Temperature-induced Lipocalin Research Articles

Crystallographic and functional studies of a plant temperature-induced lipocalin

Arabidopsis thaliana temperature-induced lipocalin (AtTIL) is a prototypical member of plant lipocalins and participates in a variety of cellular processes, particularly stress responses. Bioinformatical and physiological studies have proposed its promiscuous ligand-binding ability, but the molecular basis is yet unclear. Here, we report the 1.9-Å crystal structure of AtTIL in complex with heme. Spectrophotometric absorbance titration with heme yields a dissociation constant of ∼2 micromolar, indicating the relatively weak interaction between AtTIL and heme, which is confirmed by the AtTIL-heme structure. Although binding to retinal or biliverdin is not detected, such possibility can not be precluded as suggested by comparison with other lipocalin structures. These results show that AtTIL is a structural and functional homolog of the bacterial lipocalin Blc.

Cloning, Characterization, and Functional Analysis of EuTIL1, a Gene-Encoding Temperature-Induced Lipocalin in Eucommia ulmoides Oliv

Eucommia ulmoides Oliver is a unique tertiary relict tree species in China belonging to the Eucommia family and genus. It is a traditional and precious Chinese medicinal herb with anti-tumor, antibacterial, antioxidant, anti-inflammatory, and bidirectional blood pressure regulation effects. Eucommia ulmoides mainly grows in temperate regions of China, but due to its sensitivity to low-temperatures, it is difficult to introduce into new regions. To study the role of Eucommia ulmoides lipocalin in plants. This investigation was conducted utilizing gene cloning, bioinformatics analysis, quantitative real-time polymerase chain reaction (qRT-PCR), subcellular localization, and stable genetic transformation to transfer EuTIL1 into Nicotiana tabacum Xanthi. The wild-type transgenic vector and EuTIL1 tobacco were cold-treated, and the corresponding protective enzyme activity and cold-induced gene expression levels were measured to analyze the functions of the genes. In this study, the full-length of the temperature-induced lipocalin gene (EuTIL1) cDNA was cloned from the leaves of Eucommia ulmoides using the rapid amplification of cDNA ends (RACE) method. The sequence analysis showed that the full-length cDNA of EuTIL1 was 917 bp and encodes a protein of 188 aa residues, which is a member of the Lipocalin-2 family. Subcellular localization analysis revealed that EuTIL1 was found in the plasma membrane. The transgenic tobacco lines expressing EuTIL1 under the control of the CaMV 35S promoter had increased tolerance to cold compared to wild-type (WT) plants. The average water loss rate of EuTIL1 transgenic plants was 12.4%, the average conductivity at 24 h was 55.11%, and the malondialdehyde content at 24 h was significantly lower than that of wild-type plants. The maximum soluble sugar (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase activity of EuTIL1 plants after low-temperature treatment were 22.03 mg/g, 726.87 U/g, 1283.94 U/g, and 356.84 U/g, respectively, which are significantly higher than those of the wild-type. Meanwhile, in the EuTIL1 transgenic tobacco plants, the expression of the NtDREB1, NtDREB2, NtDREB4, and NtCOR15a elevated under the low-temperature treatment condition. In conclusion, our study demonstrates that EuTIL1 is a gene involved in the cold-stress response and has the potential to enhance cold tolerance in plants, providing a potential molecular basis for the study of Eucommia ulmoides introduction and serving as a candidate gene for evaluating cold-tolerant plants.

Temperature-induced lipocalin-mediated membrane integrity: Possible implications for vindoline accumulation in Catharanthus roseus leaves.

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co-express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature-induced lipocalin (CrTIL), which was selected for full-length cloning, tissue-specific expression profiling, in silico characterization, and upstream genomic region analysis for cis-regulatory elements. Stress-mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion-based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline.

Transcriptome analysis of thermomorphogenesis in ovules and during early seed development in Brassica napus

BackgroundPlant sexual reproduction is highly sensitive to elevated ambient temperatures, impacting seed development and production. We previously phenotyped this effect on three rapeseed cultivars (DH12075, Topas DH4079, and Westar). This work describes the transcriptional response associated with the phenotypic changes induced by heat stress during early seed development in Brassica napus.ResultsWe compared the differential transcriptional response in unfertilized ovules and seeds bearing embryos at 8-cell and globular developmental stages of the three cultivars exposed to high temperatures. We identified that all tissues and cultivars shared a common transcriptional response with the upregulation of genes linked to heat stress, protein folding and binding to heat shock proteins, and the downregulation of cell metabolism. The comparative analysis identified an enrichment for a response to reactive oxygen species (ROS) in the heat-tolerant cultivar Topas, correlating with the phenotypic changes. The highest heat-induced transcriptional response in Topas seeds was detected for genes encoding various peroxidases, temperature-induced lipocalin (TIL1), or protein SAG21/LEA5. On the contrary, the transcriptional response in the two heat-sensitive cultivars, DH12075 and Westar, was characterized by heat-induced cellular damages with the upregulation of genes involved in the photosynthesis and plant hormone signaling pathways. Particularly, the TIFY/JAZ genes involved in jasmonate signaling were induced by stress, specifically in ovules of heat-sensitive cultivars. Using a weighted gene co-expression network analysis (WGCNA), we identified key modules and hub genes involved in the heat stress response in studied tissues of either heat-tolerant or sensitive cultivars.ConclusionsOur transcriptional analysis complements a previous phenotyping analysis by characterizing the growth response to elevated temperatures during early seed development and reveals the molecular mechanisms underlying the phenotypic response. The results demonstrated that response to ROS, seed photosynthesis, and hormonal regulation might be the critical factors for stress tolerance in oilseed rape.

Membrane lipid metabolism and scavenging of reactive oxygen species are essential for cold resistance of Solanum habrochaites (LA1777): Lipidomics and transcriptomes analysis

Low temperature significantly limits the production of tomato (Solanum lycopersicum) in greenhouse. Solanum habrochaites is a wild species of tomato closely related to Solanum lycopersicum and is more tolerant to cold than S. lycopersicum. However, the mechanism by which S. habrochaites resists low temperature is unknown. This study aimed to analyze the physiology, transcriptomics, and lipidomics of LA1777 (S. habrochaites variety) after exposure to cold stress for 3 days to assess its specific mechanism to cold tolerance. A cold-sensitive cultivated tomato variety AC was used as controls. Physiological analysis showed that the two tomato varieties were significantly affected by cold stress. However, LA1777 had more stable physiological characteristics and maintained relatively good photosynthetic capacity than the AC variety. Transcriptome analysis suggested that the stability of cell membrane structure and function might promote cold tolerance of LA1777. Furthermore, 243 differentially expressed genes (DEGs) involved in lipid metabolism were identified. Particularly, the upregulation of temperature-induced lipocalin genes (TILs) and fatty acid desaturase genes (FADs) promoted cold tolerance by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing membrane lipid unsaturation, respectively. Meanwhile, lipidomics data indicated that monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) levels were significantly reduced in both plants under cold stress. MGDG levels decreased by 28% and 54% in LA1777 and AC, respectively, while DGDG decreased by 7% and 29%, respectively. Further results showed that high phosphoinositide (PI) content may increase cold tolerance in LA1777 by maintaining phosphatidylinositol signaling system. Also, results showed that adenosine triphosphate (ATP) produced via β-oxidation of fatty acids in mitochondria can maintain a higher metabolic level of wild tomato under cold stress. A metabolic model of the mechanism of cold tolerance in tomato was also constructed. This study provides a basis for further analysis of the molecular mechanism of cold resistance in tomato, which is crucial for genetic improvement.

Regulation and physiological function of proteins for heat tolerance in cowpea (Vigna unguiculata) genotypes under controlled and field conditions.

The expression of heat shock proteins is considered a central adaptive mechanism to heat stress. This study investigated the expression of heat shock proteins (HSPs) and other stress-protective proteins against heat stress in cowpea genotypes under field (IT-96D-610 and IT-16) and controlled (IT-96D-610) conditions. Heat stress response analysis of proteins at 72 h in the controlled environment showed 270 differentially regulated proteins identified using label-free quantitative proteomics in IT-96D-610 plants. These plants expressed HSPs and chaperones [BAG family molecular chaperone 6 (BAG6), Multiprotein bridging factor1c (MBF1C) and cold shock domain protein 1 (CSDP1) in the controlled environment]. However, IT-96D-610 plants expressed a wider variety of small HSPs and more HSPs in the field. IT-96D-610 plants also responded to heat stress by exclusively expressing chaperones [DnaJ chaperones, universal stress protein and heat shock binding protein (HSBP)] and non-HSP proteins (Deg1, EGY3, ROS protective proteins, temperature-induced lipocalin and succinic dehydrogenase). Photosynthesis recovery and induction of proteins related to photosynthesis were better in IT-96D-610 because of the concurrent induction of heat stress response proteins for chaperone functions, protein degradation for repair and ROS scavenging proteins and PSII operating efficiency (Fq’/Fm′) than IT-16. This study contributes to identification of thermotolerance mechanisms in cowpea that can be useful in knowledge-based crop improvement.

Comparative transcriptomic analysis and antioxidant defense mechanisms in clusterbean (Cyamopsis tetragonoloba (L.) Taub.) genotypes with contrasting drought tolerance.

To understand drought tolerance mechanism(s) in clusterbean (Cyamopsis tetragonoloba), we conducted physiological, biochemical, and de novo comparative transcriptome analysis of drought-tolerant (RGC-1002) and drought-sensitive (RGC-1066) genotypes subjected to 30days of drought stress. Relative water content (RWC) was maintained in tolerant genotype but was reduced in sensitive genotype. Leaf pigment concentrations were higher in tolerant genotype. Net photosynthesis was significantly decreased in sensitive genotype but insignificant reduction was found in tolerant genotype. Enzymatic antioxidant (GR, APX, DHAR) activities were enhanced in tolerant genotype, while there were insignificant changes in these enzymes in sensitive genotype. The ratios of antioxidant molecules (ASC/DHA and GSH/GSSG) were higher in tolerant genotype as compared to sensitive genotype. In sensitive genotype, 6625 differentially expressed genes (DEGs) were upregulated and 5365 genes were downregulated. In tolerant genotype, 5206 genes were upregulated and 2793 genes were downregulated. In tolerant genotype, transketolase family protein, phosphoenolpyruvate carboxylase 3, temperature-induced lipocalin, and cytochrome oxidase were highly upregulated. Moreover, according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the drought tolerance may be attributed to upregulated starch and sucrose metabolism-related genes in tolerant genotype. Finally, quantitative real-time PCR confirmed the reproducibility of the RNA-seq data.

A plant lipocalin promotes retinal-mediated oscillatory lateral root initiation.

In Arabidopsis, de novo organogenesis of lateral roots is patterned by an oscillatory mechanism called the root clock, which is dependent on unidentified metabolites. To determine whether retinoids regulate the root clock, we used a chemical reporter for retinaldehyde (retinal)–binding proteins. We found that retinal binding precedes the root clock and predicts sites of lateral root organogenesis. Application of retinal increased root clock oscillations and promoted lateral root formation. Expression of an Arabidopsis protein with homology to vertebrate retinoid-binding proteins, TEMPERATURE INDUCED LIPOCALIN (TIL), oscillates in the region of retinal binding to the reporter, confers retinal-binding activity in a heterologous system, and, when mutated, decreases retinal sensitivity. These results demonstrate a role for retinal and its binding partner in lateral root organogenesis.

Overexpression of Wild Arachis Lipocalin Enhances Root-Knot Nematode Resistance in Peanut Hairy Roots

Plant parasitic root-knot nematodes (RKN) have evolved sophisticated strategies for exploiting plants, and they cause devastating yield losses in many susceptible crops. The RKN Meloidogyne arenaria is the most predominant pathogenic nematode affecting cultivated peanut (Arachis hypogaea L.). Resistance against the M. arenaria has been identified in the wild peanut relative, Arachis stenosperma. Transcriptome studies on A. stenosperma–M. arenaria interaction revealed candidate genes potentially involved in the first stages of this resistance response, including a temperature-induced lipocalin (TIL) protein. Plant TILs have a protective role against harmful molecules produced in response to a number of stresses. In this study, the characterization of the RKN-responsive TIL from A. stenosperma (AsTIL) provides new insights into the role of plant lipocalins in nematode resistance. The AsTIL gene has a 2-exon/1-intron structure and encodes a 21.4 kDa polypeptide. It contains three structurally conserved regions, which are a signature for plant lipocalins. Overexpression of AsTIL in transgenic hairy roots from a susceptible peanut cultivar led to the reduction of M. arenaria galls and egg masses 60 days after inoculation. This is the first report of a possible involvement of plant lipocalins in RKN resistance. It also reveals a promising candidate gene for peanut breeding to produce cultivars resistant to M. arenaria.

Functional analyses of lipocalin proteins in tomato.

In this study, two temperature-induced lipocalin genes SlTIL1 and SlTIL2, and a chloroplastic lipocalin gene SlCHL were isolated from 'Micro-Tom' tomato. The coding sequences of SlTIL1, SlTIL2 and SlCHL were 558, 558, and 1002 bp, respectively. By TargetP analysis, no characteristic transit peptides were predicted in the proteins of SlTIL1 and SlTIL2, while a chloroplastic transit peptide was predicted in the protein of SlCHL. The subcellular localization results indicated that SlTIL1 and SlTIL2 proteins were major localized in the plasma membrane, while SlCHL was localized in chloroplast. To understand the function of lipocalins, transgenic tomato over-expressed SlTIL1, SlTIL2 and SlCHL and their virus-induced gene silencing (VIGS) plants were generated. The phenotypes were significantly affected when the SlTIL1, SlTIL2 and SlCHL were over-expressed or silenced by VIGS, which suggested that the three lipocalins played important roles in regulating the growth and development of tomato. In addition, the level of ROS (O2 - and H2O2) was low in SlTIL1, SlTIL2 and SlCHL over-expressed plants, while it was high in their silenced plants. The changes in the expression of SODs were consistent with the accumulations of ROS, which indicated that lipocalins might have an important role in abiotic oxidative stress tolerance in tomato plants. Especially SlTIL1 and SlTIL2 are localized around their membranes and protect them from ROS. The results will contribute to elucidating the functions of lipocalin in plants, and provide new strategies to improve the tolerance to abiotic stress in tomato plants.

Temperature-induced lipocalin (TIL): a shield against stress-inducing environmental shocks in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a well-established workhorse, either for recombinant or natural products, thanks to its natural traits and easily editable metabolism. However, during a bio-based industrial process it meets multiple stresses generated by operative conditions such as non-optimal temperature, pH, oxygenation and product accumulation. The development of tolerant strains is therefore indispensable for the improvement of production, yield and productivity of fermentative processes. In this regard, plants as resilient organisms are a generous source for fishing genes and/or metabolites that can help the cell factory to counteract environmental constraints. Plants possess proteins named temperature-induced lipocalins, TIL, whose levels in the cells correlates with the tolerance to sudden temperature changes and with the scavenging of reactive oxygen species. In this work, the gene encoding for the Arabidopsis thaliana TIL protein was for the first time expressed in S. cerevisiae. The recombinant strain was compared and analysed against the parental counterpart under heat shock, freezing, exposure to organic acid and oxidative agents. In all the tested conditions, TIL expression conferred a higher tolerance to the stress imposed, making this strain a promising candidate for the development of robust cell factories able to overtake the major impairments of industrial processes.

Transcriptome Analysis of a Female-sterile Mutant (fsm) in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Female-sterile mutants are ideal materials for studying pistil development in plants. Here, we identified a female-sterile mutant fsm in Chinese cabbage. This mutant, which exhibited stable inheritance, was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and ethyl methanesulfonate mutagenesis. Compared with the wild-type line ‘FT,’ the fsm plants exhibited pistil abortion, and floral organs were also relatively smaller. Genetic analysis indicated that the phenotype of fsm is controlled by a single recessive nuclear gene. Morphological observations revealed that the presence of abnormal ovules in fsm likely influenced normal fertilization process, ultimately leading to female sterility. Comparative transcriptome analysis on the flower buds of ‘FT’ and fsm using RNA-Seq revealed a total of 1,872 differentially expressed genes (DEGs). Of these, a number of genes involved in pistil development were identified, such as PRETTY FEW SEEDS 2 (PFS2), temperature-induced lipocalin (TIL), AGAMOUS-LIKE (AGL), and HECATE (HEC). Furthermore, GO and KEGG pathway enrichment analyses of the DEGs suggested that a variety of biological processes and metabolic pathways are significantly enriched during pistil development. In addition, the expression patterns of 16 DEGs, including four pistil development-related genes and 12 floral organ development-related genes, were analyzed using qRT-PCR. A total of 31,272 single nucleotide polymorphisms were specifically detected in fsm. These results contribute to shed light on the regulatory mechanisms underlying pistil development in Chinese cabbage.

Plastid Proteomic Analysis in Tomato Fruit Development.

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of ‘Micro-Tom’ fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared ‘Micro-Tom’ results with those from two other varieties, ‘Black’ and ‘White Beauty’. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of ‘Micro-Tom’, and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared ‘Micro-Tom’ fruits with ‘Black’ and ‘White Beauty’ using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the ‘Black’ and ‘White Beauty’ varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between ‘Micro-Tom’ and ‘Black’/‘White Beauty’. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

A hydrophobic proline-rich motif is involved in the intracellular targeting of temperature-induced lipocalin.

Temperature-induced lipocalins (TILs) play an essential role in the response of plants to different abiotic stresses. In agreement with their proposed role in protecting membrane lipids, TILs have been reported to be associated to cell membranes. However, TILs show an overall hydrophilic character and do not contain any signal for membrane targeting nor hydrophobic sequences that could represent transmembrane domains. Arabidopsis TIL (AtTIL) is considered the ortholog of human ApoD, a protein known to associate to membranes through a short hydrophobic loop protruding from strands 5 and 6 of the lipocalin β-barrel. An equivalent loop (referred to as HPR motif) is also present between β-strands 5 and 6 of TILs. The HPR motif, which is highly conserved among TIL proteins, extends over as short stretch of eight amino acids and contains four invariant proline residues. Subcellular localization studies have shown that TILs are targeted to a variety of cell membranes and organelles. We have also found that the HPR motif is necessary and sufficient for the intracellular targeting of TILs. Modeling studies suggest that the HPR motif may directly anchor TILs to cell membranes, favoring in this way further contact with the polar group of membrane lipids. However, some particular features of the HPR motif open the possibility that targeting of TILs to cell membranes could be mediated by interaction with other proteins. The functional analysis of the HPR motif unveils the existence of novel mechanisms involved in the intracellular targeting of proteins in plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0326-x) contains supplementary material, which is available to authorized users.

Biochemical and proteomic analysis of grape berries (Vitis labruscana) during cold storage upon postharvest salicylic acid treatment.

Salicylic acid (SA) treatment has been widely used to maintain fruit quality during postharvest storage. To elucidate the molecular mechanism related to this treatment, the effect of SA treatment on fruit quality as well as protein expression profiles of grape berries (Vitis labruscana cv. Kyoho) during the subsequent cold storage was evaluated. As expected, SA treatment inhibited postharvest loss and chilling damage by reducing fruit softening and membrane damage and slowing weight loss. A gel-based proteomic approach was designed to screen for differentially expressed proteins in SA-treated and control grape berries. A total of 69 differentially accumulated proteins were successfully identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which can be functionally classified into eight categories. Among these proteins, antioxidant enzymes including ascorbate peroxidase, oxidoreductase, and glutathione S-transferase were induced, and the abundances of several defense-related proteins, such as heat shock protein (HSP) and temperature-induced lipocalin, were up-regulated by SA treatment. In addition, proteins involved in carbohydrate catabolism and energy production were also induced by SA treatment. Interpretation of the data for differential accumulation of proteins revealed that the effect of SA on reducing postharvest losses and chilling damage of grape berries during cold storage may be due to activated defense responses and carbohydrate metabolism and higher levels of energy status.

Temperature-induced lipocalin (TIL) is translocated under salt stress and protects chloroplasts from ion toxicity

Temperature-induced lipocalins (TIL) have been invoked in the defense from heat, cold and oxidative stress. Here we document a function of TIL for basal protection from salinity stress. Heterologous expression of TIL from the salt resistant poplar Populus euphratica did not rescue growth but prevented chlorophyll b destruction in salt-exposed Arabidopsis thaliana. The protein was localized to the plasma membrane but was re-translocated to the symplast under salt stress. The A. thaliana knock out and knock down lines Attil1-1 and Attil1-2 showed stronger stress symptoms and stronger chlorophyll b degradation than the wildtype (WT) under excess salinity. They accumulated more chloride and sodium in chloroplasts than the WT. Chloroplast chloride accumulation was found even in the absence of salt stress. Since lipocalins are known to bind regulatory fatty acids of channel proteins as well as iron, we suggest that the salt-induced trafficking of TIL may be required for protection of chloroplasts by affecting ion homeostasis.

Arabidopsis lipocalins AtCHL and AtTIL have distinct but overlapping functions essential for lipid protection and seed longevity

Lipocalins are a group of multifunctional proteins, recognized as carriers of small lipophilic molecules, which have been characterized in bacteria and animals. Two true lipocalins have been recently identified in plants, the temperature-induced lipocalin (TIL) and the chloroplastic lipocalin (CHL), the expression of which is induced by various abiotic stresses. Each lipocalin appeared to be specialized in the responses to specific stress conditions in Arabidopsis thaliana, with AtTIL and AtCHL playing a protective role against heat and high light, respectively. The double mutant AtCHL KO × AtTIL KO deficient in both lipocalins was more sensitive to temperature, drought and light stresses than the single mutants, exhibiting intense lipid peroxidation. AtCHL deficiency dramatically enhanced the photosensitivity of mutants (vte1, npq1) affected in lipid protection mechanisms (tocopherols, zeaxanthin), confirming the role of lipocalins in the prevention of lipid peroxidation. Seeds of the AtCHL KO × AtTIL KO double mutant were very sensitive to natural and artificial ageing, and again this phenomenon was associated with the oxidation of polyunsaturated lipids. The presented results show that the Arabidopsis lipocalins AtTIL and AtCHL have overlapping functions in lipid protection which are essential for stress resistance and survival.

Screening Differential Expressions of Defense-related Responses in Cold-treated 'Kyoho' and 'Campbell Early' Grapevines

Low temperature is one of the major environmental factors that affect productivity including reduced growth and budding of vines, and changes of metabolic processes in grape (Vitis spp.). To screen the specific expression of abiotic stress-related genes against cold treatment in 'Kyoho' and 'Campbell Early' grapevines, expression of various defense-related genes was investigated by RT-PCR and real-time PCR. Among the 67 genes analyzed by RT-PCR and real-time PCR, 17 and 16 types of cDNA were up-regulated, while 5 and 6 types were down-regulated in cold-treated 'Kyoho' and 'Campbell Early' grapevines, respectively. Genes encoding carotene (Cart3564 and Cart4472), chalcone isomerase (CHI), cytochrome P450 (CYP), flavonol synthase (FLS), endo-<TEX>${\beta}$</TEX>-glucanase precursor (Glu), glutathione peroxidase (GPX), glutathione-S-transferase (GST), leucine-rich repeats (LRR), manganese superoxide dismutase (Mn-SOD), phenylalanine ammonia lyase (PAL), polygalacturonase-inhibiting protein (PGIP), proline rich protein 2 (PRP2), small heat shock protein (sHSP), temperature induced lipocalin (TIL), and thaumatin-like protein (TLP) were up-regulated, while those encoding CBF like transcription factor (CBF1), chitinase-like protein (CLP), cold induced protein (CIP), glycerol-3-phosphate acyltransferase (GPAT), and mitogen-activated protein kinase (MAPK) were down-regulated by low temperature treatment in both in 'Kyoho' and 'Campbell Early'.

Proteomic Identification of Differentially Expressed Proteins between Male and Female Plants in Pistacia chinensis

Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis.

Brassinolide enhances cold stress tolerance of fruit by regulating plasma membrane proteins and lipids

How to enhance fruit tolerance to cold stress is an important biological interest. In this paper, we found that mango (Mangifera indica L.) fruit treated with 10 μM brassinolide (BL) showed a higher tolerance to cold temperature of 5 °C. Further, we compared the changes in expression profiles of plasma membrane (PM) proteins and the corresponding gene expressions between BL-treated and control fruit. Fourteen differential proteins were positively identified by mass spectrometry, and were categorized into four groups, including transport, cellular biogenesis, defense and stress response, and unknown function. Among them, four proteins (remorin, abscisic stress ripening-like protein, type II SK2 dehydrin, and temperature-induced lipocalin) and genes encoding these proteins were up-regulated in BL treatment under cold stress. Moreover, we found that PM lipids in BL-treated fruit showed lower phase transition temperature and higher unsaturation degree, leading to higher fluidity under low temperature. These findings ascertain that PM proteins and lipids are involved in BL-mediated responses to cold stress in mango fruit, and provide novel evidence that BL plays an important role in regulating cold stress tolerance in fruit.