Plant Non-specific Lipid Transfer Proteins Research Articles

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)

Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

Potato Non-Specific Lipid Transfer Protein StnsLTPI.33 Is Associated with the Production of Reactive Oxygen Species, Plant Growth, and Susceptibility to Alternaria solani.

Plant non-specific lipid transfer proteins (nsLTPs) are small proteins capable of transferring phospholipids between membranes and binding non-specifically fatty acids in vitro. They constitute large gene families in plants, e.g., 83 in potato (Solanum tuberosum). Despite their recognition decades ago, very few have been functionally characterized. Here, we set out to better understand the function of one of the potato members, StnsLTPI.33. Using quantitative polymerase chain reaction, we show that StnsLTPI.33 is expressed throughout the potato plant, but at relatively higher levels in roots and leaves compared to petals, anthers, and the ovary. We also show that ectopically-expressed StnsLTPI.33 fused to green fluorescent protein colocalized with an apoplastic marker in Nicotiana benthamiana leaves, indicating that StnsLTPI.33 is targeted to the apoplast. Constitutive overexpression of the StnsLTPI.33 gene in potato led to increased levels of superoxide anions and reduced plant growth, particularly under salt stress conditions, and enhanced susceptibility to Alternaria solani. In addition, StnsLTPI.33-overexpressing plants had a depleted leaf pool of pipecolic acid, threonic acid, and glycine, while they accumulated putrescine. To our knowledge, this is the first report of an nsLTP that is associated with enhanced susceptibility to a pathogen in potato.

Sequence Analysis and Biochemical Characteristics of Two Non-specific Lipid Transfer Proteins from Tartary Buckwheat Seeds.

Plant non-specific lipid transfer proteins (nsLTPs) play an important role in plant resistance to various stresses, and show potential applications in agriculture, industrial manufacturing, and medicine. In addition, as more and more nsLTPs are identified as allergens, nsLTPs have attracted interest due to their allergenicity. Two nsLTPs from Tartary buckwheat have been isolated and identified. There is a need to study their biochemical characteristics and allergenicity. The study aims to investigate the biochemical characteristics of two nsLTPs from Tartary buckwheat seeds and evaluate their potential allergenicity. Two nsLTPs derived from Tartary buckwheat, namely FtLTP1a and FtLTP1b, were produced by gene cloning, expression, and purification. Sequence analysis and biochemical characteristics of the proteins, including lipid binding ability, α-amylase inhibition activity, antifungal activity, and allergenic activity, were investigated. High-purity recombinant FtLTP1a and FtLTP1b were obtained. FtLTP1a and FtLTP1b exhibited similar lipid binding and antifungal properties. Only FtLTP1b showed weak inhibitory activity against α-amylase. FtLTP1b could specifically bind IgE in the serum allergic to buckwheat and cross-react with pollen (w6). FtLTP1b is a novel allergenic member of the lipid-transfer protein 1 family found in Tartary buckwheat.

The non-specific lipid transfer protein McLTPII.9 of Mentha canadensis is involved in peltate glandular trichome density and volatile compound metabolism.

Mentha canadensis L. is an important spice crop and medicinal herb with high economic value. The plant is covered with peltate glandular trichomes, which are responsible for the biosynthesis and secretion of volatile oils. Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family involved in various plant physiological processes. Here, we cloned and identified a non-specific lipid transfer protein gene (McLTPII.9) from M. canadensis, which may positively regulate peltate glandular trichome density and monoterpene metabolism. McLTPII.9 was expressed in most M. canadensis tissues. The GUS signal driven by the McLTPII.9 promoter in transgenic Nicotiana tabacum was observed in stems, leaves, and roots; it was also expressed in trichomes. McLTPII.9 was associated with the plasma membrane. Overexpression of McLTPII.9 in peppermint (Mentha piperita. L) significantly increased the peltate glandular trichome density and total volatile compound content compared with wild-type peppermint; it also altered the volatile oil composition. In McLTPII.9-overexpressing (OE) peppermint, the expression levels of several monoterpenoid synthase genes and glandular trichome development-related transcription factors-such as limonene synthase (LS), limonene-3-hydroxylase (L3OH), geranyl diphosphate synthase (GPPS), HD-ZIP3, and MIXTA-exhibited varying degrees of alteration. McLTPII.9 overexpression resulted in both a change in expression of genes for terpenoid biosynthetic pathways which corresponded with an altered terpenoid profile in OE plants. In addition, peltate glandular trichome density was altered in the OE plants as well as the expression of genes for transcription factors that were shown to be involved in trichome development in plants.

Non-specific LIPID TRANSFER PROTEIN 1 enhances immunity against tobacco mosaic virus in Nicotiana benthamiana.

Plant non-specific lipid transfer proteins (nsLTPs) are small, cysteine-rich proteins that play significant roles in biotic and abiotic stress responses; however, the molecular mechanism of their functions against viral infections remains unclear. In this study, we employed virus-induced gene-silencing and transgenic overexpression to functionally analyse a type-I nsLTP in Nicotiana benthamiana, NbLTP1, in the immunity response against tobacco mosaic virus (TMV). NbLTP1 was inducible by TMV infection, and its silencing increased TMV-induced oxidative damage and the production of reactive oxygen species (ROS), compromised local and systemic resistance to TMV, and inactivated the biosynthesis of salicylic acid (SA) and its downstream signaling pathway. The effects of NbLTP1-silencing were partially restored by application of exogenous SA. Overexpressing NbLTP1 activated genes related to ROS scavenging to increase cell membrane stability and maintain redox homeostasis, confirming that an early ROS burst followed by ROS suppression at the later phases of pathogenesis is essential for resistance to TMV infection. The cell-wall localization of NbLTP1 was beneficial to viral resistance. Overall, our results showed that NbLTP1 positively regulates plant immunity against viral infection through up-regulating SA biosynthesis and its downstream signaling component, NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), which in turn activates pathogenesis-related genes, and by suppressing ROS accumulation at the later phases of viral pathogenesis.

Genome-wide characterization, chromosome localization, and expression profile analysis of poplar non-specific lipid transfer proteins

Plant non-specific lipid transfer proteins (nsLTPs) are small and have a broad biological function involved in reproductive development and abiotic stress resistance. Although a small part of plant nsLTPs have been identified, these proteins have not been characterized in poplar at the genomic level. A genome-wide characterization and expression identification of poplar nsLTP members were performed in this study. A total of 42 poplar nsLTP genes were identified from the poplar genome. A comprehensive analysis of poplar nsLTPs was conducted by a phylogenetic tree, duplication events, gene structures, and conserved motifs. The cis-elements of poplar nsLTPs were predicted to respond to light, hormone, and abiotic stress. Many transcription factors (TFs) were identified to interact with poplar nsLTP cis-elements. The tested poplar nsLTPs were expressed in leaves, stems, and roots, but their expression levels differed among tested tissues. Most poplar nsLTP expression levels were changed by abiotic stress, implying that poplar nsLTP may be involved in abiotic stress resistance. Network analysis showed that poplar nsLTPs are putative genes involved in fatty acid (FA) metabolism. This research provides sight into the further study to explain the regulatory mechanism of the poplar nsLTPs.

Genome-Wide Identification of Common Bean PvLTP Family Genes and Expression Profiling Analysis in Response to Drought Stress.

Common bean is one of the most important legume crops for human consumption. Its yield is adversely affected by environmental stress. Plant non-specific lipid transfer proteins (nsLTPs) are essential for plant growth, development, and resistance to abiotic stress, such as salt, drought, and alkali. However, changes in nsLTP family genes responding to drought stress are less known. The PvLTP gene family in the common bean was identified by a comprehensive genome-wide analysis. Molecular weights, theoretical isoelectric points, phylogenetic tree, conserved motifs, gene structures, gene duplications, chromosome localization, and expression profiles were analyzed by SignalP 5.0, ExPASy, ClustalX 2.1, MEGA 7.0, NCBI-CDD, MEME, Weblogo, and TBtools 1.09876, respectively. Heatmap and qRT-PCR analyses were performed to validate the expression profiles of PvLTP genes in different organs. In addition, the expression patterns of nine PvLTP genes in common beans treated with drought stress were investigated by qRT-PCR. We obtained 58 putative PvLTP genes in the common bean genome via genome-wide analyses. Based on the diversity of the eight-cysteine motif (ECM), these genes were categorized into five types (I, II, IV, V, and VIII). The signal peptides of the PvLTP precursors were predicted to be from 16 to 42 amino acid residues. PvLTPs had a predicated theoretical isoelectric point of 3.94-10.34 and a molecular weight of 7.15-12.17 kDa. The phylogenetic analysis showed that PvLTPs were closer to AtLTPs than OsLTPs. Conserved motif and gene structure analyses indicated that PvLTPs were randomly distributed on all chromosomes except chromosome 9. In addition, 23 tandem duplicates of PvLTP genes were arranged in 10 gene clusters on chromosomes 1 and 2. The heatmap and qRT-PCR showed that PvLTP expression significantly varied in different tissues. Moreover, 9 PvLTP genes were up-regulated under drought treatment. Our results reveal that PvLTPs play potentially vital roles in plants and provide a comprehensive reference for studies on PvLTP genes and a theoretical basis for further analysis of regulatory mechanisms influencing drought tolerance in the common bean.

Genome-wide identification, molecular evolution and expression analysis of the non-specific lipid transfer protein (nsLTP) family in Setaria italica

BackgroundFoxtail millet (Setaria italica L.) is a millet species with high tolerance to stressful environments. Plant non-specific lipid transfer proteins (nsLTPs) are a kind of small, basic proteins involved in many biological processes. So far, the genome of S. italica has been fully sequenced, and a comprehensive understanding of the evolution and expression of the nsLTP family is still lacking in foxtail millet.ResultsForty-five nsLTP genes were identified in S. italica and clustered into 5 subfamilies except three single genes (SinsLTP38, SinsLTP7, and SinsLTP44). The proportion of SinsLTPs was different in each subfamily, and members within the same subgroup shared conserved exon–intron structures. Besides, 5 SinsLTP duplication events were investigated. Both tandem and segmental duplication contributed to nsLTP expansion in S. italica, and the duplicated SinsLTPs had mainly undergone purifying selection pressure, which suggested that the function of the duplicated SinsLTPs might not diverge much. Moreover, we identified the nsLTP members in 5 other monocots, and 41, 13, 10, 4, and 1 orthologous gene pairs were identified between S. italica and S. viridis, S. bicolor, Z. mays, O. sativa, and B. distachyon, respectively. The functional divergence within the nsLTP orthologous genes might be limited. In addition, the tissue-specific expression patterns of the SinsLTPs were investigated, and the expression profiles of the SinsLTPs in response to abiotic stress were analyzed, all the 10 selected SinsLTPs were responsive to drought, salt, and cold stress. Among the selected SinsLTPs, 2 paired duplicated genes shared almost equivalent expression profiles, suggesting that these duplicated genes might retain some essential functions during subsequent evolution.ConclusionsThe present study provided the first systematic analysis for the phylogenetic classification, conserved domain and gene structure, expansion pattern, and expression profile of the nsLTP family in S. italica. These findings could pave a way for further comparative genomic and evolution analysis of nsLTP family in foxtail millet and related monocots, and lay the foundation for the functional analysis of the nsLTPs in S. italica.

Systematic and functional analysis of non-specific lipid transfer protein family genes in sugarcane under Xanthomonas albilineans infection and salicylic acid treatment.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins that play a significant regulatory role in a wide range of physiological processes. To date, no genome-wide survey and expression analysis of this gene family in sugarcane has been performed. In this study we identified the nsLTP gene family in Saccharum spontaneum and carried out expression profiling of nsLTPs in two sugarcane cultivars (Saccharum spp.) that have different resistance to leaf scald caused by Xanthomonas albilineans (Xa) infection. The effect of stress related to exogenous salicylic acid (SA) treatment was also examined. At a genome-wide level, S. spontaneum AP85-441 had 71 SsnsLTP genes including 66 alleles. Tandem (9 gene pairs) and segmental (36 gene pairs) duplication events contributed to SsnsLTP gene family expansion. Five SsnsLTP proteins were predicted to interact with five other proteins. Expression of ShnsLTPI.8/10/Gb.1 genes was significantly upregulated in LCP85-384 (resistant cultivar), but downregulated in ROC20 (susceptible cultivar), suggesting that these genes play a positive regulatory role in response of sugarcane to Xa infection. Conversely, ShnsLTPGa.4/Ge.3 appears to act as a negative regulator in response Xa infection. The majority (16/17) of tested genes were positively induced in LCP85-384 72 h after SA treatment. In both cultivars, but particularly in LCP85-384, ShnsLTPIV.3/VIII.1 genes were upregulated at all time-points, suggesting that the two genes might act as positive regulators under SA stress. Meanwhile, both cultivars showed downregulated ShnsLTPGb.1 gene expression, indicating its potential negative role in SA treatment responses. Notably, the ShnsLTPGb.1 gene had contrasting effects, with positive regulation of gene expression in response to Xa infection and negative regulation induced by SA stress. Together, our results provide valuable information for elucidating the function of ShnsLTP family members under two stressors and identified novel gene sources for development of sugarcane that are tolerant of environmental stimuli.

Genomic characterisation, phylogenetic comparison and candidate gene identification of the lipid transfer proteins gene family in pear (Pyrus bretschneideri Rehd.) and other Rosaceae species

ABSTRACT Plant non-specific lipid transfer proteins (nsLTPs) are a group of small, cryptic proteins that can reversibly bind and transport hydrophobic molecules. Studies have shown that nsLTPs can play important roles in various plant physiological processes, such as the transport of cuticular lipids, cutin synthesis, pathogen defence and abiotic stress responses. To date, a comprehensive analysis of the nsLTP gene family is still lacking in pear and other sequenced Rosaceae species. Here, 338 nsLTP genes were identified in eight Rosaceae fruit species, including 58 nsLTPs in Chinese white pear. The nsLTP family genes could be divided into four groups based on a detailed phylogenetic analysis and classification criteria in model plants. Multiple characteristics of 58 nsLTP genes in Chinese white pear were analysed, including their phylogeny, conserved motifs, chromosome locations, and cis-elements in the promoter sequences. Based on both RNA-Seq and qRT–PCR analyses, it was predicted that Pbr041771.1 might participate in cuticular wax translocation. The subcellular localisation results showed that Pbr041771.1 was localised in the plasma membrane. All results provide valuable information enhancing our understanding of the evolution and functional roles of nsLTP family genes.

The non-specific lipid transfer protein GmLtpI.3 is involved in drought and salt tolerance in soybean

Plant non-specific lipid transfer proteins (nsLTPs/LTPs) play important roles in plant growth, development and abiotic stress responses. However, little is known about LTPs in soybean. In this study, 131 LTPs were identified and divided into ten types (I, II, III, IV, V, VI, VII, VIII, IX, and Y), and each GmLtp has the characteristic eight-cysteine motif (8 CM) of plant LTPs. RNA-seq data showed that most GmLtps responded to a variety of abiotic stresses. Among the GmLtp members, GmLtpⅠ.3 was significantly induced under drought and salt treatments. Overexpressing GmLtpⅠ.3 improved tolerance to drought and salt stresses in transgenic Arabidopsis and soybean. The results showed that the accumulation of reactive oxygen species (ROS) in the roots of GmLtpⅠ.3 -overexpressing (OE -GmLtpⅠ.3 ) plants decreased significantly, compare with GmLtpⅠ.3 RNA interference (RNAi- GmLtpⅠ.3 ) and empty vector (EV-Control) plants. Furthermore, compared with EV-Control, the expression level of GmSOD1 and GmSOD2 increased in OE- GmLtpI.3 . Contrarily, the expression of GmRBOHA and GmRBOHB decreased in OE- GmLtpI.3 . The results of this study provide the basis for further comprehensive analyses of LTP genes and insights into abiotic stress response mechanisms in soybean. • 131 plant non-specific lipid transfer proteins genes were identified and analyzed in soybean. • Overexpression of GmLtpⅠ.3 can improve Arabidopsis and soybean stress tolerance. • GmLtpⅠ.3 regulates soybean tolerance to drought and salt stresses at the molecular levels.

Characteristics, expression profile, and function of non-specific lipid transfer proteins of Populus trichocarpa

Plant non-specific lipid transfer proteins (nsLTPs) are involved in various physiological processes. However, the characteristics and function of LTPs in Populus trichocarpa are unclear. Here, we report the functional properties of type IV, V, and VI P. trichocarpa nsLTPs (PtLTPs). The IV, V, and VI PtLTPs clustered in the same clade shared similar gene structures and motif and distributions. Also, collinearity analysis revealed 2 and 7 gene pairs have tandem duplication and segmental duplication events, respectively. The expression patterns of type IV, V, and VI PtLTPs differed among poplar tissues. We investigated the effects of various stresses on the Potri.010G100600, Potri.010G196300, and Potri.016G104300 (type V LTPs) mRNA levels, and type V LTPs can respond to multiple stresses. Potri.008G061800 was localized to the cell wall, extracellular space, and plasma membrane. Glutathione-S-transferase-Potri.008G061800 obtained by prokaryotic expression had weakly inhibited the growth of Septotis populiperda in vitro. Taken together, our data show that type IV, V, and VI PtLTPs may be thought as novel regulators of plant stresses. They could be considered an effective genetic resource for molecular breeding in poplar.

Systematic analysis of the non-specific lipid transfer protein gene family in Nicotiana tabacum reveal its potential roles in stress responses

Plant non-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone stabilized by four disulfide bonds; these proteins can bind or transfer lipids. NsLTPs play important roles in plant growth and development, and in the responses to abiotic and biotic stresses. In this study, 50, 51, and 100 nsLTPs from Nicotiana sylvestris, N. tomentosiformis, and their descendant N. tabacum, respectively, were identified and classified into six types (I, II, IV, V, VII, and VIII). The phylogeny, gene structures, motifs, tertiary structures, gene duplications and expression patterns were systematically analyzed. The intron/exon patterns and the conserved motifs were highly similar among the same types of nsLTP genes. Purifying selection and segmental duplication dominated the expansion of the nsLTPs family during evolution. Cis-regulatory elements of the NtLTP promoters were involved in light responsiveness, abiotic stress, and phytohormone responsiveness. Expression pattern analysis using RNA-seq and qPCR revealed that NtLTP family genes exhibited tissue-specific expression patterns and they have potential roles in response to abiotic and biotic stresses, especially drought stress, and resistance to black shank and bacterial wilt. Furthermore, overexpression of NtLTPI.38 in tobacco increased drought tolerance by improving the antioxidant defense ability, through reducing O2•— and H2O2 accumulation and increasing the number of lateral roots. These results provide a comprehensive overview of this gene family and provide valuable insights for the functional characterization of nsLTP family genes.

Identification and analysis of non-specific lipid transfer protein family in tobacco

Plant non-specific lipid transfer proteins (nsLTPs) can transfer lipids in vitro , regulate plant growth and development, and respond to environmental abiotic and biotic stresses. In this study, 74 nsLTPs genes were identified from the genome of Nicotiana tabacum variety K326, and we analyzed multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, protein domains, chromosome locations, cis -elements in the promoter sequences, 3D structure, and the expression patterns under different hormones and abiotic stresses. The results revealed that nsLTPs in tobacco could be divided into eight types, including type I, II, III, IV, V, VII, VIII, and XIII, according to the interval and sequence similarity between the eight cysteines. The same types of NtLTPs had similar intron-exon patterns and conserved motifs, motif 2 and motif 3 were the characteristic motifs of NtLTPs family. In the process of evolution, fragment duplication dominated the expansion of the NtLTPs family. RNA-seq analysis after drought treatment revealed that the functional differentiation patterns of repeat gene pairs were diverse during evolution period. Promoter analysis showed that they contained a variety of cis-acting elements in response to light response, hormones, and abiotic stress. Furthermore, qRT-PCR demonstrated that NtLTPs family genes had different expression patterns in different tissues and organs of tobacco plants, which could respond to abiotic stresses such as drought, salt, and hormone treatments (IAA, GA, and SA etc.). These results provide a theoretical reference for the in-depth analysis of the functions of NtLTPs family genes and molecular breeding.

Comprehensive classification of the plant non-specific lipid transfer protein superfamily towards its sequence-structure-function analysis.

BackgroundNon-specific Lipid Transfer Proteins (nsLTPs) are widely distributed in the plant kingdom and constitute a superfamily of related proteins. Several hundreds of different nsLTP sequences—and counting—have been characterized so far, but their biological functions remain unclear. It has been clear for years that they present a certain interest for agronomic and nutritional issues. Deciphering their functions means collecting and analyzing a variety of data from gene sequence to protein structure, from cellular localization to the physiological role. As a huge and growing number of new protein sequences are available nowadays, extracting meaningful knowledge from sequence–structure–function relationships calls for the development of new tools and approaches. As nsLTPs show high evolutionary divergence, but a conserved common right handed superhelix structural fold, and as they are involved in a large number of key roles in plant development and defense, they are a stimulating case study for validating such an approach.MethodsIn this study, we comprehensively investigated 797 nsLTP protein sequences, including a phylogenetic analysis on canonical protein sequences, three-dimensional structure modeling and functional annotation using several well-established bioinformatics programs. Additionally, two integrative methodologies using original tools were developed. The first was a new method for the detection of (i) conserved amino acid residues involved in structure stabilization and (ii) residues potentially involved in ligand interaction. The second was a structure–function classification based on the evolutionary trace display method using a new tree visualization interface. We also present a new tool for visualizing phylogenetic trees.ResultsFollowing this new protocol, an updated classification of the nsLTP superfamily was established and a new functional hypothesis for key residues is suggested. Lastly, this work allows a better representation of the diversity of plant nsLTPs in terms of sequence, structure and function.

Impact of lipid binding on the tertiary structure and allergenic potential of Jug r 3, the non-specific lipid transfer protein from walnut

Plant non-specific lipid transfer proteins type 1 (nsLTP1) are small basic proteins with a hydrophobic cavity able to host a number of different ligands: i.e. fatty acids, fatty acyl-CoA, phospholipids, glycolipids, and hydroxylated fatty acids. However, ligand binding specificity differs among nsLTPs. Within this protein family, Jug r 3 from walnut has been identified as a major allergen. So far, data on the structural characterization of Jug r 3 and its lipid binding capacity are lacking. We report the results from a fluorescence-based ligand-binding assay and ligand-based NMR experiments, to study the binding interactions between Jug r 3 and the 18-carbon monounsaturated oleic acid. Furthermore, protein-based NMR experiments were employed to detect the oleate binding site of Jug r 3. The NMR data were used to dock the oleate molecule into the structural model of Jug r 3. Finally, the impact of the interaction on the allergenic potential of Jug r 3 was investigated by IgE ELISA with 6 sera from walnut allergic patients. Our data corroborate the hypothesis of direct impact of food-derived matrix on the IgE reactivity of nsLTPs.

Mesorhizobium huakuii HtpG Interaction with nsLTP AsE246 Is Required for Symbiotic Nitrogen Fixation.

Plant nonspecific lipid transfer proteins (nsLTPs) are involved in a number of biological processes including root nodule symbiosis. However, the role of nsLTPs in legume-rhizobium symbiosis remains poorly understood, and no rhizobia proteins that interact with nsLTPs have been reported to date. In this study, we used a bacteria two-hybrid system and identified the high temperature protein G (HtpG) from Mesorhizobium huakuii that interacts with the nsLTP AsE246. The interaction between HtpG and AsE246 was confirmed by far-Western blotting and bimolecular fluorescence complementation. Our results indicated that the heat shock protein 90 (HSP90) domain of HtpG mediates the HtpG-AsE246 interaction. Immunofluorescence assay showed that HtpG was colocalized with AsE246 in infected nodule cells and symbiosome membranes. Expression of the htpG gene was relatively higher in young nodules and was highly expressed in the infection zones. Further investigation showed that htpG expression affects lipid abundance and profiles in root nodules and plays an essential role in nodule development and nitrogen fixation. Our findings provide further insights into the functional mechanisms behind the transport of symbiosome lipids via nsLTPs in root nodules.

Genome-wide identification and characterization of non-specific lipid transfer proteins in cabbage.

Plant non-specific lipid transfer proteins (nsLTPs) are a group of small, secreted proteins that can reversibly bind and transport hydrophobic molecules. NsLTPs play an important role in plant development and resistance to stress. To date, little is known about the nsLTP family in cabbage. In this study, a total of 89 nsLTP genes were identified via comprehensive research on the cabbage genome. These cabbage nsLTPs were classified into six types (1, 2, C, D, E and G). The gene structure, physical and chemical characteristics, homology, conserved motifs, subcellular localization, tertiary structure and phylogeny of the cabbage nsLTPs were comprehensively investigated. Spatial expression analysis revealed that most of the identified nsLTP genes were positively expressed in cabbage, and many of them exhibited patterns of differential and tissue-specific expression. The expression patterns of the nsLTP genes in response to biotic and abiotic stresses were also investigated. Numerous nsLTP genes in cabbage were found to be related to the resistance to stress. Moreover, the expression patterns of some nsLTP paralogs in cabbage showed evident divergence. This study promotes the understanding of nsLTPs characteristics in cabbage and lays the foundation for further functional studies investigating cabbage nsLTPs.

Overexpression of BraLTP2, a Lipid Transfer Protein of Brassica napus, Results in Increased Trichome Density and Altered Concentration of Secondary Metabolites.

Plant non-specific lipid transfer proteins (nsLTPs) belong to a large multigene family that possesses complex physiological functions. Trichomes are present on the aerial surfaces of most plants and include both glandular secretory hairs and non-glandular hairs. In this study, BraLTP2 was isolated from Brassica rapa (B. rapa) and its function was characterized in the important oilseed crop Brassica napus (B. napus). B. rapa lipid transfer protein 2 (BraLTP2) belongs to the little-known Y class of nsLTPs and encodes a predicted secretory protein. In ProBraLTP2::GUS (β-glucuronidase) transgenic plants, strong GUS activity was observed in young leaves and roots, while low activity was observed in the anther. It is noteworthy that strong GUS activity was observed in trichomes of the first four leaves of 4-week-old and 8-week-old seedings, however, it disappeared in 12-week-old seedings. In transgenic plants expressing a BraLTP2::GFP (green fluorescent protein) fusion protein, GFP fluorescence localized in the extracellular space of epidermal cells and trichomes. Overexpression of BraLTP2 in B. napus caused an increase in trichome number and altered the accumulation of secondary metabolites in leaves, including 43 upregulated secondary metabolites. Moreover, transgenic plants showed significantly increased activities of antioxidant enzymes. These results suggest that BraLTP2, a new nsLTP gene, may play a role in trichome development and the accumulation of secondary metabolites.

Molecular cloning and characterization of a lipid transfer protein gene (PsLTP1) from Pinus sylvestris (L.)

Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules. Despite extensive research on nsLTP in different plant species, mostly angiosperm, and the great diversity of physiological processes in which they seem to be involved, their exact functions still remain unclear. Also, very limited experimental data are available on nsLTP in gymnosperm. In this study, we report for the first time on the molecular cloning of nsLTP, from Pinus sylvestris L. (PsLTP1, GenBank accession JN980402.1) and the expression pattern of PsLTP1 during ontogenesis and in response to environmental stress conditions. Total RNA from roots of 7-day old pine seedlings was used to isolate the cDNA clone, corresponding to Scots pine lipid transfer protein. The open reading frame of PsLTP1 consists of 372 bp encoding a protein of 123 amino acids. Amino acid sequence alignment revealed that mature PsLTP1 shares high level of similarity with nsLTP from other conifers and with well-studied nsLTPs from angiosperms. The PsLTP1 contains a 27-amino-acid N-terminal signal sequence and presents all the features of a plant nsLTP. Amino acid comparison analysis and 3D structure prediction showed that PsLTP1 is a type 1 nsLTP. The results of the expression analysis of Scots pine PsLTP1 gene revealed that its transcripts accumulate in actively growing tissues. Furthermore, transcription of PsLTP1 was upregulated in response to cold and salt treatments, and downregulated during acidic, osmotic and water stresses.