with the CLb561A::green fluorescent protein fusion construct showed clear fluorescence on the plasma membrane of onion epidermal cells. The CLb561A protein was enriched in the plasma membrane fraction in leaves of transgenic tobacco expressing CLb561A. Moreover, the high activity of apoplastic ascorbate oxidase (AO), which was able to dispose of cyt b561-transferred reducing equivalents, increased in leaves of wild watermelon grown at high light intensity, but not lower light intensities. Taken together, these observations suggest the occurrence of a novel pathway for excess light energy dissipation in wild watermelon leaves, where excessive energy absorbed by chloroplasts can be transported to and dissipated safely in the apoplasts through the cooperative action of cyt b561 and AO.

Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves.

Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.

[Effect of low temperature stress on chilling tolerance and protective system against active oxygen of grafted watermelon].

This paper studied the chilling tolerance and the activity of protective system against active oxygen of own-rooted and grafted watermelon seedlings. The results showed that under low temperature stress, the chilling tolerance of the watermelon seedlings grafted on figleaf was higher than that of the seedlings grafted on Chaofeng F1, and that of the own-rooted seedlings was the lowest. The decrease of chlorophyll, ascorbate and GSH contents and superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase activities as well as the increase of MDA content in the leaves of both own-rooted and grafted watermelon seedlings indicated an injury on the protective system against active oxygen, and induced lipid peroxidation under low temperature stress. However, the activity of protective system against active oxygen was higher in grafted watermelon seedlings than in own-rooted watermelon seedlings. The activity of protective system against active oxygen in figleaf gourd-grafted watermelon seedlings with stronger chilling tolerance was higher than that of Chaofeng F1-grafted watermelon seedlings with weaker chilling tolerance. It could be concluded that the enhancement of chilling tolerance in grafted watermelon seedlings was related to the increase of antioxidants and antioxidative enzyme activities.

Ethylene-induced gene expression, enzyme activities, and water soaking in immature and ripe watermelon (Citrullus lanatus) fruit.

Watermelon fruit exhibit acute softening and placental-tissue water soaking following short exposure to exogenous ethylene. Experiments were performed to address transcript abundance and activities of cell wall and membrane hydrolases in placental tissue in response to treatment of watermelon fruit with ethylene. Watermelon fruit were harvested at immature and full-ripe stages and exposed to 50 microL L(-1) ethylene for 6 days at 20 degrees C. Ethylene affected the abundance of transcripts for PME (EC 3.2.1.11), and alpha-(EC 3.2.1.22) and beta-GAL (EC 3.2.1.23) but these effects were dependent on fruit maturity and appeared not to be associated with the water-soaking syndrome. PG (EC 3.2.1.15) and EXP mRNAs accumulated significantly in response to ethylene exposure. Additionally, the levels of mRNA and activities of LOX (EC 1.13.11.12), PLC (EC 3.1.4.3) and PLD (EC 3.1.4.4) were elevated in fruit of both maturity classes exposed to ethylene and were temporally associated with the visible symptoms of water soaking. The activity trends and transcript abundance in ethylene- compared with air-treated fruit indicate that PG, EXP, LOX, PLC and PLD levels increase with the onset and development of the water-soaking disorder and support the view that catabolic reactions targeting the membranes and cell-walls contribute to the disorder.