isabel diaz - Academia.edu (original) (raw)

Papers by isabel diaz

Research paper thumbnail of The GAMYB protein from barley interacts with the DOF transcription factor BPBF and activates endosperm-specific genes during seed development

Plant Journal, 2002

HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in ... more HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in response to gibberellin during germination, also has an important role in gene regulation during endosperm development. The mRNA was detected early (10 DAF) in the seeds where it accumulates, not only in the aleurone layer, starchy endosperm, nucellar projection and vascular tissue, but also in the immature embryo as shown by in situ hybridization analysis. The HvGAMYB protein, expressed in bacteria, binds to oligonucleotides containing the 5¢-TAACAAC-3¢ or 5¢-CAACTAAC-3¢ sequences, derived from the promoter regions of the endosperm-speci®c genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. Binding is prevented when these motifs are mutated to 5¢-TgACAAg-3¢ and 5¢-CgACTgAC-3¢. Transient expression experiments in co-bombarded developing endosperms demonstrate that HvGAMYB trans-activates transcription from native Hor2 and Itr1 promoters through binding to the intact motifs described above. Trans-activation of the Hor2 promoter also requires an intact prolamine box (PB). This suggests that HvGAMYB interacts in developing barley endosperms with the PB-binding factor BPBF, an endosperm-speci®c DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast twohybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.

Research paper thumbnail of The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties

Journal of Experimental Botany, 2005

An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragaria... more An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragaria3ananassa cv. Elsanta has been characterized. The corresponding gene (Cyf1) had three introns interrupting its ORF that codes for a protein (FaCPI-1) of 235 amino acid residues with a putative signal peptide of 29 residues and an estimated molecular mass for the mature protein of 23.1 kDa. This protein contains, besides a C-terminal extension, several motifs conserved in all members of the PhyCys superfamily: (i) a GG and LARFAV-like motifs towards the N-terminal part of the protein; (ii) the reactive site QVVAG, and (iii) a conserved PW, downstream of the reactive site. Northern blot and in situ hybridization analyses indicated that the Cyf1 gene was expressed in fully expanded leaves, in roots and in achenes, but not in the receptacle (pseudocarp) during fruit development. The recombinant FaCPI-1 protein expressed in E. coli efficiently inhibited papain (K i 1.9310 29 M) and less so cathepsin H (K i 4.73 10 27 M) and cathepsin B (K i 3.3310 26 M), and was a good inhibitor of the in vitro growth of phytopathogenic fungi Botrytis cinerea (EC 50 : 1.90 lM) and Fusarium oxysporum (EC 50 : 2.28 lM).

Research paper thumbnail of Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Molecular Breeding, 1999

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous ... more Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.

Research paper thumbnail of Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Transgenic Research, 2000

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under... more Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.

Research paper thumbnail of Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

Molecular and General Genetics, 1996

The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less t... more The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reported Ltp4 cDNA (now Ltp4.1). Southern blot analysis indicated the existence of three or more Ltp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genes HvLtp4.2 and HvLtp4.3 folio wing transformation by particle bombardment, using promoter fusions to the j9-glucuronidase repórter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ssl) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except that Ltp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using the Zíp4-specific probé, indicated that Xanthomonas campestris pv. translucens induced an increase over basal levéis of Ltp4 mRNA, while Pseudomonas syringae pv. japónica caused a decrease. The Ltp4.3-Gus promoter fusión also responded in opposite ways to these two compatible bacterial pathogens, whereas the Ltp4.2-Gus construction did not respond to infection.

Research paper thumbnail of SAD: a new DOF protein from barley that activates transcription of a cathepsin B-like thiol protease gene in the aleurone of germinating seeds

Plant Journal, 2003

Functional analysis of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells fo... more Functional 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.

Research paper thumbnail of Stephen hw

Research paper thumbnail of The GAMYB protein from barley interacts with the DOF transcription factor BPBF and activates endosperm-specific genes during seed development

Plant Journal, 2002

HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in ... more HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in response to gibberellin during germination, also has an important role in gene regulation during endosperm development. The mRNA was detected early (10 DAF) in the seeds where it accumulates, not only in the aleurone layer, starchy endosperm, nucellar projection and vascular tissue, but also in the immature embryo as shown by in situ hybridization analysis. The HvGAMYB protein, expressed in bacteria, binds to oligonucleotides containing the 5¢-TAACAAC-3¢ or 5¢-CAACTAAC-3¢ sequences, derived from the promoter regions of the endosperm-speci®c genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. Binding is prevented when these motifs are mutated to 5¢-TgACAAg-3¢ and 5¢-CgACTgAC-3¢. Transient expression experiments in co-bombarded developing endosperms demonstrate that HvGAMYB trans-activates transcription from native Hor2 and Itr1 promoters through binding to the intact motifs described above. Trans-activation of the Hor2 promoter also requires an intact prolamine box (PB). This suggests that HvGAMYB interacts in developing barley endosperms with the PB-binding factor BPBF, an endosperm-speci®c DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast twohybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.

Research paper thumbnail of The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties

Journal of Experimental Botany, 2005

An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragaria... more An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragaria3ananassa cv. Elsanta has been characterized. The corresponding gene (Cyf1) had three introns interrupting its ORF that codes for a protein (FaCPI-1) of 235 amino acid residues with a putative signal peptide of 29 residues and an estimated molecular mass for the mature protein of 23.1 kDa. This protein contains, besides a C-terminal extension, several motifs conserved in all members of the PhyCys superfamily: (i) a GG and LARFAV-like motifs towards the N-terminal part of the protein; (ii) the reactive site QVVAG, and (iii) a conserved PW, downstream of the reactive site. Northern blot and in situ hybridization analyses indicated that the Cyf1 gene was expressed in fully expanded leaves, in roots and in achenes, but not in the receptacle (pseudocarp) during fruit development. The recombinant FaCPI-1 protein expressed in E. coli efficiently inhibited papain (K i 1.9310 29 M) and less so cathepsin H (K i 4.73 10 27 M) and cathepsin B (K i 3.3310 26 M), and was a good inhibitor of the in vitro growth of phytopathogenic fungi Botrytis cinerea (EC 50 : 1.90 lM) and Fusarium oxysporum (EC 50 : 2.28 lM).

Research paper thumbnail of Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Molecular Breeding, 1999

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous ... more Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.

Research paper thumbnail of Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Transgenic Research, 2000

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under... more Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.

Research paper thumbnail of Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

Molecular and General Genetics, 1996

The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less t... more The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reported Ltp4 cDNA (now Ltp4.1). Southern blot analysis indicated the existence of three or more Ltp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genes HvLtp4.2 and HvLtp4.3 folio wing transformation by particle bombardment, using promoter fusions to the j9-glucuronidase repórter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ssl) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except that Ltp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using the Zíp4-specific probé, indicated that Xanthomonas campestris pv. translucens induced an increase over basal levéis of Ltp4 mRNA, while Pseudomonas syringae pv. japónica caused a decrease. The Ltp4.3-Gus promoter fusión also responded in opposite ways to these two compatible bacterial pathogens, whereas the Ltp4.2-Gus construction did not respond to infection.

Research paper thumbnail of SAD: a new DOF protein from barley that activates transcription of a cathepsin B-like thiol protease gene in the aleurone of germinating seeds

Plant Journal, 2003

Functional analysis of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells fo... more Functional 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.

Research paper thumbnail of Stephen hw