Metabolic engineering and mechanical investigation of enhanced plant autoluminescence.

The fungal bioluminescence pathway (FBP) was identified from glowing fungi, which releases self-sustained visible green luminescence. However, weak bioluminescence limits the potential application of the bioluminescence system. Here, we screened and characterized a C3'H1 (4-coumaroyl shikimate/quinate 3'-hydroxylase) gene from Brassica napus, which efficiently converts p-coumaroyl shikimate to caffeic acid and hispidin. Simultaneous expression of BnC3'H1 and NPGA (null-pigment mutant in A. nidulans) produces more caffeic acid and hispidin as the natural precursor of luciferin and significantly intensifies the original fungal bioluminescence pathway (oFBP). Thus, we successfully created enhanced FBP (eFBP) plants emitting 3 × 1011 photons/min/cm2 , sufficient to illuminate its surroundings and visualize words clearly in the dark. The glowing plants provide sustainable and bio-renewable illumination for the naked eyes, and manifest distinct responses to diverse environmental conditions via caffeic acid biosynthesis pathway. Importantly, we revealed that the biosynthesis of caffeic acid and hispidin in eFBP plants derived from the sugar pathway, and the inhibitors of the energy production system significantly reduced the luminescence signal rapidly from eFBP plants, suggesting that the FBP system coupled with the luciferin metabolic flux functions in an energy-driven way. These findings lay the groundwork for genetically creating stronger eFBP plants and developing more powerful biological tools with the FBP system.

SlMPK1- and SlMPK2-mediated SlBBX17 phosphorylation positively regulates CBF-dependent cold tolerance in tomato.

B-box (BBX) proteins are an important class of zinc finger transcription factors that play a critical role in plant growth and stress response. However, the mechanisms of how BBX proteins participate in the cold response in tomato remain unclear. Here, using approaches of reverse genetics, biochemical and molecular biology we characterized a BBX transcription factor, SlBBX17, which positively regulates cold tolerance in tomato (Solanum lycopersicum). Overexpressing SlBBX17 enhanced C-repeat binding factor (CBF)-dependent cold tolerance in tomato plants, whereas silencing SlBBX17 increased plant susceptibility to cold stress. Crucially, the positive role of SlBBX17 in CBF-dependent cold tolerance was dependent on ELONGATED HYPOCOTYL5 (HY5). SlBBX17 physically interacted with SlHY5 to directly promote the protein stability of SlHY5 and subsequently increased the transcriptional activity of SlHY5 on SlCBF genes under cold stress. Further experiments showed that cold-activated mitogen-activated protein kinases, SlMPK1 and SlMPK2, also physically interact with and phosphorylate SlBBX17 to enhance the interaction between SlBBX17 and SlHY5, leading to enhanced CBF-dependent cold tolerance. Collectively, the study unveiled a mechanistic framework by which SlMPK1/2-SlBBX17-SlHY5 regulated transcription of SlCBFs to enhance cold tolerance, thereby shedding light on the molecular mechanisms of how plants respond to cold stress via multiple transcription factors.

SEC1-C3H39 module fine-tunes cold tolerance by mediating its target mRNA degradation in tomato.

Plants adapt to cold stress at the physiological and biochemical levels, thus enabling them to maintain growth and development. However, the molecular mechanism of fine-tuning cold signals remains largely unknown. We addressed the function of SlSEC1-SlC3H39 module in cold tolerance by using SlSEC1 and SlC3H39 knockout and overexpression tomato lines. A tandem CCCH zinc-finger protein SlC3H39 negatively modulates cold tolerance in tomato. SlC3H39 binds to AU-rich elements in the 3'-untranslated region (UTR) to induce mRNA degradation and regulates gene expression post-transcriptionally. We further validate that SlC3H39 participates in post-transcriptional regulation of a variety of cold-responsive genes. An O-linked N-acetylglucosamine transferase SlSEC1 physically interacts with SlC3H39 proteins and negatively regulates cold tolerance in tomato. Further study shows that SlSEC1 is essential for SlC3H39 protein stability and maintains SlC3H39 function in cold tolerance. Genetic analysis shows that SlC3H39 is epistatic to SlSEC1 in cold tolerance. The findings indicate that SlC3H39 negatively modulates plant cold tolerance through post-transcriptional regulation by binding to cold-responding mRNA 3'-UTR and reducing those transcripts. SlSEC1 promotes the O-GlcNAclation status of SlC3H39 and maintains SlC3H39 function in cold tolerance. Taken together, we propose a SlSEC1-SlC3H39 module, which allows plants to balance defense responses and growth processes.

Robust predictions of specialized metabolism genes through machine learning.

Plant specialized metabolism (SM) enzymes produce lineage-specific metabolites with important ecological, evolutionary, and biotechnological implications. Using Arabidopsis thaliana as a model, we identified distinguishing characteristics of SM and GM (general metabolism, traditionally referred to as primary metabolism) genes through a detailed study of features including duplication pattern, sequence conservation, transcription, protein domain content, and gene network properties. Analysis of multiple sets of benchmark genes revealed that SM genes tend to be tandemly duplicated, coexpressed with their paralogs, narrowly expressed at lower levels, less conserved, and less well connected in gene networks relative to GM genes. Although the values of each of these features significantly differed between SM and GM genes, any single feature was ineffective at predicting SM from GM genes. Using machine learning methods to integrate all features, a prediction model was established with a true positive rate of 87% and a true negative rate of 71%. In addition, 86% of known SM genes not used to create the machine learning model were predicted. We also demonstrated that the model could be further improved when we distinguished between SM, GM, and junction genes responsible for reactions shared by SM and GM pathways, indicating that topological considerations may further improve the SM prediction model. Application of the prediction model led to the identification of 1,220 A. thaliana genes with previously unknown functions, each assigned a confidence measure called an SM score, providing a global estimate of SM gene content in a plant genome.

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network.

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330--or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)--and Solyc04g012020 (Sl-ASAT2). These enzymes were used--in concert with two previously identified BAHD acyltransferases--to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

Functionally divergent alleles and duplicated Loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes.

Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

MAIN CONCLUSION: The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

BACKGROUND: microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far. RESULTS: Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE. CONCLUSIONS: Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable lignin biosynthesis.

Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ-aminobutyric acid metabolic pathway.

γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress.

H(+) -pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat.

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H(+) -PPase is involved in salt-stimulated NO3 (-) uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H(+) -PPase from S. europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K(+) /Na(+) ratio in leaves and exhibited increased NO3 (-) uptake, inorganic pyrophosphate-dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up-regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up-regulation of H(+) -PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.