Non-specific Lipid Transfer Protein Gene Research Articles

Soybean transcription factor GmNF-YB20 confers resistance to stripe rust in transgenic wheat by regulating nonspecific lipid transfer protein genes.

Worldwide food security is severely threatened by the devastating wheat stripe rust disease. The utilization of resistant wheat cultivars represents the most cost-effective and efficient strategy for combating this disease. However, the lack of resistant resources has been a major bottleneck in breeding for wheat disease resistance. Therefore, revealing novel gene resources for combating stripe rust and elucidating the underlying resistance mechanism is of utmost urgency. In this study, we identified that the soybean NF-YB transcription factor GmNF-YB20 in wheat provides resistance to the stripe rust fungus (Puccinia striiformis f. sp. tritici, Pst). Wheat lines with stable overexpression of the GmNF-YB20 enhanced resistance against multiple Pst races. Transcriptome profiling of GmNF-YB20 transgenic wheat under Pst infection unveiled its involvement in the lipid signaling pathway. RT-qPCR assays suggested that GmNF-YB20 increased transcript levels of multiple nonspecific lipid transfer protein (LTP) genes during wheat-Pst interaction, luciferase reporter analysis illustrates that it activates the transcription of TaLTP1.50 in wheat protoplast, and GmNF-YB20 overexpressed wheat plants had higher total LTP content in vivo during Pst infection. Overexpression of TaLTP1.50 in wheat significantly increased resistance to Pst, whereas knockdown of TaLTP1.50 exhibited the opposite trends, indicating that TaLTP1.50 plays a positive role in wheat resistance. Taken together, our findings provide perspective regarding the molecular mechanism of GmNF-YB20 in wheat and highlight the potential use for wheat breeding.

A Novel Non-Specific Lipid Transfer Protein Gene, CmnsLTP6.9, Enhanced Osmotic and Drought Tolerance by Regulating ROS Scavenging and Remodeling Lipid Profiles in Chinese Chestnut (Castanea mollissima Blume).

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5'-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.

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.

Overexpression of a cotton nonspecific lipid transfer protein gene, GhLTP4, enhances drought tolerance by remodeling lipid profiles, regulating abscisic acid homeostasis and improving tricarboxylic acid cycle in cotton

Drought is a major abiotic stress that causes severely crop yield loss worldwide. Lipid transfer proteins (LTPs) play an essential role during plant growth and cell signaling transduction associated with stress responses. However, the regulatory circuits involved in drought stress remain largely unresolved. Here, we reported that a nonspecific lipid transfer protein, namely GhLTP4 , positively modulated drought stress tolerance in cotton. GhLTP4 (synonym Pfs6 , GenBank: ABO42261) was preferentially expressed in fiber cells, while, it was also strongly induced by phytohormone abscisic acid (ABA) and drought stress treatments. Compared with wild type, over- and down-expression of GhLTP4 in cotton led to the increased and decreased drought tolerance, respectively. Overexpression of GhLTP4 remarkably increased lipid components and the accumulation of culticular waxes in transgenic cotton leaves under normal condition. Under drought stress, GhLTP4 -overexpressing transgenic cotton exhibited the remodeled lipid profiles, more ABA contents, and the improved tricarboxylic acid cycle process. Taken together, we reveal the function and underlying mechanisms of GhLTP4 in regulating cotton drought tolerance and create valuable genetic accessions for cotton drought-tolerant breeding. • Overexpression of GhLTP4 enhances significantly cotton drought tolerance. • Overexpression of GhLTP4 alters the lipid profiles of cotton leaf tissue. • Overexpression of GhLTP4 improves the drought tolerance by remodeling lipid profiles and ABA homeostasis. • Overexpression of GhLTP4 promotes tricarboxylic acid metabolism under drought stress.

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.

Genome-wide identification and expression profile under abiotic stress of the barley non-specific lipid transfer protein gene family and its Qingke Orthologues

BackgroundPlant non-specific lipid transfer proteins (nsLTPs), a group of small, basic ubiquitous proteins to participate in lipid transfer, cuticle formation and stress response, are involved in the regulation of plant growth and development. To date, although the nsLTP gene family of barley (Hordeum vulgare L.) has been preliminarily identified, it is still unclear in the recently completed genome database of barley and Qingke, and its transcriptional profiling under abiotic stress has not been elucidated as well.ResultsWe identified 40 barley nsLTP (HvLTP) genes through a strict screening strategy based on the latest barley genome and 35 Qingke nsLTP (HtLTP) orthologues using blastp, and these LTP genes were divided into four types (1, 2, D and G). At the same time, a comprehensive analysis of the physical and chemical characteristics, homology alignment, conserved motifs, gene structure and evolution of HvLTPs and HtLTPs further supported their similar nsLTP characteristics and classification. The genomic location of HvLTPs and HtLTPs showed that these genes were unevenly distributed, and obvious HvLTP and HtLTP gene clusters were found on the 7 chromosomes including six pairs of tandem repeats and one pair of segment repeats in the barley genome, indicating that these genes may be co-evolutionary and co-regulated. A spatial expression analysis showed that most HvLTPs and HtLTPs had different tissue-specific expression patterns. Moreover, the upstream cis-element analysis of HvLTPs and HtLTPs showed that there were many different stress-related transcriptional regulatory elements, and the expression pattern of HvLTPs and HtLTPs under abiotic stress also indicated that numerous HvLTP and HtLTP genes were related to the abiotic stress response. Taken together, these results may be due to the differences in promoters rather than by genes themselves resulting in different expression patterns under abiotic stress.ConclusionDue to a stringent screening and comprehensive analysis of the nsLTP gene family in barley and Qingke and its expression profile under abiotic stress, this study can be considered a useful source for the future studies of nsLTP genes in either barley or Qingke or for comparisons of different plant species.

Tissue-specific expression of GhnsLTPs identified via GWAS sophisticatedly coordinates disease and insect resistance by regulating metabolic flux redirection in cotton.

Cotton (Gossypium hirsutum) is constantly attacked by pathogens and insects. The most efficient control strategy is to develop resistant varieties using broad-spectrum gene resources. Several resistance loci harboured by superior varieties have been identified through genome-wide association studies. However, the key genes and/or loci have not been functionally identified. In this study, we identified a locus significantly associated with Verticillium wilt (VW) resistance, and within a 145.5-kb linkage disequilibrium, two non-specific lipid transfer protein genes (named GhnsLTPsA10) were highly expressed under Verticillium pathogen stress. The expression of GhnsLTPsA10 significantly increased in roots upon Verticillium dahliae stress but significantly decreased in leaves under insect attack. Furthermore, GhnsLTPsA10 played antagonistic roles in positively regulating VW and Fusarium wilt resistance and negatively mediating aphid and bollworm resistance in transgenic Arabidopsis and silenced cotton. By combining transcriptomic, histological and physiological analyses, we determined that GhnsLTPsA10-mediated phenylpropanoid metabolism further affected the balance of the downstream metabolic flux of flavonoid and lignin biosynthesis. The divergent expression of GhnsLTPsA10 in roots and leaves coordinated resistance of cotton against fungal pathogens and insects via the redirection of metabolic flux. In addition, GhnsLTPsA10 contributed to reactive oxygen species accumulation. Therefore, in this study, we elucidated the novel function of GhnsLTP and the molecular association between disease resistance and insect resistance, balanced by GhnsLTPsA10. This broadens our knowledge of the biological function of GhnsLTPsA10 in crops and provides a useful locus for genetic improvement of cotton.

Identification of non-specific Lipid Transfer Protein gene family members in Solanum lycopersicum and insights into the features of Sola l 3 protein

Non-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone that is stabilized by four disulphide bonds. The strong interest towards this protein family is mainly due to the fact that nsLTPs are involved in many biological processes and have been identified as major human allergens. Since tomato (Solanum lycopersicum L.) is one of the most consumed and allergenic vegetables, a full characterization of this family is needed. In this study, hidden Markov model profiles were used to identify nsLTPs within the tomato protein complement. Following manual curation, 64 nsLTP genes were classified into six sub-families. Furthermore, nsLTP gene structure, distribution and arrangement along tomato chromosomes were investigated. Available RNA-seq expression profile data and Real-Time PCR analyses were used to derive expression patterns of tomato nsLTPs in different tissues/organs. Non-specific LTP genes with high level of expression in tomato fruits were filtered out since they could play a key role in tomato allergenicity. Among these genes was Solyc10g075090 that encodes the allergen Sola l 3. Finally, cloning, heterologous expression, purification and biochemical characterization of the recombinant protein Sola l 3 was performed.

Genome-wide analysis of the barley non-specific lipid transfer protein gene family

Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley (Hordeum vulgare L.) (HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes, which can be divided into five types (1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.

Genomic Identification and Comparative Expansion Analysis of the Non-Specific Lipid Transfer Protein Gene Family in Gossypium.

Plant non-specific lipid transfer proteins (nsLTPs) are involved in many biological processes. In this study, 51, 47 and 91 nsLTPs were identified in Gossypium arboreum, G. raimondii and their descendant allotetraploid G. hirsutum, respectively. All the nsLTPs were phylogenetically divided into 8 distinct subfamilies. Besides, the recent duplication, which is considered cotton-specific whole genome duplication, may have led to nsLTP expansion in Gossypium. Both tandem and segmental duplication contributed to nsLTP expansion in G. arboreum and G. hirsutum, while tandem duplication was the dominant pattern in G. raimondii. Additionally, the interspecific orthologous gene pairs in Gossypium were identified. Some GaLTPs and GrLTPs lost their orthologs in the At and Dt subgenomes, respectively, of G. hirsutum. The distribution of these GrLTPs and GaLTPs within each subfamily was complementary, suggesting that the loss and retention of nsLTPs in G. hirsutum might not be random. Moreover, the nsLTPs in the At and Dt subgenomes might have evolved symmetrically. Furthermore, both intraspecific and interspecific orthologous genes showed considerable expression variation, suggesting that their functions were strongly differentiated. Our results lay an important foundation for expansion and evolutionary analysis of the nsLTP family in Gossypium, and advance nsLTP studies in other plants, especially polyploid plants.

A Novel Non-specific Lipid Transfer Protein Gene from Sugarcane (NsLTPs), Obviously Responded to Abiotic Stresses and Signaling Molecules of SA and MeJA

Non-specific lipid transfer proteins (NsLTPs) are soluble, small, basic proteins in plant, which have been reported to be involved in plant physiological functions such as the catalyzing transfer of phospholipids and play an important role in plant defense and stress responses. In this study, a member of NsLTP family gene (ScNsLTP, Acc. No. KR259657) was isolated from a full-length cDNA library of sugarcane stalk. The cDNA of ScNsLTP was 671 bp long and contained a 312 bp open reading frame (ORF), which can encode a protein of 103 amino acid residues with molecular weight of 10.66 kDa. The ScNsLTP transcript levels in sugarcane seedlings decreased in response to SA, whereas it increased under MeJA treatment, suggesting an antagonistic regulatory mechanism between the signaling molecules of SA and MeJA. The transcript levels of ScNsLTP were obviously up-regulated under chilling and PEG stresses, implying that the ScNsLTP gene was involved in response to abiotic stresses and playing a positive role in adaption to low temperature and drought stresses. These results provide important information for further functional studies of plant NsLTPs gene.

Function of non-specific lipid transfer protein gene PsnsLTP in Polygonum sibiricum Laxm.

Function of non-specific lipid transfer protein gene PsnsLTP in Polygonum sibiricum Laxm.

Cloning, Expression and Characterization of a Non-Specific Lipid Transfer Protein Gene from Potato

Plant non-specific lipid transfer proteins(nsLTPs) are abundantlipid binding proteins with small molecules that play several biological roles including antimicrobial defense,signaling,cell wall loosening,proteinase inhibiting and so on. A new cDNA clone,named StLTPa1,corresponding to potato nsLTP gene was isolated from a cultivated potato(Solanum tuberosum) infected with Ralstonia solanacearum. The sequence analysis showed that the cloned cDNA contained 636 bp nucleotide which encoded a type I nsLTP of 91 amino acids. The StLTPa1 gene is well conserved in the coding region with 60–92% identity at the amino acid level,and 75–85% identity at the nucleotide sequence level compared with other Solanaceae nsLTPs. The transcripts of the gene accumulated in potato leaf and stem tissues induced by R. solanacearum and showed dominant differential expression between resistant and susceptible genotypes. In situ hybridization results showed that the StLTPa1 mRNA was localized in phloem cells of vascular tissues in potato leaf and stem tissues after pathogen infection. Salicylic acid,methyl jasmonate and abscisic acid induced StLTPa1 gene expression in different manners. These three signal molecules could produce a long-term effect on the StLTPa1 gene expression during the early stages of potato—R. solanacearum interaction. These results thus showed that the StLTPa1 may be pathogen-responsive gene in plant defenses and involve in a complex gene regulation network.

Proteinase Inhibitor from Ginkgo Seeds Is a Member of the Plant Nonspecific Lipid Transfer Protein Gene Family

A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10(-5)-10(-4) m). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the alpha-helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.

Expression of 9-kDa non-specific lipid transfer protein genes in developing wheat grain is enhanced by high temperatures but not by post-anthesis fertilizer

A survey of EST databases identified 16 9-kDa non-specific lipid transfer proteins (nsLTPs) expressed in developing grain from the US spring wheat Butte 86. Two of the most abundant sequences encoded nsLTPs similar to proteins identified previously by two-dimensional gel electrophoresis/mass spectrometry (2-DE/MS). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to examine the expression of these nsLTPs in developing grain or endosperm produced under controlled temperature and fertilizer regimens. Under a moderate (24 °C/17 °C day/night) temperature regimen, transcripts accumulated late in grain development and achieved highest levels as grain reached maximum dry weight. When high temperature regimens (37 °C/28 °C day/night) were imposed from anthesis or 15 days post-anthesis (DPA) until maturity, maximum transcript levels were as much as 9-fold higher. Post-anthesis fertilizer had relatively small effects on transcript levels. Accumulation profiles for 9-kDa nsLTPs generated by 2-DE were consistent with transcript profiles. The nsLTPs increased 2 to 3.5-fold in amount in endosperm in response to high temperatures but showed little change in response to fertilizer. Flour from grain produced under the high temperature regimens also contained greater amounts of nsLTPs. Increases in the levels of nsLTPs in flour as a result of environmental conditions during grain fill may have important implications for flour quality and allergenic potential.

Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds

Nine cDNA clones encoding non-specific lipid transfer proteins (nsLTPs) were isolated from Triticum aestivum and Triticum durum cDNA libraries and characterized. One cDNA is predicted to encode a type 2 nsLTP (7 kDa) while others encode type 1 nsLTPs (9 kDa). All encoded proteins contain an N-terminal signal sequence and possess the characteristic features of nsLTPs. The genomic structures of the wheat nsLtp genes show that type 2 TaLtp7.1a, TaLtp7.2a and type 1 TaLtp9.2b genes lack introns while the other type 1 genes consist of one intron. Construction of a phylogenic tree of Poaceae nsLTPs shows that wheat nsLTPs can be divided into eleven distinct groups and are closely related to barley sequences. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression patterns of nine nsLtp genes were studied during wheat seed development and germination. We identified three different profiles of nsLtp gene transcript accumulation. Whereas TdLtp7.1a, TdLtp9.4a and TdLtp9.7a transcripts were detected during all maturation stages, TdLtp7.2a, TdLtp9.2a, TdLtp9.3a, TdLtp9.5a and TdLtp9.6a transcripts were only present in the first and TdLtp9.1a in the last stages of seed development. Moreover, these nine wheat nsLtp genes are not seed-specific and are also expressed in the coleoptile of young seedlings. The present study revealed the complexity of the wheat nsLtp gene family and showed that the expression of nsLtp genes is developmentally regulated in the seeds, suggesting a specific function for each of the corresponding proteins.

Identification of a 49-bp fragment of the HvLTP2 promoter directing aleurone cell specific expression

Identification of regulatory elements directing definite and specific spatiotemporal expression patterns is a prerequisite to the next generation of transgenic plants with commercial and ethical feasibility for producing plantibodies or other pharmaceutically important compounds. Here we describe the functional dissection of the barley nonspecific lipid transfer protein gene promoter, HvLTP2. The gene is specifically expressed in aleurone cells of cereals and used as an aleurone marker in maize and rice. The transcript is uniformly localised in the barley aleurone cells from around 10 DAP. Patchy expression in aleurone cells of transgenic rice has been reported and explained by silencing of transgenes. We have performed deletion analyses of the 801-bp HvLTP2 promoter to gain insight into the molecular basis of its regulation and the presence of putative regulatory elements. From the deletion studies, a 49-bp promoter region directing aleurone-specific expression was identified. Simultaneously, in vivo footprinting was carried out to identify promoter elements bound by putative regulatory proteins. Within the 49-bp fragment, the most promising candidate for a minimal cis-acting regulatory region directing aleurone specificity is the ds-sequence. Based on our results, we hypothesise that the ds-sequence directs aleurone specificity, possibly through a concerted action with elements directing general expression in the seed. Moreover, we present an overview of LTP2 elements putatively involved in directing seed, endosperm, and aleurone expression. Additionally, we report HvLTP2 expression in the embryo, not previously detected. The regulatory element(s) directing expression in embryo is located downstream of the 49-bp fragment directing aleurone specificity, thus demonstrating independent control of aleurone and embryo-localised expression. Finally, we discuss the existence of several endosperm-specific boxes and whether alternative promoter elements and combinations of them may direct aleurone expression, explaining why comparing different genes expressed in aleurone fail to identify only one required, common promoter element.