In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA). We reported previously that MYC and MYB recognition sites in the rd22 promoter region function as cis-acting elements in the drought- and ABA-induced gene expression of rd22. bHLH- and MYB-related transcription factors, rd22BP1 (renamed AtMYC2) and AtMYB2, interact specifically with the MYC and MYB recognition sites, respectively, in vitro and activate the transcription of the β-glucuronidase reporter gene driven by the MYC and MYB recognition sites in Arabidopsis leaf protoplasts. Here, we show that transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA. The ABA-induced gene expression of rd22 and AtADH1 was enhanced in these transgenic plants. Microarray analysis of the transgenic plants overexpressing both AtMYC2 and AtMYB2 cDNAs revealed that several ABA-inducible genes also are upregulated in the transgenic plants. By contrast, a Ds insertion mutant of the AtMYC2 gene was less sensitive to ABA and showed significantly decreased ABA-induced gene expression of rd22 and AtADH1. These results indicate that both AtMYC2 and AtMYB2 proteins function as transcriptional activators in ABA-inducible gene expression under drought stress in plants.

Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

/pdf/virus-resistance-and-gene-silencing-in-plants-can-be-induced-dwautat2u4.pdf

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Cytokinins (CKs) play a crucial role in various phases of plant growth and development, but the basic molecular mechanisms of their biosynthesis and signal transduction only recently became clear. The progress was achieved by identifying a series of key genes encoding enzymes and proteins controlling critical steps in biosynthesis, translocation, and signaling. Basic schemes for CK homeostasis and root/shoot communication at the whole-plant level can now be devised. This review summarizes recent findings on the relationship between CK structural variation and activity, distinct features in CK biosynthesis between higher plants and Agrobacterium infected plants, CK translocation at whole-plant and cellular levels, and CKs as signaling molecules for nutrient status via root-shoot communication.

CYTOKININS: Activity, Biosynthesis, and Translocation

In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene.

Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

Basic helix-loop-helix (bHLH) transcription factors (TFs) belong to a family of transcriptional regulators present in three eukaryotic kingdoms. Many different functions have been identified for these proteins in animals, including the control of cell proliferation and development of specific cell lineages. Their mechanism for controlling gene transcription often involves homodimerization or heterodimerization. In plants, little is known about the bHLH family, but we have determined that there are 133 bHLH genes in Arabidopsis thaliana and have confirmed that at least 113 of them are expressed. The AtbHLH genes constitute one of the largest families of transcription factors in A. thaliana with significantly more members than are found in most animal species and about an equivalent number to those in vertebrates. Comparisons with animal sequences suggest that the majority of plant bHLH genes have evolved from the ancestral group B class of bHLH genes. By studying the AtbHLH genes collectively, twelve subfamilies have been identified. Within each of these main groups, there are conserved amino acid sequence motifs outside the DNA binding domain. Potential gene redundancy among members of smaller subgroups has been analyzed, and the resulting information is presented to provide a simplified visual interpretation of the gene family, identifying related genes that are likely to share similar functions. Based on the current characterization of a limited number of plant bHLH proteins, we predict that this family of TFs has a range of different roles in plant cell and tissue development as well as plant metabolism.

/pdf/the-basic-helix-loop-helix-transcription-factor-family-in-3svujnnhic.pdf

The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L) cultivars Nipponbare and Taipei 309 Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse Two Taipei 309 and 35 Nipponbare plants set viable seeds Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed

A Polyethylene Glycol-Mediated Protoplast Transformation System for Production of Fertile Transgenic Rice Plants'

Nucleotide sequence of a mutant acetolactate synthase gene from an imidazolinone-resistant Arabidopsis thaliana var. Columbia

Acetolactate synthase (ALS), a key enzyme in the biosynthesis of valine, leucine and isoleucine in plants is inhibited by herbicides comprising imidazolinones. A mutant gene encoding imidazolinone-resistant ALS has been isolated from an imidazolinone-resistant Arabidopsis thaliana (GH-90) and stably maintained in a plasmid pKS1. When compared with the gene coding for imidazolinone-sensitive ALS, the imidazolinone-resistant ALS gene contains a point mutation, wherein adenosine is substituted for guanosine at nucleotide 1958, resulting in the substitution of asparagine for serine at amino acid residue 653. The mutant ALS gene can be used to impart imidazolinone resistance to a crop plant; thereby permitting the utilization of the imidazolinone or analogous herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

Mutant acetolactate synthase gene from Ararbidopsis thaliana for conferring imidazolinone resistance to crop plants

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.

/pdf/molecular-basis-of-imidazolinone-herbicide-resistance-in-2iuih1ljzy.pdf

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia.

Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice seed storage protein glutelin gene Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -51 to -18 kb region The functional importance of proximal elements in the context of 18 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217) A simultaneous mutation of five protein-binding sites (-410/-86) essentially eliminated the activity of the 18 kb promoter Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -51 kb to -155 bp, spatial control of the glutelin gene was altered when the 51 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants

Norimoto Murai

Papers

A Polyethylene Glycol-Mediated Protoplast Transformation System for Production of Fertile Transgenic Rice Plants'

Nucleotide sequence of a mutant acetolactate synthase gene from an imidazolinone-resistant Arabidopsis thaliana var. Columbia

Mutant acetolactate synthase gene from Ararbidopsis thaliana for conferring imidazolinone resistance to crop plants

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia.

5′ distal and proximal cis‐acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene