Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

Background

Plant systemic signaling characterized by the long distance transport of molecules across plant organs involves the xylem and phloem conduits. Root-microbe interactions generate systemic signals that are transported to aerial organs via the xylem sap. We analyzed the xylem sap proteome of soybean seedlings in response to pathogenic and symbiotic interactions to identify systemic signaling proteins and other differentially expressed proteins.

Results

We observed the increase of a serine protease and peroxidase in the xylem sap in response to Phytophthora sojae elicitor treatment. The high molecular weight fraction of soybean xylem sap was found to promote the growth of Neurospora crassa in vitro at lower concentrations and inhibit growth at higher concentrations. Sap from soybean plants treated with a P. sojae elicitor had a significantly higher inhibitory effect than sap from control soybean plants. When soybean seedlings were inoculated with the symbiont Bradyrhizobium japonicum, the abundance of a xyloglucan transendoglycosyl transferase protein increased in the xylem sap. However, RNAi-mediated silencing of the corresponding gene did not significantly affect nodulation in soybean hairy root composite plants.

Conclusion

Our study identified a number of sap proteins from soybean that are differentially induced in response to B. japonicum and P. sojae elicitor treatments and a majority of them were secreted proteins.     

Proteomic analysis of hybrid poplar xylem sap

Xylem sap collected from Populus trichocarpa × Populus deltoides using root pressure was estimated to contain more than 100 proteins. Ninety-seven of these proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These proteins were classified into 10 functional categories including metabolism, signaling, stress response and cell wall functions. The majority of xylem sap proteins were metabolic enzymes involved in processes including translation, proteolysis, and glycolysis. Stress-related proteins were also prevalent. In contrast to xylem sap proteins collected from annual plants, the majority of poplar xylem sap proteins do not appear to be classically secreted since only 33 proteins were predicted to have an N-terminal signal peptide targeting them to the secretory pathway. Of the remaining 64 proteins, 27 were predicted to be secreted non-classically. While a number of proteins identified here have been previously reported in xylem sap proteomes of annual plants, many xylem sap proteins were identified in poplar which may reflect functions specific to perennial plants.     

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.     

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

Nitrogen Under-and Over-supply Induces Distinct Protein Responses in Maize Xylem Sap

Xylem sap primarily transports water and mineral nutrients such as nitrogen(N)from roots to shoots in vascular plants.However,it remains largely unknown how nitrogenous compounds,especially proteins in xylem sap,respond to N under-or over-supply.We found that reducing N supply increased amino-N percentage of total N in maize(Zea mays L.)xylem sap.Proteomic analysis showed that 23 proteins in the xylem sap of maize plants,including 12 newly identified ones,differentially accumulated in response to various N supplies.Fifteen of these 23 proteins were primarily involved in general abiotic or biotic stress responses,whereas the other five proteins appeared to respond largely to N under-or over-supply,suggesting distinct protein responses in maize xylem upon N under-and over-supply.Furthermore,one putative xylanase inhibitor and two putative O-glycosyl hydrolases had preferential gene expression in shoots.     

Characterization of the maize xylem sap proteome

The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.     

The mixed xylem sap proteome of Fusarium oxysporum-infected tomato plants

Secreted proteins are known to play decisive roles in plant–fungus interactions. To study the molecular details of the interaction between the xylem-colonizing, plant-pathogenic fungus Fusarium oxysporum and tomato, the composition of the xylem sap proteome of infected tomato plants was investigated and compared with that of healthy plants. Two-dimensional gel separation and mass spectrometry yielded peptide masses and peptide sequences of 33 different proteins. Despite the absence of complete genome sequences of either tomato or F. oxysporum , 21 proteins were identified as tomato proteins and seven as fungal proteins. Thirteen of the tomato proteins were specific for infected plants. Sixteen tomato proteins were found in xylem sap for the first time, four of which were identified based on matches to expressed sequences only. Coding sequences for new proteins from F. oxysporum were identified through either direct matching to a database sequence, matching of peptide sequences to genome or expressed sequence tag databases of other Fusarium species, or PCR with degenerate primers on cDNA derived from infected plants followed by screening of a F. oxysporum BAC library. Together, these findings provide an excellent basis for further exploration of the interaction between xylem-colonizing pathogens and their hosts.     

Changes to the proteome and targeted metabolites of xylem sap in Brassica oleracea in response to salt stress

Root-to-shoot signalling via xylem sap is an important mechanism by which plants respond to stress. This signalling could be mediated by alteration in the concentrations of inorganic and/or organic molecules. The effect of salt stress on the contents of xylem sap in Brassica olarecea has been analysed by mass spectrometry in order to quantify these changes. Subcellular location of arabinogalactan proteins (AGPs) by immunogold labelling and peroxidase isozymes was also analysed by isoelectrofocusing. The xylem sap metabolome analysis demonstrated the presence of many organic compounds such as sugars, organic acids and amino acids. Of these, amino acid concentrations, particularly that of glutamine, the major amino acid in the sap, were substantially reduced by salt stress. The xylem sap proteome analysis demonstrated the accumulation of enzymes involved in xylem differentiation and lignification, such as cystein proteinases, acid peroxidases, and a putative hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase under salt stress. The peroxidase isozyme pattern showed that salt stress induced a high accumulation of an acid isoform. These results suggest that xylem differentiation and lignification is induced by salt stress. The combination of different methods to analyse the xylem sap composition provides new insights into mechanisms in plant development and signalling under salt stress.     

转Bt 基因抗虫棉(Gossyposium)伤流中Bt 毒蛋白的运输

本文以常规棉品种‘石远321’和抗虫棉品种‘SGK321’与‘99B’为材料, 通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量。结果发现, 转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉, 并在抗虫棉伤流中检测到了Bt毒蛋白, 而常规棉伤流中没有Bt毒蛋白的存在。通过嫁接实验研究发现, 将常规棉嫁接到转Bt基因抗虫棉砧木上, 叶片中也有Bt毒蛋白的积累。这些结果说明, Bt毒蛋白可以通过木质部伤流液向地上部运输, 并在叶片中积累, Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的。     

The tomato xylem sap protein XSP10 is required for full susceptibility to Fusarium wilt disease

XSP10 is an abundant 10 kDa protein found in the xylem sap of tomato. The protein displays structural similarity to plant lipid transfer proteins (LTPs). LTPs are involved in various physiological processes, including disease resistance, and some are able to bind and transfer diverse lipid molecules. XSP10 abundance in xylem sap declines upon infection with Fusarium oxysporum f. sp. lycopersici (Fol), implying involvement of XSP10 in the plant-pathogen interaction. Here, the biochemical characterization of XSP10 with respect to fatty acid-binding properties is reported; a weak but significant binding to saturated fatty acids was found. Furthermore, XSP10-silenced tomato plants were engineered and it was found that these plants exhibited reduced disease symptom development upon infection with a virulent strain of Fol. Interestingly, the reduced symptoms observed did not correlate with an altered expression profile for known reporter genes of plant defence (PR-1 and WIPI). This work demonstrates that XSP10 has lipid-binding properties and is required for full susceptibility of tomato to Fusarium wilt.     

Biochemical Changes in Root Exudate and Xylem Sap of Tomato Plants Infected with Meloidogyne incognita

Under two monoxenic culture techniques of growing plants (filter paper and silica sand cultures), sugar in root exudate from Meloidogyne incognita-infected tomato increased 133 to 836% over controls. In contrast, amino acids were moderately reduced 52 to 56%. Chromatographic analysis showed that galled root exudate contained three sugars, twelve amino acids, and three organic acids, whereas healthy root exudate contained four sugars, fifteen amino acids, and four organic acids. Polysaccharide was responsible for the large increase of sugars in galled root exudates. The concn and the absolute amount of total sugars in the infected plant xylem sap were greater than in healthy plant xylem sap up to 6 wk after inoculation, whereas amino acids were moderately lower than in controls throughout the test period. Chromatographic analysis showed that xylem sap from both healthy and infected plants at 4 wk after inoculation contained four sugars and five organic acids. We identified 18 and 17 amino acids in the healthy and infected plant xylem sap, respectively. The concn of sugar increased as the nematode inoculum increased at 2, 4 and 6 wk after inoculation. The amino acids in all samples from the infected plant moderately decreased with an increase of nematode inoculum. We suggest that changes in total sugars and amino acids, of infected plant xylem sap and root exudate are a probable mechanism by which tomato plants are predisposed to Fusarium wilt.     

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N-glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC-MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty-one proteins were identified in the N-glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido-reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.     

Analysis of sugars in squash xylem sap

Xylem sap contains organic and inorganic compounds that might be involved in root-to-shoot communication. To clarify the physiological functions of sugars in xylem sap, we characterized the sugar compounds of the xylem sap. The 80% ethanol-soluble fraction of xylem sap contained mainly myo-inositol and oligosaccharides. The 80% ethanol precipitate was solubilized with cyclohexanediamine tetraacetate and fractionated using anion exchange chromatography. The non-bound fraction from the anion-exchange column reacted with Yariv reagent and was rich in arabinogalactan, indicating the presence of arabinogalactan proteins (AGP). The bound fraction eluted with 50 mM ammonium formate buffer and separated using size exclusion chromatography producing the pectins rhamnogaracturonan (RG)-I and RG-II with apparent molecular masses of 15000 and 11000, respectively. These results indicate that the AGP, RG-I, borate cross-linked RG-II dimer and oligosaccharides produced by root tissues are transported to above-ground organs via xylem sap.     

Accumulation of weathered pp'-DDE in xylem sap of grafted watermelon

Movement of weathered p,p'-dichlorodiphenyldichloroethane (p,p'-DDE) from contaminated soil to the rhizosphere pore water to the xylem sap of grafted watermelon was studied under green house conditions. p,p'-DDE concentrations in pore water and xylem sap was compared in intact plants, homografted, and compatible heterografts of Cucurbita pepo spp. pepo and Citrullus lanatus plants. An average p,p'-DDE concentrations in pore water of contaminated soil ranged from 0.36 microg/L to 0.55 microg/L and there were no statistically significant among the cultivars. Conversely, the xylem sap p,p'-DDE concentration of heterografted watermelon having a zucchini rootstock and watermelon scion was 71 microg/L and it was greater than intact watermelon plants (0.49 microg/L) but less than that of intact plants of zucchini (141 microg/L). Homografting showed no effect on xylem sap p,p'-DDE concentrations of the identical cultivars. The bio-concentration factors (BCFs) which is an average p,p'-DDE concentration in xylem sap over average p,p'-DDE in pore water were 344, 325, 197, 1.28, and 0.89 for intact plant of zucchini, homografted zucchini, heterografted watermelon, homografted watermelon, and intact plant of watermelon, respectively. Xylem sap p,p'-DDE concentrations of the heterografted watermelon plants were clearly influenced by plant phylogeny and enhanced by the zucchini rootstock compared to intact watermelon plants.     

Induction of xylem sap methylglycine by a drought and rewatering treatment and its inhibitory effects on the growth and development of plant organs

In higher plants, the xylem vessels functionally connect the roots with the above-ground organs. The xylem sap transports various organic compounds, such as proteins and amino acids. We examined drought and rewatering-inducible changes in the amino acid composition of root xylem sap collected from Cucurbita maxima roots. The major free amino acids in C . maxima root xylem sap were methylglycine (MeGly; sarcosine) and glutamine (Gln), but MeGly was not detected in the xylem sap of cucumber. MeGly is an intermediate compound in the metabolism of trimethylglycine (TMG; betaine), but its physiological effects in plants are unknown. Drought and rewatering treatment resulted in an increase in the concentration of MeGly in root xylem sap to 2.5 m M . After flowering, the MeGly concentration in the xylem sap dropped significantly, whereas the concentration of Gln decreased only after fruit ripening. One milli molar MeGly inhibited the formation of adventitious roots and their elongation in C . maxima , but glycine, dimethylglycine, or TMG had no effect. Similar effects and the inhibition of stem elongation were observed in shoot cuttings of cucumber and Phaseolus angularis . These observations seem to imply a possible involvement of xylem sap MeGly in the physiological responses of C . maxima plants to drought stress.     

Use of Gold Nanoparticles to Detect Water Uptake in Vascular Plants

Direct visualization of water-conducting pathways and sap flows in xylem vessels is important for understanding the physiology of vascular plants and their sap ascent. Gold nanoparticles (AuNPs) combined with synchrotron X-ray imaging technique is a new promising tool for investigating plant hydraulics in opaque xylem vessels of vascular plants. However, in practical applications of AuNPs for real-time quantitative visualization of sap flows, their interaction with a vascular network needs to be verified in advance. In this study, the effect of AuNPs on the water-refilling function of xylem vessels is experimentally investigated with three monocot species. Discrepancy in the water uptakes starts to appear at about 20 min to 40 min after the supply of AuNP solution to the test plant by the possible gradual accumulation of AuNPs on the internal structures of vasculature. However conclusively, it is observed that the water-refilling speeds in individual xylem vessels are virtually unaffected by hydrophilically surface-modified AuNPs (diameter ∼20 nm). Therefore, the AuNPs can be effectively used as flow tracers in the xylem vessels in the first 20∼30 min without any physiological barrier. As a result, AuNPs are found to be useful for visualizing various fluid dynamic phenomena occurring in vascular plants.     

Changes in the photosynthetic apparatus during fusarium wilt of tomato

Development of fusarium wilt was studied in 4-to 6-month-old tomato plants (Lycopersicon esculentum L., cv. Kunera). It was shown that the development of this disease could follow two patterns. When the wilt developed slowly (type I disease), the mycelium of Fusarium oxysporum fungus partly blocked the xylem and grew extensively within parenchyma. When the wilt developed fast (type II syndrome), the occlusion of both xylem and phloem was observed; the xylem sap circulation was suppressed and, consequently, tomato plant tissues were dehydrated. The development of type I and type II diseases led to suppression of photosynthetic activity in plants. In the case of slow wilt (type I), both light and dark stages of photosynthesis were damaged. This was evident from the decrease in the effectiveness of light harvesting and charge separations in the reaction centers of photosystem II (PSII), suppression of electron transport at the acceptor side of PSII, and the decrease in activity of Rubisco. In the case of fast wilt (type II), the Rubisco activity did not change, and photochemical activity of chloroplasts was suppressed to a smaller degree than during type I fusarium wilt. The decrease in the rate of linear electron transport in tomato leaves was mostly due to inhibition of electron flow at the acceptor side of PSII. The data obtained suggest that photosynthetic activity in tomato plants is suppressed by different mechanisms depending on the developmental pattern of fusarium wilt.     

Metabolomic and proteomic changes in the xylem sap of maize under drought

Plants produce compounds in roots that are transported to shoots via the xylem sap. Some of these compounds are vital for signalling and adaptation to environmental stress such as drought. In this study, we screened the xylem sap using mass spectrometry to quantify the changes in new and previously identified sap constituents under extended drought. We detected and quantified the changes in the concentration of 31 compounds present in the xylem sap under progressively increasing drought stress. We found changes in the hormones abscisic acid (ABA) and cytokinin, and the presence of high concentrations of the aromatic cytokinin 6-benzylaminopurine (BAP). Several phenylpropanoid compounds (coumaric, caffeic and ferulic acids) were found in xylem sap. The concentrations of some of these phenylpropanoid compounds changed under drought. In parallel, an analysis of the xylem sap proteome was conducted. We found a higher abundance of cationic peroxidases, which with the increase in phenylpropanoids may lead to a reduction in lignin biosynthesis in the xylem vessels and could induce cell wall stiffening. The application of new methodologies provides insights into the range of compounds in sap and how alterations in composition may lead to changes in development and signalling during adaptation to drought.