Urolithin A exerts a protective effect on lipopolysaccharide-induced acute lung injury by regulating HMGB1-mediated MAPK and NF-κB signaling pathways.

Acute lung injury (ALI) is a severe inflammatory disorder that has a high morbidity and mortality rate. Urolithin A (UA) is reported to have anti-inflammatory and anti-oxidative effects in ALI. However, its molecular mechanisms in ALI remain to be explored. Mice and BEAS-2B cells were administrated with lipopolysaccharide (LPS) to mimic the ALI model in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to detect the pathological injury of lung tissues. The levels of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and culture supernatant and the levels of oxidative stress markers in lung tissues were measured using ELISA. DCFH-DA probe was used to assess the reactive oxygen species (ROS) level. TUNEL staining and flow cytometry were performed to determine cell apoptosis. The key targets and pathways were confirmed by immunohistochemistry (IHC) and western blot. UA suppressed the pathologic damage, wet/dry weight ratio, and total protein and inflammatory cells in BALF. UA decreased neutrophil infiltration and proinflammatory cytokines production. UA reduced the level of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in pulmonary tissues. UA also inhibited cell apoptosis in lung tissues by decreasing Bax expression and increasing Bcl-2 expression. In addition, UA suppressed LPS-induced inflammatory factor production, ROS level, and cell apoptosis in BEAS-2B. Importantly, UA decreased the expression of HMGB1 in LPS-treated mice and BEAS-2B cells. HMGB1 overexpression greatly abrogated the inhibition of UA on inflammation, ROS, and cell apoptosis in LPS-administrated BEAS-2B. Furthermore, UA treatment suppressed the phosphorylated levels of p38, JNK, ERK, and p65 in LPS-administrated mice and BEAS-2B cells. UA alleviated lung inflammation, oxidative stress, and apoptosis in ALI by targeting HMGB1 to inactivate the MAPK/NF-κB signaling, suggesting the potential of UA to treat ALI.

Knockout of SlYTH2, encoding a YTH domain-containing protein, caused plant dwarfing, delayed fruit internal ripening, and increased seed abortion rate in tomato.

The m6A is one of the most abundant and widespread modifications in eukaryotic mRNAs, which regulates gene expression at the post-transcriptional level and plays key roles in many physiological processes. The YT521-B homologous (YTH) domain-containing proteins act as m6A readers to regulate m6A-RNA metabolism processes and mediate the various functions of m6A modification. Previously, we reported tomato contains 9 YTH genes, among which SlYTH2 is relatively highly expressed. This study reports the physiological functions of SlYTH2 in tomato. SlYTH2 protein is distributed in the nucleus and cytoplasm, and its three-dimensional structure is highly similar to human HsYTHDF1. SlYTH2 knockout through Crispr/Cas9 gene editing technology leaded to pleiotropic phenotypes, including dwarfing plant, delayed fruit internal ripening process, and increased seed abortion rate. The deletion of SlYTH2 gene increased the accumulation of endogenous ABA, decreased the content of endogenous GA3, and enhanced the sensitivity of seed germination to exogenous ABA and seedling growth to exogenous GA3. RNA-Seq data showed that the expression levels of multiple hormone-related genes were altered in SlYTH2 knockout line. These facts suggested SlYTH2 plays its physiological roles related to ABA, gibberellin and other hormones in tomato.

Co-production of biogas and humic acid using rice straw and pig manure as substrates through solid-state anaerobic fermentation and subsequent aerobic composting.

Compared with wet anaerobic digestion, solid-state fermentation possesses many merits such as low water consumption, high biogas yield and low processing cost. In this work, co-producing biogas and humic acid (HA) by two-step solid-state fermentation was innovatively investigated using rice straw and pig manure as materials. The result indicates that C/N ratio, straw particle size, and total solid content (TS%) caused significant effects on the solid-state fermentation process. At the first step for anaerobic biogas fermentation, the optimal fermentation conditions included C/N ratio of 27.5, straw particle size of 0.85 mm and TS% of 25%. The maximal biogas productivity and methane content were up to 0.43 m3/(m3·d) and 64.88%, respectively. This means that biogas production was significantly improved by adjusting C/N ratio during the co-fermentation of rice straw and pig manure. Following, the digested residue was aerobically composted for HA biosynthesis to improve the fertilizer efficiency of the fermented substrate. The optimal aeration rate of 0.75 L/min was obtained, and the volatile solid (VS) degradation rate, HA content, and the germination index (GI) value were up to 19.16%, 100.89 mg/g, and 103.07%, respectively, which indicates that HA biosynthesis and compost maturity were significantly enhanced. Therefore, the co-production of biogas and HA using rice straw and pig manure as fermentation materials was achieved by adopting the two-step solid-state fermentation, and the bioconversion efficiencies of livestock manure and straw were significantly improved.

Tomato SlYTH1 encoding a putative RNA m6A reader affects plant growth and fruit shape.

N6-methyladenosine (m6A), the most abundant and common modification on eukaryotic mRNA, plays crucial roles in multiple biological processes through controlling endogenous gene activity in organisms. The m6A reader specifically recognizes the m6A mark to mediate the regulation of m6A on mRNA, and determines the fate of its target mRNA. In plants, the currently confirmed m6A readers are YTH (YT521B homology) domain-containing proteins. We previously reported that tomato contains 9 YTH genes, of which SlYTH1 has the strongest expression. The present study reports the functional characterization of SlYTH1 in tomato. SlYTH1 mutants generated via CRISPR/Cas9 technology exhibited pleiotropic phenotypes, including low seed germination rate, shortened seedling root, retarded plant growth and development during vegetative development, and elongated and longitudinally flattened fruit with reduced the locule number. SlYTH1 knockout reduced GA3 content and downregulated the expression of related genes in gibberellin biosynthesis pathway. Moreover, exogenous GA3 application could partially restore the phenotypic defects caused by SlYTH1 mutations. SlYTH1 knockout could alleviate the inhibition of seedling root elongation by exogenous GA3 application at relatively low concentration. These facts indicated SlYTH1 is involved in regulating gibberellin biosynthesis and plays important roles in multiple physiological processes in tomato.

Genome-wide identification and characterization of YTH domain-containing genes, encoding the m6A readers, and their expression in tomato.

KEY MESSAGE: 9 YTH genes in tomato were identified and cloned, and their expression patterns were comprehensively analyzed, which reveal potential multiple roles in development and fruit ripening. N6-methyladenosine (m6A) is an abundant and pervasive post-transcriptional modification in eukaryotic mRNAs. The YTH domain-containing proteins act as m6A readers to read m6A marks and transduce their downstream regulatory effects by altering m6A-mRNA metabolism processes. Identification of YTH proteins is essential for understanding the regulatory mechanisms of m6A in physiological processes, but little is known about YTH proteins in tomato, a model system for fruit development. Here, we report that tomato genomes contain a total of 9 SlYTH genes. While YTH proteins of both tomato and Arabidopsis can be classified into two subfamilies, the member distributions in subfamilies are very different between the two species. Homology modeling exhibited the similar three-dimensional structures of SlYTH proteins to human YTHDF1 or YTHDC1. Multiple hormone-response elements locating on the promoters of SlYTH genes indicate that they are involved in the physiological processes related to phytohormone. SlYTH genes are ubiquitous and spatiotemporal dynamic expression in tomato. Eight SlTYH genes have the strongest expression in stamens among the parts of flowers. Throughout fruit ontogeny, most of the SlYTH genes display obvious high mRNA levels during the developmental phases (4 dpa to mature green); moreover, SlYTH1 and SlYTH2 have absolute predominant expressions demonstrated by RNA-seq. The results lay a foundation for future characterizations on the functions of YTH proteins and m6A regulatory mechanism in tomato.

A novel microRNA, SlymiR208, promotes leaf senescence via regulating cytokinin biosynthesis in tomato.

Leaf senescence is a highly-programmed developmental process during the plant life cycle. Cytokinin (CK) has been widely acknowledged as a negative regulator to delay leaf senescence. MiRNAs play key roles in a variety of developmental and physiological processes through negatively regulating their target gene expression. However, to date, the roles of microRNAs (miRNAs) in CK biosynthesis remain unclear, and the knowledge on miRNA regulation of leaf senescence is still very limited. Isopentenyltransferases (IPTs) catalyze the initial and rate-limiting step of CK biosynthesis in higher plants. Our previous work uncovered that silencing of SlIPT4 expression in tomato resulted in premature leaf senescence. Here, we identified a novel tomato miRNA, SlymiR208, which regulates the expression of SlIPT2 and SlIPT4 at the post-transcriptional level. SlymiR208 expression is ubiquitous in tomato and exhibits an opposite transition to its target transcripts in aged leaf. SlymiR208 overexpression in tomato sharply reduced the transcript levels of SlIPT2 and SlIPT4, and the concentrations of endogenous CKs in leaves. The early leaf senescence caused by SlymiR208 overexpression was consistent with the phenotype of SlIPT4-silenced lines. The data demonstrated that SlymiR208 is a positive regulator in leaf senescence through negatively regulating CK biosynthesis via targeting SlIPT2 and SlIPT4 in tomato. This study indicated that post-transcriptional regulation via miRNA is a control point of CK biosynthesis and added a new layer to the understanding of the regulation of CK biosynthesis in tomato and a new factual proof to support that miRNAs are involved in leaf senescence.

SlCAND1, encoding cullin-associated Nedd8-dissociated protein 1, regulates plant height, flowering time, seed germination, and root architecture in tomato.

KEY MESSAGE: Silencing of SlCAND1 expression resulted in dwarfish, loss of apical dominance, early flowering, suppression of seed germination, and abnormal root architecture in tomato Cullin-RING E3 ligases (CRLs)-dependent ubiquitin proteasome system mediates degradation of numerous proteins that controls a wide range of developmental and physiological processes in eukaryotes. Cullin-associated Nedd8-dissociated protein 1 (CAND1) acts as an exchange factor allowing substrate recognition part exchange and plays a vital role in reactivating CRLs. The present study reports on the identification of SlCAND1, the only one CAND gene in tomato. SlCAND1 expression is ubiquitous and positively regulated by multiple plant hormones. Silencing of SlCAND1 expression using RNAi strategy resulted in a pleiotropic and gibberellin/auxin-associated phenotypes, including dwarf plant with reduced internode length, loss of apical dominance, early flowering, low seed germination percentage, delayed seed germination speed, short primary root, and increased lateral root proliferation and elongation. Moreover, application of exogenous GA3 or IAA could partly rescue some SlCAND1-silenced phenotypes, and the expression levels of gibberellin/auxin-related genes were altered in SlCAND1-RNAi lines. These facts revealed that SlCAND1 is required for gibberellin/auxin-associated regulatory network in tomato. Although SlCAND1 is crucial for multiple developmental processes during vegetative growth stage, SlCAND1-RNAi lines didn't exhibit visible effect on fruit development and ripening. Meanwhile, we discussed that multiple physiological functions of SlCAND1 in tomato are different to previous report of its ortholog in Arabidopsis. Our study adds a new perspective on the functional roles of CAND1 in plants, and strongly supports the hypothesis that CAND1 and its regulated ubiquitin proteasome system are pivotal for plant vegetative growth but possibly have different roles in diverse plant species.

Response of lipid biosynthesis in Chlorella pyrenoidosa to intracellular reactive oxygen species level under stress conditions.

An increase in the total lipid content in algal cells under stress conditions is often accompanied by an increase in reactive oxygen species (ROS). However, the link between these two events is unclear. In this study, the regulatory mechanism of ROS formation on lipid accumulation in C. pyrenoidosa was investigated using a Fenton-like reaction. A high Spearman correlation coefficient of 0.901 was obtained between the Hydroxyl radical (OH) level and lipid content. Importantly, the increase in the total lipid content was clearly coupled with a significant increase in the intracellular OH concentration rather than increases in the H2O2 and O2- concentrations. Transcriptome data confirms that most of the differential expression genes (DEGs) involved in fatty acid and glycerolipid biosynthesis were up-regulated by the increased OH under stress conditions. These results reveal that lipid accumulation in algal cells was promoted by OH.

Tomato (Solanum lycopersicum) SlIPT4, encoding an isopentenyltransferase, is involved in leaf senescence and lycopene biosynthesis during fruit ripening.

BACKGROUND: Lycopene is an important carotenoid pigment in red fruits and vegetables, especially in tomato. Although lycopene biosynthesis and catabolism have been found to be regulated by multiple factors including phytohormones, little is known about their regulatory mechanism. Cytokinins are crucial to various aspects of plant growth. Isopentenyltransferases (IPTs) catalyze the initial rate-limiting step of cytokinins biosynthesis, however, their roles in fruit ripening remain unclear. RESULTS: Here, the functions of SlIPT4, encoding an isopentenyltransferase, were characterized via RNAi-mediated gene silencing in tomato. As we expected, silencing of SlIPT4 expression resulted in accelerated leaf senescence. However, down-expression of SlIPT4 generated never-red orange fruits, corresponding with a dramatic reduction of lycopene. Among lycopene biosynthesis-related genes, the fact of remarkable decrease of ZISO transcript and upregulation of other genes, revealed that SlIPT4 regulates positively lycopene biosynthesis via directly affecting ZISO expression, and also supported the existence of regulatory loops in lycopene biosynthesis pathway. Meanwhile, the accumulation of abscisic acid (ABA) was reduced and the transcripts PSY1 were increased in SlIPT4-RNAi fruits, supporting the feedback regulation between ABA and lycopene biosynthesis. CONCLUSION: The study revealed the crucial roles of SlIPT4 in leaf senescence and the regulatory network of lycopene biosynthesis in tomato, providing a new light on the lycopene biosynthesis and fruit ripening.

Overexpression of an EIN3-binding F-box protein2-like gene caused elongated fruit shape and delayed fruit development and ripening in tomato.

Ethylene signaling converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-like (EIL) transcription factors to regulate a wide range of developmental processes in plants. EBF1/2 (EIN3-binding F-box protein 1 and 2) negatively regulate the ethylene signaling pathway by mediating the degradation of EIN3/EIL proteins. We uncovered previously that SlEBF1 and SlEBF2 are involved in ethylene response, plant senescence, and fruit ripening in tomato. The present study reports on the identification of a novel tomato F-box gene, designated as SlEBF2-like due that its encoded protein is greater similarity to SlEBF2. The SlEBF2-like promoter region contains three ethylene-response elements (EREs). SlEBF2-like is upregulated by ethylene and downregulated by ethylene inhibitors in tomato seedlings. It is dynamically expressed in flowers during bud-to-anthesis and anthesis-to-post-anthesis transitions, and at the onset of fruit ripening, suggesting its role in these situations where ethylene is required for flower opening and fruit ripening. SlEBF2-like overexpression leaded to fruit elongation, caused ripening and color change to start from fruit bottom and expand gradually to the pedicel, and strongly delayed fruit development and ripening in tomato. Our study indicates that the novel EBF gene, SlEBF2-like, is involved in fruit development and ripening via regulating the ethylene response in tomato.

miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato.

In Arabidopsis thaliana, Argonaute1 (AGO1) interacts with miR168 to modulate the small RNA regulatory pathway. However, the underlying mechanism of regulation and relationship between AGO1 and miR168 is poorly understood in the cash crop Solanum lycopersicum (tomato). We previously found that SlAGO1A and SlAGO1B were cleaved by miR168 in tomato. In this study, we show that SlAGO1A and SlAGO1B accumulate in miR168-sponge transgenic plants, and that expression of miR168-resistant SlAGO1A (4m-SlAGO1A) and SlAGO1B (4m-SlAGO1B) in tomato results in a series of defects affecting growth rate, floral timing, leaves, and fruit. Accumulation of miR156 was found when 4m-SlAGO1A was at an early developmental stage compared to the wild type and original SlAGO1A transgenic plants, and miR172 was highly expressed in adult 4m-SlAGO1A compared to the controls. In addition, the expression of multiple small RNAs was altered in 4m-SlAGO1A. Taken together, our data provide novel insights into the interaction between SlAGO1s and miR168 in determining growth rate, phase change, leaf epinasty, fruit initiation and expansion, and other developmental processes in tomato.

Molecular cloning and characterisation of SlAGO family in tomato.

BACKGROUND: AGO (Argonaute) protein participates in plant developmental processes and virus defense as a core element of transcriptional regulator or/and post-transcriptional regulator in RNA induced silencing complex (RISC), which is guided by small RNAs to repress target genes expression. Previously, it was revealed that 15 putative AGO genes in tomato genome. RESULTS: In present study, out of 15 detected SlAGO genes, only SlAGO4C and SlAGO15 couldn't be detected in roots, stems, leaves, buds, flowers and fruit of tomato by 30 cycles of PCR. SlAGO7 could be detected in early stage of fruit (-2 dpa, 0 dpa and 4 dpa), but it was significantly down-regulated in fruit collected on the 6 days post anthesis. Moreover, SlAGO5 could only be detected in reproductive tissues and SlAGO4D was specifically detected in fruit. According to blast result with miRNA database, three SlAGO genes harbored complementary sequences to miR168 (SlAGO1A and SlAGO1B) or miR403 (SlAGO2A). 5' RACE (Rapid amplification of cDNA ends) mapping was used to detect the 3' cleavage products of SlAGO mRNAs. In addition, subcellular localization of SlAGO proteins was detected. Our results showed that most SlAGO proteins localized to nucleus and cytoplasm. Importantly, nuclear membrane localization of AGO proteins was observed. Furthermore, mutated miR168 complementary site of SlAGO1A resulted in expanded localization of SlAGO1A, indicating that miR168 regulated localization of SlAGO1A. CONCLUSIONS: Our results contribute to demonstration of potential roles of these newly isolated AGO family in tomato developmental processes and proved the conserved relationships between AGO genes and miRNAs in tomato, which might play important roles in tomato development and virus defense.

The tomato SlIAA15 is involved in trichome formation and axillary shoot development.

The Aux/IAA genes encode a large family of short-lived proteins known to regulate auxin signalling in plants. Functional characterization of SlIAA15, a member of the tomato (Solanum lycopersicum) Aux/IAA family, shows that the encoded protein acts as a strong repressor of auxin-dependent transcription. The physiological significance of SlIAA15 was addressed by a reverse genetics approach, revealing that SlIAA15 plays multiple roles in plant developmental processes. The SlIAA15 down-regulated lines display lower trichome number, reduced apical dominance with associated modified pattern of axillary shoot development, increased lateral root formation and decreased fruit set. Moreover, the leaves of SlIAA15-inhibited plants are dark green and thick, with larger pavement cells, longer palisade cells and larger intercellular space of spongy mesophyll cells. The SlIAA15-suppressed plants exhibit a strong reduction in type I, V and VI trichome formation, suggesting that auxin-dependent transcriptional regulation is required for trichome initiation. Concomitant with reduced trichome formation, the expression of some R2R3 MYB genes, putatively involved in the control of trichome differentiation, is altered. These phenotypes uncover novel and specialized roles for Aux/IAAs in plant developmental processes, clearly indicating that members of the Aux/IAA gene family in tomato perform both overlapping and specific functions.

A conserved phosphorylation site regulates the transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato.

ETHYLENE-INSENSITIVE3/ETHYLENE-INSENSITIVE3-like (EIN3/EIL) transcription factors are important downstream components of the ethylene transduction pathway known to regulate the transcription of early ethylene-responsive genes in plants. Previous studies have shown that phosphorylation can repress their transcriptional activity by promoting protein degradation. The present study identifies a new phosphorylation region named EPR1 (EIN3/EIL phosphorylation region 1) in tomato EIL1 proteins. The functional significance of EPR1 was tested by introducing mutations in this region of the Sl-EIL1 gene and by expressing these mutated versions in transgenic tomato plants. Transient expression data and phenotypic analysis of the transgenic lines indicated that EPR1 is essential for the transcriptional activity of Sl-EIL1. Moreover, mutation in the EPR1 site that prevents phosphorylation abolishes ethylene constitutive responses normally displayed by the Sl-EIL1-overexpressing lines. Bimolecular fluorescence complementation (BiFC) studies showed that the presence of a functional phosphorylation site within EPR1 is instrumental in the dimerization of Sl-EIL1 proteins. The results illuminate a new molecular mechanism for the control of EIN3/EIL activity and propose a model where phosphorylation within the EPR1 promotes the dimerization process allowing the initiation of EIL-mediated transcription of early ethylene-regulated genes.

Functional identification of a novel F-box/FBA gene in tomato.

In plants and animals, the SCF-type ubiquitin protein ligases play an important role in many different physiological processes by regulating protein stability such as S-RNase-based self-compatibility, flower development, hormone responses and meiosis. This study identified an SlFbf gene in tomato that encodes 381 amino acid residues containing a typical F-box motif and an FBA_1 motif associated proteasome pathway; the transcripts of SlFbf was detected in all the tissues (root, stem, leaf, sepal, petal, stamen, pistil, green fruit, breaker fruit and red fruit), with the highest in stamen specifically during flowering stage; SlFbf responded to gibberellins, abscisic acid and light. Suppressed SlFbf leads to bigger pollen and less seeds showing that SlFbf might have an effect on fertilization through regulating stamen development. These findings provide more information about the functions of Fbf gene family.

The auxin receptor homologue in Solanum lycopersicum stimulates tomato fruit set and leaf morphogenesis.

TIR1 and its homologues act as auxin receptors and play a crucial role in auxin-mediated plant development. While the functions of auxin receptor genes have been widely studied in Arabidopsis thaliana, there has been no report on the consequences of TIR1 overexpression in plants that regulate fruit development. Here a putative tomato auxin receptor gene, homologous to Arabidopsis AtTIR1, is reported. This gene, designated as Solanum lycopersicum TIR1 (SlTIR1), was found to be expressed in all the parts of floral buds and flowers at anthesis stages. From bud to anthesis, SlTIR1 expression increases slightly in sepal tissue and decreases dramatically in stamen. From anthesis to post-anthesis when fruit set is expected to occur, the expression of SlTIR1 declines in the ovary and sepal. Overexpression of SlTIR1 results in a pleiotropic phenotype including parthenocarpic fruit formation and leaf morphology. Furthermore, SlTIR1 overexpression altered transcript levels of a number of auxin-responsive genes. The present data demonstrate that the tomato SlTIR1 gene plays an important role at the stages of flower-to-fruit transition and leaf formation.

Isolation and characterization of a novel lignocellulose decomposing fungal strain.

A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70 degrees C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28 degrees C (pH 6.0) in flasks stirred at 150 x g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.

Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato.

The hormone ethylene regulates a wide range of plant developmental processes and EBF (EIN3-binding F-box) proteins were shown to negatively regulate the ethylene signalling pathway via mediating the degradation of EIN3/EIL proteins. The present study reports on the identification of two tomato F-box genes, Sl-EBF1 and Sl-EBF2 from the EBF subfamily. The two genes display contrasting expression patterns in reproductive and vegetative tissues and in response to ethylene and auxin treatment. Sl-EBF1 and Sl-EBF2 genes are actively regulated at crucial stages in the development of the reproductive organs. Their dynamic expression in flowers during bud-to-anthesis and anthesis-to-post-anthesis transitions, and at the onset of fruit ripening, suggests their role in situations where ethylene is required for stimulating flower opening and triggering fruit ripening. VIGS-mediated silencing of a single tomato EBF gene uncovered a compensation mechanism that tends to maintain a threshold level of Sl-EBF expression via enhancing the expression of the second Sl-EBF gene. In line with this compensation, tomato plants silenced for either of the Sl-EBF genes were indistinguishable from control plants, indicating functional redundancy among Sl-EBF genes. By contrast, co-silencing of both Sl-EBFs resulted in ethylene-associated phenotypes. While reports on EBF genes to date have focused on their role in modulating ethylene responses in Arabidopsis, the present study uncovered their role in regulating crucial stages of flower and fruit development in tomato. The data support the hypothesis that protein degradation via the ubiquitin/26S proteasome pathway is a control point of fruit ripening and open new leads for engineering fruit quality.

Isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display.

Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.

Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance.

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.