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Chanyong Jang,
Eun-Young Seo,
Jiryun Nam, Hanhong Bae,
Yeong Guk Gim,
Hong Gi Kim,
In Sook Cho,
Zee-Won Lee,
Gary R Bauchan,
John Hammond,
Hyoun-Sub Lim
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ABSTRACT: Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.
Frontiers in plant science. 01/2013; 4:5.
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ABSTRACT: Endophytic Trichoderma isolates collected in tropical environments were evaluated for biocontrol activity against Phytophthora capsici in hot pepper (Capsicum annuum). Six isolates were tested for parasitic and antimicrobial activity against P. capsici and for endophytic and induced resistance capabilities in pepper. Isolates DIS 70a, DIS 219b, and DIS 376f were P. capsici parasites, while DIS 70a, DIS 259j, DIS 320c, and DIS 376f metabolites inhibited P. capsici. All six isolates colonized roots but were inefficient stem colonizers. DIS 259j, DIS 320c, and DIS 376f induced defense-related expressed sequence tags (EST) in 32-day-old peppers. DIS 70a, DIS 259j, and DIS 376f delayed disease development. Initial colonization of roots by DIS 259j or DIS 376f induced EST with potential to impact Trichoderma endophytic colonization and disease development, including multiple lipid transferase protein (LTP)-like family members. The timing and intensity of induction varied between isolates. Expression of CaLTP-N, encoding a LTP-like protein in pepper, in N. benthamiana leaves reduced disease development in response to P. nicotianae inoculation, suggesting LTP are functional components of resistance induced by Trichoderma species. Trichoderma isolates were endophytic on pepper roots in which, depending on the isolate, they delayed disease development by P. capsici and induced strong and divergent defense reactions.
Molecular Plant-Microbe Interactions 03/2011; 24(3):336-51. · 4.43 Impact Factor
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ABSTRACT: Theobroma cacao (cacao) is a tropical understory tree with sensitivity to drought. Cacao responds to drought by decreases in net photosynthesis,
PS II efficiency, stomatal conductance, water potential and changes in leaf florescence. Drought also alters cacao gene expression
as well as leaf glucose and free amino acid content. In recent years an incredible diversity of fungal endophytes has been
identified in association with cacao. These endophytes are being studied for the benefits they provide to cacao including
tolerance to biotic and abiotic stresses. During establishment of the endophytic association between cacao and fungal endophytes
both plant and fungal gene expression are altered. The endophytic Trichoderma hamatum isolate DIS 219b delays the onset of drought stress in cacao. This delay manifests itself through enhanced root growth, maintenance
of stomatal conductance, water potential, net photosynthesis, and PSII efficiency, changes in free amino acid concentrations,
and a delay in drought-induced changes in leaf gene expression. The cacao plant and DIS 219b adapt to each other and this
adaptation may contribute to the observed plant growth promotion and the delay in onset of drought stress. The increase in
root growth is thought to increase water uptake and availability, delaying the time point where the water supply becomes limiting
and drought stress occurs.
12/2010: pages 157-172;
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ABSTRACT: Transgenic tobacco plants expressing the hyperthermostable beta-glucosidase BglB of Thermotoga maritima were generated with the goal of cost-effective production of the enzyme for the application in bioconversion of lignocellulosic biomass. The enzyme was targeted to the cytosol and chloroplasts, where it accumulated to level of 4.5% and 5.8% of total soluble protein, respectively. The optimal temperature and pH of the plant-expressed BglB was 80 degrees C and 4.5, respectively. BglB activity was preserved in leaves after lyophilization, but decreased by over 70% with drying at room temperature. When BglB was synergistically supplied in a 1% (w/v) rice straw with Cel5A for efficient cellulase conversion, a 37% increase in glucose was observed. This report demonstrates the potential of utilizing transgenic tobacco for mass production of BglB.
Bioresource technology 09/2010; 101(18):7155-61. · 4.25 Impact Factor
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ABSTRACT: Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.
Journal of General Virology 08/2010; 91(Pt 8):2102-15. · 3.36 Impact Factor
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ABSTRACT: Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 degrees C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 degrees C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the 'core' Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P(1110),R(1121),K(1255)]+TGB1(L(88))] always induced systemic necrosis at 15 degrees C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1(P(88)) significantly reducing silencing suppression. The difference in silencing suppression between TGB1(P(88)) and TGB1(L(88)) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1(P(88)) and TGB1(L(88)) resulted in interference in the suppression of silencing by TGB1(L(88)). Yeast two-hybrid analysis confirmed that TGB1(P(88)) and TGB1(L(88)) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.
Journal of General Virology 01/2010; 91(Pt 1):277-87. · 3.36 Impact Factor
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ABSTRACT: Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The impact of the endophytic fungus Trichoderma hamatum isolate DIS 219b on cacao's response to drought was studied. Colonization by DIS 219b delayed drought-induced changes in stomatal conductance, net photosynthesis, and green fluorescence emissions. The altered expression of 19 expressed sequence tags (ESTs) (seven in leaves and 17 in roots with some overlap) by drought was detected using quantitative real-time reverse transcription PCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of seven ESTs being observed after 7 d of withholding water. Changes in gene expression in leaves were not observed until after 10 d of withholding water. DIS 219b colonization delayed the drought-altered expression of all seven ESTs responsive to drought in leaves by > or = 3 d, but had less influence on the expression pattern of the drought-responsive ESTs in roots. DIS 219b colonization had minimal direct influence on the expression of drought-responsive ESTs in 32-d-old seedlings. By contrast, DIS 219b colonization of 9-d-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. Drought induced an increase in the concentration of many amino acids in cacao leaves, while DIS 219b colonization caused a decrease in aspartic acid and glutamic acid concentrations and an increase in alanine and gamma-aminobutyric acid concentrations. With or without exposure to drought conditions, colonization by DIS 219b promoted seedling growth, the most consistent effects being an increase in root fresh weight, root dry weight, and root water content. Colonized seedlings were slower to wilt in response to drought as measured by a decrease in the leaf angle drop. The primary direct effect of DIS 219b colonization was promotion of root growth, regardless of water status, and an increase in water content which it is proposed caused a delay in many aspects of the drought response of cacao.
Journal of Experimental Botany 07/2009; 60(11):3279-95. · 5.36 Impact Factor
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ABSTRACT: Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC), an arginine decarboxylase (TcADC), an S-adenosylmethionine decarboxylase (TcSAMDC), a spermidine synthase (TcSPDS), and a spermine synthase (TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5-3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.
Plant Physiology and Biochemistry 03/2008; 46(2):174-88. · 2.84 Impact Factor
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ABSTRACT: Rice spotted leaf 6 (spl6) mutant produces lesions caused by spontaneous cell death in the absence of pathogenic infection. Expression of this genetic trait was developmentally programmed. After the tillering stage, small red and brown lesions were initiated in groups on the leaf blade. Eventually, the lesions formed parallel lines along the midrib of the leaf. Under light and transmission electron microscopy, we observed that thylakoid membranes of mesophyll chloroplasts were progressively damaged in the nonspotted section of the mutant leaf. However, chloroplasts were absent in the mesophyll cells of the spotted area of the spl6 mutant. These results indicated that lesion formation of the spl6 mutant might be caused by oxidative burst. Proteome analysis revealed that 159 protein spots were up or downregulated in comparison between spotted leaves of the spl6 mutant plants and normal leaves of the wild type. Among them, protein disulfide isomerase (PDI), transketolase, thioredoxin peroxidase (TPX), ATP synthase, RuBisCO large subunit, and RuBisCO activase small subunit were not identified in the spl6 mutant but were abundant in the wild type. Especially, the absence of TPX and PDI might be the cause of the failure to protect cells against oxidative burst resulting in degradation of the thylakoid membranes and leading to programmed cell death and lesion development.
PROTEOMICS 08/2007; 7(14):2447-58. · 4.51 Impact Factor
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ABSTRACT: In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.
Journal of Plant Physiology 07/2007; 164(6):756-63. · 2.79 Impact Factor
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ABSTRACT: Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five
different levels of soybean seed proteins (75, 100, 125, 150, and 200μg) by 2D-PAGE. Following 2D-PAGE and spot excision,
proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase
in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3)
precursors] increased linearly between 75 and 125μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between
75 and 150μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer,
increased linearly with an increase in the amount of protein through 200μg, whereas spot intensity of β-conglycinin β-homotrimer
and the allergen Gly m bd 28K increased linearly until 150μg and did not increase further at 200μg. These results suggest
that 150μg protein was a suitable amount for the separation of abundant proteins, and 200μg protein was suitable for the
separation of less abundant proteins prepared from soybean seeds.
Plant Molecular Biology Reporter 05/2007; 25(1):55-62. · 2.45 Impact Factor
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ABSTRACT: Treatment of Arabidopsis (Arabidopsis thaliana) with a necrosis- and ethylene-inducing peptide (Nep1) from Fusarium oxysporum inhibited both root and cotyledon growth and triggered cell death, thereby generating necrotic spots. Nep1-like proteins are produced by divergent microbes, many of which are plant pathogens. Nep1 in the plant was localized to the cell wall and cytosol based on immunolocalization results. The ratio of chlorophyll a fluorescence (F685 nm/F730 nm) significantly decreased after 75-min treatment with Nep1 in comparison to the control. This suggested that a short-term compensation of photosynthesis occurred in response to localized damage to cells. The concentrations of most water-soluble metabolites analyzed were reduced in Arabidopsis seedlings after 6 h of Nep1 treatment, indicating that the integrity of cellular membranes had failed. Microarray results showed that short-term treatment with Nep1 altered expression of numerous genes encoding proteins putatively localized to organelles, especially the chloroplast and mitochondria. Short-term treatment with Nep1 induced multiple classes of genes involved in reactive oxygen species production, signal transduction, ethylene biosynthesis, membrane modification, apoptosis, and stress. Quantitative PCR was used to confirm the induction of genes localized in the chloroplast, mitochondria, and plasma membrane, and genes responsive to calcium/calmodulin complexes, ethylene, jasmonate, ethylene biosynthesis, WRKY, and cell death. The majority of Nep1-induced genes has been associated with general stress responses but has not been critically linked to resistance to plant disease. These results are consistent with Nep1 facilitating cell death as a component of diseases caused by necrotrophic plant pathogens.
Plant physiology 08/2006; 141(3):1056-67. · 6.53 Impact Factor
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ABSTRACT: A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.
Journal of Agricultural and Food Chemistry 05/2006; 54(8):3114-20. · 2.82 Impact Factor
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ABSTRACT: The effects of CO2 enrichment on the growth and physiology of maize were investigated at the molecular, biochemical, leaf, and canopy levels. Maize plants were grown in sunlit soil–plant–atmosphere research (SPAR) chambers at ambient (370 μmol mol−1) or elevated (750 μmol mol−1) atmospheric carbon dioxide concentration (Ca) under well-watered and fertilized conditions. Canopy gas exchange rates and leaf temperatures were monitored continuously during the growing season. CO2 enrichment did not enhance the growth or canopy photosynthesis of maize plants. However, canopy evapotranspiration rates decreased by 22% and daytime leaf temperatures were increased about 1°C in response to CO2 enrichment. Leaf carboxylation efficiency and leaf nitrogen concentration also decreased at elevated Ca. Transcription profiling using maize cDNA microarrays revealed that approximately 5% of tested genes responded to CO2 enrichment. Of the altered transcripts, several were known to encode proteins involved in stomatal development or photosynthesis. For the majority of the altered transcripts, however, it was difficult to link their functions with specific physiological factors partly because many of these genes encoded unknown proteins. We conclude that maize did not exhibit enhanced growth or photosynthesis in response to CO2 enrichment but a number of molecular and physiological processes including those involved in stomatal relations were affected by growth in elevated Ca.
Global Change Biology 02/2006; 12(3):588 - 600. · 6.86 Impact Factor
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ABSTRACT: Phvytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.
Mycological Research 01/2006; 109(Pt 12):1373-85. · 2.81 Impact Factor
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ABSTRACT: Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.
Plant Physiology and Biochemistry 07/2005; 43(6):611-22. · 2.84 Impact Factor
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ABSTRACT: Yield of Theobroma cacao (cacao), the source of chocolate, is limited by disease and insect pests. Developmental stage influences the resistance of cacao leaves to disease and insect pests. Mechanical wounding, ethylene, and methyl jasmonate induce resistance to pests in many plant species. The effects of mechanical wounding, ethylene, and methyl jasmonate on gene expression was studied in cacao leaves at two developmental stages, young red (YR) and mature green (MG). Differential expression was observed for genes putatively encoding a DNA binding protein (TcWRKY-1), a protein regulating cell division (TcORFX-1), a Type III peroxidase (TcPer-1), an endo-1,4-β-glucanase (TcGlu-1), a class VII chitinase (TcChiB), a caffeine synthase (TcCaf-1), and a light-harvesting complex protein (TcLhca-1). Wounding induced TcWRKY-1 and TcORFX-1 in YR and MG leaves. Elevated TcPer-1 mRNA levels were detected in YR and MG leaves after wounding. Wounding induced TcChiB in YR leaves and repressed TcLhca-1 in MG leaves. Ethylene (12 μL L−1) induced TcPer-1 in YR and MG leaves but induced TcGlu-1 in MG leaves only. Ethylene repressed TcLhca-1 and TcCaf-1 in YR leaves. Ethylene repressed TcLhca-1 and TcChiB in MG leaves. Methyl jasmonate (0.2 mM) induced TcCaf-1 and TcChiB in YR leaves and TcPer-1 and TcChiB in MG leaves. Ethylene/methyl jasmonate combined induced TcChiB in YR leaves and TcGlu-1 in MG leaves. 1-Methylcyclopropene, an inhibitor of ethylene action, blocked ethylene induced responses but did not block responses to wounding or methyl jasmonate. The cacao response to wounding, ethylene, and/or methyl jasmonate was influenced by developmental stage. Cross-talk between ethylene and methyl jasmonate action on cacao gene expression resulted in synergistic and antagonistic responses. It is critical to account for tissue developmental stage when studying the molecular responses of cacao to mechanical wounding, ethylene, and methyl jasmonate. The constituative and inducible defense strategies used by cacao are dependent on the developmental stage of the tissues involved.
Plant Science.
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ABSTRACT: Transgenic tobacco plants expressing the hyperthermostable β-glucosidase BglB of Thermotoga maritima were generated with the goal of cost-effective production of the enzyme for the application in bioconversion of lignocellulosic biomass. The enzyme was targeted to the cytosol and chloroplasts, where it accumulated to level of 4.5% and 5.8% of total soluble protein, respectively. The optimal temperature and pH of the plant-expressed BglB was 80 °C and 4.5, respectively. BglB activity was preserved in leaves after lyophilization, but decreased by over 70% with drying at room temperature. When BglB was synergistically supplied in a 1% (w/v) rice straw with Cel5A for efficient cellulase conversion, a 37% increase in glucose was observed. This report demonstrates the potential of utilizing transgenic tobacco for mass production of BglB.
Bioresource Technology.
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ABSTRACT: We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.
Plant Physiology and Biochemistry 45(6-7):436-44. · 2.84 Impact Factor
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ABSTRACT: Plant responses to elevated atmospheric CO2 vary with species and with environmental conditions. Rates of dry matter formation were initially enhanced in response to CO2 enrichment but these accelerated growth rates typically were not maintained over long periods of time. The objective of this study was to better understand the basis for this acclimation process. Changes of metabolite levels and of total protein expression in response to CO2 enrichment were studied using biochemical assays and two-dimensional gel electrophoresis. Arabidopsis thaliana (L.) Henyh. (Columbia ecotype) plants were grown for 2–6 weeks in controlled environment chambers providing 36 (ambient) or 100 (elevated) Pa CO2. Averaged over all harvest dates above-ground biomass was greater (P < 0.05) in the elevated than in the ambient CO2 treatment but shoot biomass did not differ between treatments on the final harvest. Flowering was delayed by CO2 enrichment. One or more flowers were observed for 52% and 100% of the elevated and ambient CO2 grown plants, respectively, after 4-weeks growth. Starch and sucrose levels were increased 132 and 43%, respectively, in leaves of 6-week-old plants in response to CO2 enrichment. Nitrate varied with plant age, although mean nitrate levels in rosettes were decreased 31% by CO2 enrichment when averaged over all harvest dates. Chlorophyll, the chlorophyll a/b ratio, carotenoids and total soluble protein did not differ between CO2 treatments. Total Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity decreased with plant age and was lower (P < 0.01) in the elevated compared to the ambient CO2 treatment. The above results suggested that acclimation to elevated CO2 occurred in Arabidopsis without developing symptoms of N-deficiency. A total of 400 major proteins were separated and compared by two-dimensional gel electrophoresis. No proteins appeared de novo or disappeared in response to CO2 enrichment, although pixel densities for 13 protein spots differed significantly between CO2 treatments on at least one harvest date. Six of these proteins were identified by mass spectrometry. Three of these identified proteins were involved in plant growth and development or were associated with stress. Two other proteins were encoded by genes with putative functions. Only one protein, the 23 kDa subunit of the oxygen evolving complex (OEC23), was involved in photosynthesis. It was concluded that long-term plant growth in elevated CO2 caused only small changes in the Arabidopsis proteome.
Field Crops Research.
