bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase
Authors: Koiwa Hisashi , D'Urzo Matilde Paino , Assfalg‐Machleidt Irmgard , Zhu‐Salzman Keyan , Shade Richard E. , An Haejung , Murdock Larry L. , Machleidt Werner , Bressan Ray A. , Hasegawa Paul M. 
Published: March 2003

SummaryTwo hairpin‐loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain‐like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin‐loop regions were constructed. Approximately 1010−1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin‐loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild‐type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pm) than parental scN, due to its increased association rate constant (2.6 ± 0.09 × 107 m−1sec−1). These results define residues in the first and second hairpin‐loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.
The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase‐like protein sinapoylglucose:choline sinapoyltransferase
Authors: Shirley Amber M. , McMichael Colleen M. , Chapple Clint 
Published: March 2003

SummarySerine carboxypeptidase‐like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). Cloning of the SNG2 gene by a combination of map‐based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.
Reduced expression of α‐tubulin genes in Arabidopsis thaliana specifically affects root growth and morphology, root hair development and root gravitropism
Authors: Bao Yiqun , Kost Benedikt , Chua Nam‐Hai 
Published: March 2003

SummaryDifferent α‐tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of α‐tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.
Inhibition of systemic onset of post‐transcriptional gene silencing by non‐toxic concentrations of cadmium
Authors: Ueki Shoko , Citovsky Vitaly 
Published: March 2003

SummaryPost‐transcriptional gene silencing (PTGS) is an important mechanism for regulation of plant gene expression and virus–plant interactions. To better understand this process, the heavy metal cadmium was identified as a specific inhibitor in two different PTGS systems, constitutive and inducible. The pattern of cadmium‐induced inhibition of PTGS allowed several insights into PTGS development. First, cadmium treatment prevented only systemic but not local onset of PTGS, uncoupling between these two modes of PTGS. Second, non‐toxic, but not toxic, levels of cadmium inhibited PTGS, suggesting induction of a pathway that interferes with PTGS. Third, cadmium effects on PTGS closely paralleled those on the movement of tobamoviruses, suggesting that both processes may share common steps in their systemic transport pathways. Interestingly, these effects of cadmium do not represent a general property of toxic metal ions because two other such elements, that is zinc and aluminum, did not interfere with PTGS and viral systemic movement.
The role of auxin‐binding protein 1 in the expansion of tobacco leaf cells
Authors: Chen Jin‐Gui , Shimomura Shoji , Sitbon Folke , Sandberg Göran , Jones Alan M 
Published: March 2003

SummaryTobacco leaf was used to investigate the mechanism of action of auxin‐binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin‐inducible growth were each determined in control tobacco leaves and leaves over‐expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1‐induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1‐induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin‐inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY‐2) in culture displayed two dose‐dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]‐thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.
Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato
Authors: Han Yuanhuai , Grierson Don 
Published: March 2003

SummaryWe investigated the occurrence and properties of small sense and antisense RNAs and aberrant transcripts in transgenic tomato plants exhibiting post‐transcriptional silencing of the endogenous polygalacturonase (PG) sense gene and a truncated homologous transgene. Small antisense RNAs, about 23 nucleotides in size, were present in the leaves and immature green fruit where the transgene was transcribed in the absence of transcripts from the fruit ripening‐specific endogenous PG gene. This suggests that the small antisense RNAs were produced from the transgene, which was supported by their failure to hybridise with the 3′ half of the endogenous PG gene, the region that was excluded from the transgene. The features of two aberrant RNAs, corresponding to the 5′ and 3′ halves of the endogenous gene, strongly suggest that they arise by endonucleolytic cleavage of endogenous PG mRNA. The region where cleavage occurs is associated with the preferential production of small RNAs from the 3′ end of the transgene. These results suggest a model where small antisense RNAs generated from the 3′ end of the transgene anneal to the endogenous mRNA, resulting in cleavage at or near the annealing regions.
The Arabidopsis gain‐of‐function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease
Authors: Pilloff Rachel K. , Devadas Sendil K. , Enyedi Alexander , Raina Ramesh 
Published: March 2003

SummaryWe describe the characterization of a novel gain‐of‐function Arabidopsis mutant, dll1 (disease‐like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense‐related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non‐pathogenic to wild‐type Col‐0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance‐ and disease‐associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.
Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis
Authors: Schrick Kathrin , Mayer Ulrike , Martin Gottfried , Bellini Catherine , Kuhnt Christine , Schmidt Jürgen , Jürgens Gerd 
Published: March 2003

SummaryThe sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post‐embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C‐14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk‐like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C‐24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C‐8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15‐azasterol, an inhibitor of sterol C‐14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post‐embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC–MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.
Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double‐strand breaks in vivo
Authors: West Christopher E. , Waterworth Wanda M. , Story Geraint W. , Sunderland Paul A. , Jiang Qing , Bray Clifford M. 
Published: March 2003

SummaryDouble‐strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by γ‐irradiation, and represent one of the most serious threats to genomic integrity. Non‐homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T‐DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high affinity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana, and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is specific for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA‐damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis.
The VERNALIZATION INDEPENDENCE 4 gene encodes a novel regulator of FLOWERING LOCUS C
Authors: Zhang Hua , Van Nocker Steven 
Published: March 2003

SummaryThe late‐flowering, vernalization‐responsive habit of many Arabidopsis ecotypes is mediated predominantly through repression of the floral programme by the FLOWERING LOCUS C (FLC) gene. To better understand this repressive mechanism, we have taken a genetic approach to identify novel genes that positively regulate FLC expression. We identified recessive mutations in a gene designated VERNALIZATION INDEPENDENCE 4 (VIP4), that confer early flowering and loss of FLC expression in the absence of cold. We cloned the VIP4 gene and found that it encodes a highly hydrophilic protein with similarity to proteins from yeasts, Drosophila, and Caenorhabditis elegans. Consistent with a proposed role as a direct activator of FLC, VIP4 is expressed throughout the plant in a pattern similar to that of FLC. However, unlike FLC, VIP4 RNA expression is not down‐regulated in vernalized plants, suggesting that VIP4 is probably not sufficient to activate FLC, and that VIP4 is probably not directly involved in a vernalization mechanism. Epistasis analysis suggests that VIP4 could act in a separate pathway from previously identified FLC regulators, including FRIGIDA and the autonomous flowering promotion pathway gene LUMINIDEPENDENS. Mutants lacking detectable VIP4 expression flower earlier than FLC null mutants, suggesting that VIP4 regulates flowering‐time genes in addition to FLC. Floral morphology is also disrupted in vip4 mutants; thus, VIP4 has multiple roles in development.
TIR‐X and TIR‐NBS proteins: two new families related to disease resistance TIR‐NBS‐LRR proteins encoded in Arabidopsis and other plant genomes
Authors: Meyers Blake C. , Morgante Michele , Michelmore Richard W. 
Published: March 2003

SummaryThe Toll/interleukin‐1 receptor (TIR) domain is found in one of the two large families of homologues of plant disease resistance proteins (R proteins) in Arabidopsis and other dicotyledonous plants. In addition to these TIR‐NBS‐LRR (TNL) R proteins, we identified two families of TIR‐containing proteins encoded in the Arabidopsis Col‐0 genome. The TIR‐X (TX) family of proteins lacks both the nucleotide‐binding site (NBS) and the leucine rich repeats (LRRs) that are characteristic of the R proteins, while the TIR‐NBS (TN) proteins contain much of the NBS, but lack the LRR. In Col‐0, the TX family is encoded by 27 genes and three pseudogenes; the TN family is encoded by 20 genes and one pseudogene. Using massively parallel signature sequencing (MPSS), expression was detected at low levels for approximately 85% of the TN‐encoding genes. Expression was detected for only approximately 40% of the TX‐encoding genes, again at low levels. Physical map data and phylogenetic analysis indicated that multiple genomic duplication events have increased the numbers of TX and TN genes in Arabidopsis. Genes encoding TX, TN and TNL proteins were demonstrated in conifers; TX and TN genes are present in very low numbers in grass genomes. The expression, prevalence, and diversity of TX and TN genes suggests that these genes encode functional proteins rather than resulting from degradation or deletions of TNL genes. These TX and TN proteins could be plant analogues of small TIR‐adapter proteins that function in mammalian innate immune responses such as MyD88 and Mal.
Acyl CoA profiles of transgenic plants that accumulate medium‐chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds
Authors: Larson Tony R. , Edgell Teresa , Byrne James , Dehesh Katayoon , Graham Ian A. 
Published: March 2003

SummarySeveral Brassica napus lines transformed with genes responsible for the synthesis of medium‐ or long‐chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long‐chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium‐chain thioesterase and 3‐ketoacyl‐ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, β‐oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium‐chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.
SAD: a new DOF protein from barley that activates transcription of a cathepsin B‐like thiol protease gene in the aleurone of germinating seeds
Authors: Isabel‐LaMoneda Inés , Diaz Isabel , Martinez Manuel , Mena Montaña , Carbonero Pilar 
Published: March 2003

SummaryFunctional analysis of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, has identified a GA‐responsive complex (GARC) as a tripartite element containing a pyrimidine‐box motif 5′‐CCTTTT‐3′. We describe here the characterization of a new barley gene (Sad gene) encoding a transcription factor (SAD) of the DNA binding with One Finger (DOF) class that binds to the pyrimidine box in vitro and activates transcription of a GA‐induced protease promoter in bombarded aleurone layers. RT‐PCR and in situ hybridization analyses showed that the Sad transcripts accumulated in all tissues analysed, being especially abundant in the scutellum and aleurone cells upon seed germination. The SAD protein, expressed in bacteria, binds in a specific manner to two oligonucleotides containing the sequence 5′‐G/CCTTTT/C‐3′, derived from the promoter region of the Al21 gene encoding a cathepsin B‐like cysteine protease. Although the Sad transcript accumulation did not respond to external GA‐incubation in aleurone cells, in transient expression experiments in co‐bombarded aleurone layers, SAD trans‐activated transcription from the Al21 promoter in a similar manner as did GAMYB, a MYB protein previously shown to respond to GA and to activate several hydrolase gene promoters in barley aleurone cells. In vivo interaction between the GAMYB and SAD proteins was shown in the yeast two‐hybrid system, where GAMYB potentiates the SAD trans‐activation capacity through interaction with its C‐terminal domain.
Functional complementation in yeast reveals a protective role of chloroplast 2‐Cys peroxiredoxin against reactive nitrogen species
Authors: Sakamoto Atsushi , Tsukamoto Shigefumi , Yamamoto Hiroshi , Ueda–Hashimoto Manami , Takahashi Misa , Suzuki Hitomi , Morikawa Hiromichi 
Published: March 2003

SummaryThe importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO‐derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS‐induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite‐scavenging activity in plants of 2‐Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide‐reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast‐localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino‐terminally hexahistidine‐tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite‐induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite‐mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS‐mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.