Comprehensive Analysis of Major Latex-Like Protein Family Genes in Cucumber (Cucumis sativus L.) and Their Potential Roles in Phytophthora Blight Resistance.

Major latex-like proteins (MLPs) play crucial roles in abiotic and biotic stresses. However, little was known about this gene family in cucumbers. In this study, a total of 37 putative cucumber MLP genes were identified on a genome-wide level and classified into three groups by sequence homologous comparison with Arabidopsis thaliana. Chromosome mapping suggested that only tandem duplication occurred in evolution. The multiple regulatory cis-elements related to stress, hormone, light and growth response were found in the promoter region of these CsMLP genes, indicating that CsMLPs might be widely involved in the process of plant growth, development and various stress conditions. Transcriptome analysis indicated a strong reprogramming of MLPs expression in response to Phytophthora melonis infection in cucumber. Knockdown of CsMLP1 reduced the P. melonis tolerance, while transient overexpression of CsMLP1 improved disease tolerance in cucumber. Conversely, the silence of CsMLP5 decreased the lesion area caused by P. melonis in the cotyledons, and overexpression of CsMLP5 promoted lesion expansion. Taken together, our results provide a comprehensive basis for further mining the function of CsMLP members and will also be significant for elucidating the evolutionary relationship in cucumber.

Genome-wide identification and characterization of major latex-like protein genes responsible for crop contamination in Cucurbita pepo.

BACKGROUND: Zucchini plants (Cucurbita pepo) accumulate persistent organic pollutants (POPs) at high concentrations in their aerial parts, and major latex-like proteins (MLPs) play crucial roles in their accumulation. MLPs bind to POPs in root cells, MLP-POP complexes are then translocated into xylem vessels, and POPs are transported to the aerial parts. We previously identified three CpMLP genes (MLP-PG1, MLP-GR1, and MLP-GR3) as transporting factors for POPs; however, other studies have shown that the genomes of several plant species contain more than 10 MLP genes, thus, further MLP genes responsible for POP accumulation may have been overlooked. METHODS AND RESULTS: Here, we investigated the number of CpMLP genes by performing a hidden Markov model search against the C. pepo genome database and characterized their effects on POP accumulation by performing the expression analysis in the organs and in silico structural analysis. The C. pepo genome contained 21 CpMLP genes, and several CpMLP genes, including MLP-PG1 and MLP-GR3, were highly expressed in roots. 3D structural prediction showed that all examined CpMLPs contained a cavity with a hydrophobic region, which facilitated binding to POPs. CONCLUSIONS: The present study provides insights regarding CpMLP genes responsible for POP accumulation.

MLP-PG1, a major latex-like protein identified in Cucurbita pepo, confers resistance through the induction of pathogenesis-related genes.

MAIN CONCLUSION: MLP-PG1, identified in Cucurbita pepo, plays a crucial role in resistance against fungal pathogens through the induction of pathogenesis-related genes. ASTRACT: MLP-PG1, a major latex-like protein (MLP) from zucchini (Cucurbita pepo), was identified as a transporting factor for hydrophobic organic pollutants. MLPs are members of the Bet v 1 family, similar to pathogenesis-related class 10 proteins (PR-10s). However, the biological functions of MLPs remain unclear. Herein, we show that MLP-PG1 induces the expression of pathogenesis-related (PR) genes and indirectly promotes resistance against pathogens. The activity of the MLP-PG1 promoter in leaves of transgenic tobacco plants was significantly enhanced by inoculation with Pseudomonas syringae pv. tabaci. However, MLP-PG1 did not induce direct resistance through RNase activity. Therefore, we examined the possibility that MLP-PG1 is indirectly involved in resistance; indeed, we found that MLP-PG1 induced the expression of defense-related genes. Overexpression of MLP-PG1 highly upregulated PR-2 and PR-5 and decreased the area of lesions caused by Botrytis cinerea in the leaves of transgenic tobacco plants. Our results demonstrate that MLP-PG1 is involved in indirect resistance against plant diseases, especially caused by fungal pathogens, through the induction of PR genes. This study is the first report to show the induction of PR genes by the expression of MLP from the RNA sequencing analysis and the involvement of MLP-PG1 in the resistance.

Structure of ginseng major latex-like protein 151 and its proposed lysophosphatidic acid-binding mechanism.

Lysophosphatidic acid (LPA) is a phospholipid growth factor with myriad effects on biological systems. LPA is usually present bound to animal plasma proteins such as albumin or gelsolin. When LPA complexes with plasma proteins, it binds to its cognate receptors with higher affinity than when it is free. Recently, gintonin from ginseng was found to bind to LPA and to activate mammalian LPA receptors. Gintonin contains two components: ginseng major latex-like protein 151 (GLP) and ginseng ribonuclease-like storage protein. Here, the crystal structure of GLP is reported, which belongs to the plant Bet v 1 superfamily, and a model is proposed for how GLP binds LPA. Amino-acid residues of GLP recognizing LPA were identified using site-directed mutagenesis and isothermal titration calorimetry. The resulting GLP mutants were used to study the activation of LPA receptor-dependent signalling pathways. In contrast to wild-type GLP, the H147A mutant did not bind LPA, elicit intracellular Ca(2+) transients in neuronal cells or activate Ca(2+)-dependent Cl(-) channels in Xenopus oocytes. Based on these results, a mechanism by which GLP recognizes LPA and its requirement to activate G protein-coupled LPA receptors to elicit diverse biological responses were proposed.

Major latex-like proteins show pH dependency in their binding to hydrophobic organic pollutants.

The Cucurbitaceae family accumulates hydrophobic organic pollutants in its aerial parts at high concentrations. Major latex-like proteins (MLPs) were identified in zucchini (Cucurbita pepo) as a transporting factor for hydrophobic organic pollutants. MLPs bind to hydrophobic organic pollutants in the roots, are secreted to xylem vessels as complexes, and are transported to the aerial parts. However, the suitable conditions for binding MLPs to hydrophobic organic pollutants remain elusive. In the present study, we show that MLPs bind to the hydrophobic organic pollutant pyrene with higher affinity under acidic conditions. Our results demonstrated that pH regulates the binding of MLPs to hydrophobic organic pollutants.

Cucurbita pepo contains characteristic proteins without a signal peptide in the xylem sap.

Xylem sap is a fluid that transfers water and nutrients from the rhizosphere. This sap contains relatively low concentrations of proteins that originate from the extracellular space among the root cells. One of the characteristic proteins in the xylem sap of the Cucurbitaceae family, which includes cucumber and zucchini, is a major latex-like protein (MLP). MLPs are responsible for crop contamination through the transport of hydrophobic pollutants from the roots. However, detailed information on the content of MLPs in the xylem sap is not available. Proteomic analysis of root and xylem sap proteins from the Cucurbita pepo cultivars Patty Green (PG) and Raven (RA) showed that the xylem sap of cv. RA, a high accumulator of hydrophobic pollutants, contained four MLPs that accounted for over 85% of the total xylem sap proteins in this cultivar. The xylem sap of PG, a low accumulator, mainly contained an uncharacterized protein. The amount of each root protein between the PG and RA cultivars was significantly and positively correlated in spite of being with and without a signal peptide (SP). However, the amount of xylem sap proteins without an SP was not correlated. These results suggest that cv. RA is characterized by MLPs in the xylem sap.

A20/AN1 zinc-finger proteins positively regulate major latex-like proteins, transporting factors toward dioxin-like compounds in Cucurbita pepo.

The Cucurbitaceae family accumulates dioxin-like compounds in its fruits. We previously showed that A20/AN1 zinc finger protein (ZFP) genes were highly expressed in the zucchini (Cucurbita pepo) subspecies pepo, which accumulates dioxin-like compounds at high concentrations. Transgenic tobacco (Nicotiana tabacum) plants overexpressing A20/AN1 ZFP genes show accumulation of dioxin-like compounds in their upper parts. However, the mechanisms underlying the accumulation of dioxin-like compounds regulated by the A20/AN1 ZFPs remain unclear. Here, we show that A20/AN1 ZFPs positively regulate the expression of the major latex-like protein (MLP) and its homolog genes in N. tabacum and C. pepo. MLPs are involved in the transport of dioxin-like compounds from the roots to the upper parts of C. pepo. Overexpression of A20/AN1 ZFP genes in N. tabacum leads to the upregulation of pathogenesis-related protein class-10 genes with the binding ability toward dioxin-like compounds. Our results demonstrated that A20/AN1 ZFPs upregulate MLP and its homolog genes in N. tabacum and C. pepo, resulting in the accumulation of dioxin-like compounds.

Review: Biological functions of major latex-like proteins in plants.

Major latex-like proteins (MLPs) have been identified in dicots and monocots. They are members of the birch pollen allergen Bet v 1 family as well as pathogenesis-related proteins class 10. MLPs have two main features. One is binding affinity toward various hydrophobic compounds, such as long-chain fatty acids, steroids, and systemic acquired resistance signals, via its internal hydrophobic cavity or hydrophobic residues on its surface. MLPs transport such compounds to other organs via phloem and xylem vessels and contribute to the expression of physiologically important ligands' activity in the particular organs. The second feature is responses to abiotic and biotic stresses. MLPs are involved in drought and salt tolerance through the mediation of plant hormone signaling pathways. MLPs generate resistance against pathogens by the induction of pathogenesis-related protein genes. Therefore, MLPs play crucial roles in drought and salt tolerance and resistance against pathogens. However, knowledge of MLPs is fragmented, and an overview of them is needed. Herein, we summarize the current knowledge of the biological functions of MLPs, which to our knowledge, is the first review about MLPs that has been reported.

Transport enhancement of hydrophobic pollutants by the expression of zucchini major latex-like protein genes in tobacco plants.

The environmental spread of hydrophobic pollutants has been receiving attention because of specific characteristics of these compounds that make them resistant to degradation, thus causing various toxic effects on humans as a result of their bioaccumulation. Here, we report the role of zucchini major-latex like proteins (MLPs) on the accumulation of hydrophobic pollutants, as consumption of contaminated crops is one of the main routes for accumulation. Transgenic tobacco plants expressing an aryl hydrocarbon receptor (AhR) gene with a ß-glucuronidase (GUS) inducible expression system were transformed with one of the three zucchini MLP genes (PG1, GR1, and GR3). MLP transgenic plants showed a significant increase in the fold induction of GUS activity compared to the parental AhR tobacco plants when one of the most toxic polychlorinated biphenyl (PCB) congeners, 3,3',4,4',5-pentachlorobiphenyl (CB126), was applied. GUS activity was detected in both aerial parts and roots after treatment with the strong carcinogen 3-methylcholanthrene. Phenotypic changes in the MLP tobacco during incubation with CB126 were also observed. The MLP transgenic plant PG1 responded to treatment with 0.32 nM CB126, whereas vector control plants significantly induced GUS activity at 200 nM CB126. Moreover, GUS activities in the MLP plants treated with other PCB congeners were significantly higher than those in the plants given the mock treatment. As GUS activities in the aerial parts of the plants were significantly correlated with the accumulation level of PCBs, these results strongly suggest that zucchini MLPs are related to the translocation of hydrophobic pollutants from the roots to the aerial parts through their binding affinity.

Suppression of the genes responsible for transporting hydrophobic pollutants leads to the production of safer crops.

Hydrophobic pollutants have become widely distributed across the world. From an agricultural perspective, their accumulation in crops from contaminated soil threatens food security and quality, leading to many diseases in humans. The Cucurbitaceae family can accumulate high concentrations of hydrophobic pollutants in their aerial parts. The Cucurbitaceae family contains major latex-like proteins (MLPs) as transporting factors for hydrophobic pollutants. MLP genes are expressed in the roots in which the MLPs bind hydrophobic pollutants. MLPs transport these hydrophobic pollutants to the aerial parts of the plant through the xylem vessels. As a result, hydrophobic pollutant contamination occurs in the Cucurbitaceae family. In this study, we suppressed the expression of MLP genes in the roots and reduced the amounts of MLPs with pesticide treatments. First, the fungicides Benlate and Daconil that deceased the hydrophobic pollutant, perylene, concentration in the xylem sap of zucchini plants were selected. Daconil suppressed the transcription activity of MLP in the roots. In the Daconil treatment, the amount of MLPs in the roots and xylem sap of zucchini plants was decreased, and the concentrations of the hydrophobic pollutants, pyrene and dieldrin, were significantly decreased. Our research contributes to the production of safer crops.

Pesticide treatment reduces hydrophobic pollutant contamination in Cucurbita pepo through competitive binding to major latex-like proteins.

Hydrophobic pollutants are still present in agricultural soil. The Cucurbitaceae family accumulates hydrophobic pollutants through roots, resulting in the contamination of aerial parts. Major latex-like proteins (MLPs), found in the Cucurbitaceae family, play an important role in the contamination by binding to these hydrophobic pollutants. Thus far, efficient cultivation methods for the production of safe crops with lower concentrations of hydrophobic pollutants have not been developed. Herein, we competitively inhibited the binding of MLPs to hydrophobic pollutants, pyrene and dieldrin, in roots by using MLP binding pesticides. By conducting a chemical array screening, we found that MLPs bound compounds with indole- and quinazoline-like structures. Commercially available pesticides amisulbrom and pyrifluquinazon, which possess such structures, successfully inhibited the binding of MLPs to pyrene and dieldrin in vitro. When zucchini plants were cultivated in the contaminated soil with 1.25 mmol/kg pyrene and 12.5 µmol/kg dieldrin, the concentration of pyrene and dieldrin in xylem sap was significantly decreased by 30% and 15%, respectively. Our results demonstrate that the pesticides binding to MLPs competitively inhibited the binding of MLPs to pyrene and dieldrin in roots, resulting in the reduction of overall contamination. This study proposes a novel approach to cultivate safer crops and advances the utilization of unknown functions of pesticides.

High temperatures promote the uptake of hydrophobic pollutants by Cucurbita pepo via altered gene expression levels of major latex-like proteins.

Cucurbitaceae family members are accumulators of hydrophobic pollutants. Such pollutants have been detected in cucurbits at levels above the maximum residue limit. Since major latex-like proteins (MLPs) are involved in hydrophobic pollutant uptake, changes in MLP expression can increase or decrease contamination. MLP expression levels were altered in the roots of Cucurbita pepo 'Magda,' and MLP-PG1 was detected in the xylem sap of Magda when cultivated at a high temperature (35°C). Day length also influenced MLP expression levels but only induced minor changes in the amount of MLPs. The concentration of pyrene, a hydrophobic pollutant, significantly increased with increasing MLP levels in the xylem sap of Magda when cultivated at 35°C. Thus, high temperatures promote the pollution of cucurbits by hydrophobic pollutants. These results can be used to develop novel techniques to reduce crop contamination and establish efficient phytoremediation.

Factors regulating the differential uptake of persistent organic pollutants in cucurbits and non-cucurbits.

Contamination with persistent organic pollutants (POPs) has become a worldwide concern owing to their the toxicity to humans and wildlife. Pumpkin, cucumber, and squash (Cucurbitaceae) accumulate POPs in their shoots in concentrations higher than those in non-cucurbits; to elucidate the underlying molecular mechanisms of this accumulation, POP transporters were analyzed in the xylem sap of cucurbits and non-cucurbits. The 17-kDa xylem sap proteins detected in all cucurbits but not in non-cucurbits readily bound polychlorinated biphenyl (PCB) in all tested cucurbits, except in cucumber and loofah, and to dieldrin in all tested cucurbits. Ten genes encoding major latex-like proteins (MLPs) responsible for the accumulation of PCBs in zucchini plants were cloned from cucurbits. Phylogenetic analysis using MLP sequences identified two separate clades, one containing Cucurbitaceae MLPs and the other containing those of non-cucurbit members. Recombinant MLPs bound PCB and dieldrin. Western blotting with anti-MLP antibodies identified translocatable and non-translocatable MLPs between root and stem xylem vessels. Translocation of MLPs from the root to stem xylem vessels and POP-binding ability of MLPs are important for selective accumulation of MLPs in cucurbits. This study provides basic knowledge about phytoremediation through overexpression of MLP genes and for breeding cucurbits that accumulate less contaminants.

Panax ginseng as an adjuvant treatment for Alzheimer's disease.

Longevity in medicine can be defined as a long life without mental or physical deficits. This can be prevented by Alzheimer's disease (AD). Current conventional AD treatments only alleviate the symptoms without reversing AD progression. Recent studies demonstrated that Panax ginseng extract improves AD symptoms in patients with AD, and the two main components of ginseng might contribute to AD amelioration. Ginsenosides show various AD-related neuroprotective effects. Gintonin is a newly identified ginseng constituent that contains lysophosphatidic acids and attenuates AD-related brain neuropathies. Ginsenosides decrease amyloid ß-protein (Aß) formation by inhibiting ß- and γ-secretase activity or by activating the nonamyloidogenic pathway, inhibit acetylcholinesterase activity and Aß-induced neurotoxicity, and decrease Aß-induced production of reactive oxygen species and neuroinflammatory reactions. Oral administration of ginsenosides increases the expression levels of enzymes involved in acetylcholine synthesis in the brain and alleviates Aß-induced cholinergic deficits in AD models. Similarly, gintonin inhibits Aß-induced neurotoxicity and activates the nonamyloidogenic pathway to reduce Aß formation and to increase acetylcholine and choline acetyltransferase expression in the brain through lysophosphatidic acid receptors. Oral administration of gintonin attenuates brain amyloid plaque deposits, boosting hippocampal cholinergic systems and neurogenesis, thereby ameliorating learning and memory impairments. It also improves cognitive functions in patients with AD. Ginsenosides and gintonin attenuate AD-related neuropathology through multiple routes. This review focuses research demonstrating that ginseng constituents could be a candidate as an adjuvant for AD treatment. However, clinical investigations including efficacy and tolerability analyses may be necessary for the clinical acceptance of ginseng components in combination with conventional AD drugs.

Development of intron polymorphism markers in major latex-like protein gene for locality-level and cultivar identification of Salvia miltiorrhiza.

BACKGROUND: Salvia miltiorrhiza (Danshen) is one of the most widely used medicinal herbs in traditional Chinese medicine. Locality-level and cultivar identification is of great importance not only for protecting highest therapeutic effectiveness of Daodi Danshen, but also for the genetic conservation and utilization of existing S. miltiorrhiza populations. RESULTS: Intron polymorphisms including SNPs (single nucleotide polymorphisms) and indels were exploited in major latex-like protein (MLP) gene. Based on these markers, genetic relationships among S. miltiorrhiza cultivar and populations in different locations were evaluated by constructing a dendrogram. Moreover, S. miltiorrhiza specimens from Laiwu region were geographically distinguishable by the developed SNP marker. A 204 bp-indel marker was exploited for the first space breeding cultivar Luyuan Danshen-1 (LD-1), and an effective real-time PCR assay was successfully developed for fast screening of LD-1 among local landraces. CONCLUSIONS: MLP intron is a valuable DNA barcode for intra-specific study of S. miltiorrhiza populations, and the developed markers can serve as a useful tool for molecular identification of LD-1 cultivar and geographically distinct populations of S. miltiorrhiza.