Search

The epidermis of shoot organs in plants develops from the outermost layer (L1) of the shoot apical meristem. In Arabidopsis, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1-specific genes. atml1-1 pdf2-1 double mutants show striking defects in the differentiation of shoot epidermal cells. However, because atml1-1 and pdf2-1 have a T-DNA inserted downstream of the respective homeobox sequences, these alleles may not represent null mutations. Here we characterized additional mutant alleles that have a T-DNA insertion at different positions of each gene. Double mutants of a strong atml1-3 allele with each pdf2 allele were found to cause embryonic arrest at the globular stage. Although with low frequency, all double mutant combinations of a weak atml1-1 allele with each pdf2 allele germinated and showed phenotypes defective in shoot epidermal cell differentiation. We further confirmed that transgenic induction of PDF2 fused to the Drosophila Engrailed repressor domain temporarily interferes with epidermal cell differentiation in the wild-type background. These results indicate that ATML1 and PDF2 act redundantly as a positive regulator of shoot epidermal cell differentiation and at least one copy of these genes is essential for embryo development.

The acaulis2 (acl2) mutant of Arabidopsis thaliana shows a defect in flower stalk elongation. We identified the mutation point of acl2 by map-based cloning. The ACL2 locus is located within an approximately 320-kb region at around 100 map units on chromosome 1. One nucleotide substitution was detected in this region in the acl2 mutant, but no significant open reading frames were found around this mutation point. When wild-type DNA fragments containing the mutation point were introduced into acl2 mutant plants, some transgenic plants partially or almost completely recovered from the defect in flower stalk elongation. 3'-RACE experiments showed that bidirectional transcripts containing the acl2 mutation point were expressed, and the Plant MPSS database revealed that several small RNAs were produced from this region. Microarray analysis showed that transcription of many genes is activated in flower stalks of acl2 mutant plants. Overexpression of some of these genes caused a dwarf phenotype in wild-type plants. These results suggest the following novel mechanism for control of the elongation of flower stalks. Bidirectional non-coding RNAs are transcribed from the ACL2 locus, and small RNAs are generated from them in flower stalks. These small RNAs repress the transcription of a set of genes whose expression represses flower stalk elongation, and flower stalks are therefore fully elongated.

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28°C than at 22°C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.

Summary Development of the epidermis involves members of the class–IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

Pedicel length and orientation (angle) contribute to the diversity of inflorescence architecture, and are important for optimal positioning of the flowers. However, relatively little is known about pedicel development. We previously described the Arabidopsis CORYMBOSA1 (CRM1)/BIG gene, which affects inflorescence architecture by controlling pedicel elongation and orientation. Here, we performed a suppressor screen using the partial loss-of-function allele crm1-13 to identify genes and pathways that affect pedicel development. We identified a hypomorph allele of the meristem identity regulator LEAFY (LFY) as the suppressor. Consistent with this, crm1 pedicels had elevated LFY levels and conditional gain of LFY function produced downward-bending pedicels. Steroid activation of 35S:LFY-GR plants caused a reduction in the cortical cell length in the abaxial domain and additional defects associated with adaxialization. Further analyses of loss of LFY function revealed that LFY is required for reduced cortical cell elongation at the adaxial side of the pedicel base. Defects in conditional LFY gain-of-function pedicels were correlated with decreased BREVIPEDICELLUS (BP) expression, while ASYMMETRIC LEAVES2 (AS2), a transcriptional repressor of BP, and REVOLUTA, a promoter of adaxial cell fate, were highly and ectopically expressed in LFY gain-of-function pedicels. LFY bound to cis-regulatory regions upstream of AS2, and as2 mutations partially suppressed the pedicel length and orientation defects caused by increased LFY activity. These data suggest that LFY activity promotes adaxial cell fate and hence the proper orientation and length of the pedicel partly by directly activating AS2 expression, which suppresses BP expression.

The protein encoded by the activated disease resistance 1-like1 (ADR1-L1) gene (locus name, At4g33300) belongs to the activated disease resistance 1 (ADR1) family of coiled-coil nucleotide-binding site leucine-rich repeat-type disease resistance proteins. This family contains four proteins and they have specific features in their amino acid sequences. It has been reported that ADR1 protein belongs to the ADR1 family, which is related to not only defense response but also drought tolerance. We found that transgenic plants overexpressing the ADR1-L1 gene showed a dwarf phenotype and morphological change in leaves. The expression levels of defense-related genes and the resistance to Pseudomonas syringae pv. tomato DC3000 were increased in transgenic plants. However, enhancement of drought tolerance and activation of abiotic response genes were not observed. When the growth temperature was changed from 22°C to 28°C, the expression of defense-related genes and the enhancement of resistance to a bacterial pathogen were suppressed and the dwarf phenotype and morphological change of leaves recovered.

NAC proteins comprise one of the largest families of transcription factors in the plant genome. They are known to be involved in various aspects of plant development, but the functions of most of them have not yet been determined. ANAC036, a member of the Arabidopsis NAC transcription factor family, contains unique sequences that are conserved among various NAC proteins found in other plant species. Expression analysis of the ANAC036 gene indicated that this gene was strongly expressed in leaves. Transgenic plants overexpressing the ANAC036 gene showed a semidwarf phenotype. The lengths of leaf blades, petioles and stems of these plants were smaller than those in wild-type plants. Microscopy revealed that cell sizes in leaves and stems of these plants were smaller than those in wild-type plants. These findings suggested that ANAC036 and its orthologues are involved in the growth of leaf cells.

Comparative analysis of nucleotide sequences of the genomic region located around 100 map unit of chromosome 1 using two accessions, Columbia (Col) and Landsberg erecta (Ler), of Arabidopsis thaliana was performed. High divergence was detected between them, and the length of the Ler sequence was half of corresponding sequence of Col. This divergence occurred by tandem duplication, deletion of large regions, and insertion of unrelated sequences. These events led to the high polymorphism of plant disease resistant genes, which are located in the analyzed region. It is highly probable that two-round duplication occurred, and the insertion sequences are transposable elements. The data suggest that the analyzed region had been evolving until quite recently.

N-glycosylation is a common protein modification. Joining of polypeptide and carbohydrate elements into hybrid molecules provides an opportunity to fine-tune protein properties. However, the role of N-glycosylation on the development of multicellular organisms remains elusive. Here we report a hypomorphic allele of KNOPF/GLUCOSIDASE 1, which allows us to describe the effects of impaired α-glucosidase I on post-embryonic development of plants for the first time. This knf-101 mutation alters cell shape but does not affect cell arrangements, except for the patterning of specialized epidermal cells, delineating the significance of N-glycan processing during epidermal development in Arabidopsis.

The acaulis2 (acl2) mutant of Arabidopsis thaliana shows a defect in flower stalk elongation. We identified the mutation point of acl2 by map-based cloning. The ACL2 locus is located within an approximately 320-kb region at around 100 map units on chromosome 1. One nucleotide substitution was detected in this region in the acl2 mutant, but no significant open reading frames were found around this mutation point. When wild-type DNA fragments containing the mutation point were introduced into acl2 mutant plants, some transgenic plants partially or almost completely recovered from the defect in flower stalk elongation. 3'-RACE experiments showed that bidirectional transcripts containing the acl2 mutation point were expressed, and the Plant MPSS database revealed that several small RNAs were produced from this region. Microarray analysis showed that transcription of many genes is activated in flower stalks of acl2 mutant plants. Overexpression of some of these genes caused a dwarf phenotype in wild-type plants. These results suggest the following novel mechanism for control of the elongation of flower stalks. Bidirectional non-coding RNAs are transcribed from the ACL2 locus, and small RNAs are generated from them in flower stalks. These small RNAs repress the transcription of a set of genes whose expression represses flower stalk elongation, and flower stalks are therefore fully elongated.

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase α subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 μM l -Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5(ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis(sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5phenotype. sac51-d disrupts a short upstream open reading frame(uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-dresults in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51,which may lead to the expression of a subset of genes required for stem elongation.

Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.

We have previously developed an antibiotic-free system for the selection of plant transformants that is based on a gene (OASA1D) for a mutant α subunit of rice anthranilate synthase. The product of this gene shows a reduced sensitivity to negative feedback regulation by tryptophan. Whereas 5-methyltryptophan (5MT) is lethal for normal plant cells because it causes tryptophan deficiency, expression of OASA1D confers resistance to this tryptophan analog. We used this OASA1D-5MT system for the transformation of Arabidopsis thaliana. An expression vector containing OASA1D under the control of the 35S promoter of cauliflower mosaic virus was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants that harbored OASA1D exhibited resistance to 5MT but did not manifest any other differences in growth, morphology, or fertility. The OASA1D-5MT selection system performed as well as the HPT-hygromycin system for the transformation of Arabidopsis. Given the limited number of conventional marker genes currently available, OASA1D should prove to be a useful tool in Arabidopsis transformation, especially for the generation of plants carrying multiple transgenes.

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.

▪ Abstract In Arabidopsis thaliana, the initiation of flowering is carried out by four genetic pathways: gibberellin, autonomous, vernalization, and light-dependent pathways. These processes are integrated by the function of the genes FD, FE, FWA, PDF2, SOC1, and FT at the integration pathway. The integrated signal of the floral induction is transmitted to the floral meristem identity genes LFY and AP1, and floral morphogenesis is performed.

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of Arabidopsis . We show here that PROTODERMAL FACTOR2 ( PDF2 ), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1 , another L1-specific HD-GL2 class gene sharing the highest homology with PDF2 , display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.

The cDNA clone RXF12, which encodes a xylanase (EC 3.2.1.8), was isolated from Arabidopsis thaliana. The C-terminal half of the amino acid sequence of the deduced protein, named AtXyn1, showed similarity with the catalytic domain of barley xylanase X-1. The N-terminal half of AtXyn1 also contained three regions with sequences similar to cellulose-binding domains (CBDs). A xylanase assay revealed that transgenic A. thaliana plants expressing exogenous AtXyn1 fused with enhanced green fluorescent protein (EGFP) possessed approximately twice as much xylanase activity as wild-type plants. Observation by fluorescence microscopy of transgenic A. thaliana plants expressing a fusion protein of AtXyn1 and EGFP suggested that AtXyn1 is a cell wall protein. Analysis of the localization of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants containing a chimeric gene with the upstream sequence of the AtXyn1 gene and the GUS gene demonstrated that the AtXyn1 gene is predominantly expressed in vascular bundles, but not in vessel cells. These data suggest that AtXyn1 is involved in the secondary cell wall metabolism of vascular bundle cells. A database search revealed that four putative xylanase genes exist in the A. thaliana genome, besides the AtXyn1 gene. Of these, two also contain several regions with sequences similar to CBDs in their N-terminal regions. Comparison of the amino acid sequences of the five xylanases suggests a possible process for their molecular evolution.

Summary The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline-rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis-regulatory sequences required for the L1 layer-specific expression, a series of 5′ deletions of the PDF1 promoter were fused to the β-glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1-specific expression of PDF1 is confined within a 260-bp fragment upstream of the transcription start site. We identified an 8-bp motif in this region that is conserved between promoter regions of all the L1-specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1-specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 l1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1-specific expression.

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.

A cDNA encoding eukaryotic translation initiation factor 4A (eIF4A) was isolated from a cDNA library of rice (Oryza sativa L.). Based on this DNA sequence, a 414-amino acid protein exhibiting 67, 64 and 59% homology to the mouse, Schizosaccharomyces pombe and Saccharomyces cerevisiae eIF4A, respectively, was predicted. The deduced amino acid sequence contains the characteristic motifs shared by the DEAD box supergene family. Another cDNA of rice eIF4A was reported previously. Comparison of the coding sequences of the two rice eIF4As showed 85% homology in the nucleotide sequence and 90% homology in the amino acid sequence. The genomic clones corresponding to the two rice eIF4A cDNAs were also isolated from a genomic library of rice (Oryza sativa L.). It was found that the two genes have common patterns of exon-intron boundaries. Their coding regions are split into four exons, and there is an additional exon in the 5'-non coding region.

Receptor-like protein kinases (RLKs) are encoded by a divergent multigene family and their functions have been implicated in a wide range of signal transduction pathways. In this study, we examined the effect of salicylic acid (SA) on the expression of RLK genes in Arabidopsis thaliana. RNA gel blot analysis revealed that transcripts of RKC1 and a number of its homologs, whose translation products contain C-X8-C-X2-C motifs in the putative extracellular domain, accumulated to a higher level in response to SA treatment of plants. The chimeric fusion between the RKC1 5'-upstream region and the beta-glucuronidase (GUS) reporter gene reproduced the SA responsiveness in transgenic plants. In addition, some of RLK genes of the leucine-rich repeat (LRR) class and those of the S-domain class were also induced by SA. We found that the upstream regions of these SA-responsive RLK genes contain the TTGAC sequence, which has been suggested to be important for induced expression of many plant defense genes. These results suggest the involvement of a number of RLKs in SA-mediated defense responses.

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis ACAULIS5 ( ACL5 ) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon‐induced allele suggests a non‐cell‐autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock‐dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis . We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.

This paper describes expression profiles of the Arabidopsis HSP18.2 heat-shock gene promoter by using three different reporter genes, and the application of this promoter to a method we have developed to drive heat-shock-dependent transcription of plant genome DNA flanked by T-DNA insertions. We show that, irrespective of the location of the T-DNA insertion, an HSP18.2 promoter towards the left border of the T-DNA effectively induces transcription of flanking genome sequences in response to heat shock. If polyadenylated, tagged transcripts can be easily identified by RT-PCR.

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.

We isolated two copia -type retrotransposons from Arabidopsis thaliana . We named these elements AtRE1 ( Arabidopsis thaliana Retro Element 1) and AtRE2. Nucleotide sequence analysis revealed that both elements have long terminal repeats (LTRs), and that their internal sequences include one large open reading frame that could encode Gag protein, protease, integrase, reverse transcriptase, and RNaseH. The deduced amino acids sequences contain several domains that are conserved among a large family of retrotransposons. The primer binding site for first-strand DNA synthesis and the polypurine tract for second-strand DNA synthesis existed at corresponding positions. A 5 bp target site duplication (TSD) sequence was also found in the flanking region of LTRs. Southern hybridization and sequence determination of the flanking region demonstrated that AtRE s exist at different loci in the two A. thaliana ecotypes Columbia and Landsberg erecta . Moreover, AtRE2 exists at two loci in Landsberg erecta , in contrast to the existence of only one copy in Columbia. These findings suggest that AtREs were recently transfected via some mediators or that AtREs were transposed after differentiation of the two ecotypes. One cDNA clone derived from the transcripts of AtRE1 was isolated, and the nucleotide sequence showed that this RNA was transcribed in the antisense direction. RT-PCR analysis revealed that AtRE1 was transcribed in both directions. This result suggests that the antisense RNA controls the expression of AtRE1 at the post-transcriptional level.

A plant nuclear gene coding for a mitochondrial elongation factor G (mEF-G) was cloned from a cDNA library and genomic library of rice (Oryza sativa L.). This DNA sequence predicts a 757-amino-acid protein exhibiting 79%, 55% and 49% homology to Arabidopsis thaliana, Saccharomyces cerevisiae and rat mEF-G respectively, 53% homology to the elongation factor G in Escherichia coli and 43% homology to soybean chloroplast elongation factor G. The deduced amino acid sequence contains the characteristic motifs shared by all GTP binding proteins. Comparison of the sequence of the genomic clone to that of the cDNA clone revealed that this gene is split nineteen times by introns, although the gene of Arabidopsis is split seventeen times by introns. Some of the introns found in the rice genome are relatively long and they result in a long gene with a size of approximately 15 kb.

A mutant of Arabidopsis with reduced internodal cell length, acaulis5 (acl5), has recently been shown to have reduced transcript levels of a gene for endoxyloglucan transferase, EXGT-A1 (Y. Hanzawa, T. Takahashi, Y. Komeda [1997] Plant J 12: 863–874). In the present study, we cloned genomic fragments of five members of theEXGT gene family, EXGT-A1,EXGT-A3, EXGT-A4, XTR2, and XTR3, and examined their expression in the wild type and in a series of acl mutants. In wild-type plants, theEXGT-A3 gene showed higher expression in lower internodes (internodes between nodes bearing axillary shoots) than in upper and young internodes, in which EXGT-A1 was highly expressed. EXGT-A4 was preferentially expressed in roots and XTR3 in siliques. The XTR2 gene was constitutively expressed. In acl1, acl3, and acl4 mutants, which have a severe defect in leaf expansion as well as in internode elongation, theEXGT-A1 gene showed reduced levels of expression before bolting of plants. In contrast, XTR3 was increased in these mutant seedlings. Reduction of EXGT-A1 expression was also detected after bolting of all acl mutants except acl2, whose growth defect is restricted to lower internodes. These results suggest the involvement of each EXGT in different aspects of organ development.

In order to analyze the organization of genes located at the 100 map unit of chromosome 1, we screened cDNAs hybridized with approximately 300 kb contiguous DNA using four P1 clones and one YAC clone. A total of 40 kinds of cDNA were isolated, and their entire sequences were determined. A comparison with the GenBank/EMBL database indicated that three of the cDNAs have been found in Arabidopsis , and that similar sequences to 18 of the cDNAs had been detected in Arabidopsis or other organisms. cDNAs were aligned on a physical map of the contiguous DNA, and the transcriptional direction of each cDNA was determined. This contiguous DNA contains a large direct repeat, which contains five genes. In addition, identical or very similar sequences to two cDNAs are located in a narrow region. Thus, a total of 50 genes were identified, and the gene density was revealed to be approximately one gene every 6 kb. In addition, cDNA sequencing revealed the existence of unusual transcripts. A sequence of seven cDNAs seemed to have no significant open reading frames. Furthermore, the existence of antisense RNA and the possibility of alternative splicing were also revealed.

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed.

Those mutants were studied whose defects resulted in the morphological changes of inflorescences inArabidopsis thaliana. We characterized newly isolatedcorymbosa mutants andacaulis5 mutants. Thecorymbosa1-1 mutation was caused by the defects in the elongation of pedicels and the previously identifiederecta mutation belonged to this class. Thecorymbosa2-1 mutation was caused mainly by the increase of the number of the floral buds in the inflorescence. The expression of theERECTA gene whose defect resulted to the corymbose inflorescence was analyzed. TheERECTA gene was expressed in subsets of cells in both the peripheral zone and central zone and was thought to have important role for the development of inflorescences. The phenotypes of theacaulis5 mutation was pronounced just after the transition from the vegetative to reproductive growth phase. We found that the expressions of the genes for EXGT-A1 and γ-TIP were drastically reduced in theacaulis5 mutants.

In rosette plants, the formation of initial flowers is closely linked to the lengthening of internodes (bolting). In order to clarify the molecular basis of bolting, mutants with reduced lengths of internodes were screened. This paper presents the identification and characterization of recessive mutations in ACAULIS5 (ACL5), a gene required for internodal growth in Arabidopsis thaliana. Unlike previously described mutants with reduced size of organs, the acl5 mutant has a severe defect that is restricted to the process of cell elongation after transition to the reproductive stage and shows no phenotype before floral induction. The results of RNA blot hybridizations showed that the acl5 mutation causes a striking reduction in the transcript levels of genes encoding the tonoplast intrinsic protein (gamma-TIP) and the endoxyloglucan transferase (EXGT-A1), both of which have recently been suggested to be important for cell elongation. Furthermore, our morphological study indicates that the mutation also causes proliferative arrest of the apical inflorescence meristem. These results strongly suggest that, during the reproductive phase, the wild-type ACL5 gene product has a critical function not only in the control of elongation growth of organs but also in the continued maintenance of the proliferative activity of flower-producing meristems.

DCECP63 is a carrot embryogenic cell protein. To perform its genetic analysis, we isolated an Arabidopsis thaliana (At) ECP63 cDNA. The cDNA encoded a polypeptide of 449 amino acids (aa). Its deduced aa sequence showed extensive similarity with DCECP63, DCDC8 and BPBP8 which are embryonic proteins isolated from carrot and birch, respectively. The aa sequence contained eight well-conserved tyrosine phosphorylation sites and 16 repeats of 11 aa. Southern analysis showed that the AtECP63 gene might belong to a small multigene family. The AtECP63 transcripts accumulated specifically in mature seeds, and exogenous abscisic acid (ABA) induced its expression in immature siliques, but not in vegetative tissues. These results suggested that the AtECP63 gene encoding a putative phosphotyrosine protein belonging to late embryogenesis abundant (LEA) protein in group 3 might be involved in maturation and desiccation tolerance of seeds. Restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) mapping showed that AtECP63 gene was present in the South part of chromosome 4.

The developmental regulation of pedicel and internode ensures that plants of the same species have similar inflorescence architecture. In Arabidopsis, the elongation of pedicels and internodes must be temporally and spatially regulated. We previously isolated the corymbosa1 (crm1) mutant, which has a corymb-like inflorescence because of shortened pedicels and internodes. CRM1/BIG encodes a membrane-associated protein and is required for auxin transport. Although CRM1/BIG and auxin have important roles in the development of pedicels and internodes, the developmental changes in cell growth in pedicels and internodes have not been examined. Here, we investigated the role of auxin in the cell growth of pedicels and internodes. Wild-type plants had rapid pedicel and internodal elongation that resulted from the temporal control of cell division and elongation. The crm1 mutants had defects in cell division and elongation. Auxin signaling and cell cycle gene expression in crm1 inflorescences were lower than those in the wild type. Moreover, wild-type plants treated with an auxin transport inhibitor and mutants defective in auxin signaling had shorter pedicels and internodes, with fewer and smaller cells. Our results suggest that auxin transport and signaling have important roles in controlling the proliferation and elongation of pedicel and internodal cells.

Using carrot ECP31 cDNA as a probe, we isolated AtECP31 cDNA from a seed library of Arabidopsis thaliana. The AtECP31 cDNA contained an open reading frame of 256 amino acids. Its deduced amino acid sequence showed more than 60% identity with those of DCECP31, ZMRAB28 and GHLEA D-34 which are late embryogenesis abundant (LEA) proteins of carrot, maize and cotton, respectively. Northern blot analysis showed that the transcripts of AtECP31 accumulated specifically in mature seeds. The results suggest that AtECP31 cDNA, which encodes a LEA protein in group 5, might be involved in maturation and desiccation tolerance of seeds. Restriction fragment length polymorphism (RFLP) mapping showed that AtECP31 gene was present on chromosome 3. Cleaved amplified polymorphic sequence (CAPS) mapping allowed us to map more accurately the AtECP31 gene in the region of 28.3-29.6 cM of chromosome 3.

The intergenic spacer (IGS) region of rDNA in Daucus carota contains at least eight kinds of repeated sequence. One sequence may act as a genuine initiation site for a stable transcript and another may act as a spacer promoter that may be an entry site for proteins required for transcription.

A system for the controlled expression of a foreign gene in cultured tobacco cells (Nicotiana tabacum, BY2) by temperature shift was constructed. A 925-base-pair (bp) DNA fragment containing the 5'-flanking region of a low-molecular-mass heat-shock protein gene (HSP18.2) of Arabidopsis thaliana was inserted upstream of the beta-glucuronidase reporter gene (GUS). The resulting HSP18.2-GUS construct was introduced into BY2 cells by electroporation or Agrobacterium-mediated transformation. Transient expression of the HSP18.2 promoter in protoplasts was very low regardless of the heat shock. Although expression of the HSP18.2-GUS chimeric gene in the stable transformants of BY2 was hardly detected in culture at 25 degrees C, the expression increased rapidly on the transcriptional level when the incubation temperature was shifted to 35-37 degrees C. The optimal temperature for heat-shock induction was 37 degrees C. After a 2-h incubation at 37 degrees C, GUS activity was about 1000-fold greater than that before heat shock. The amount of GUS mRNA was maximum 2 h after heat shock, and then decreased gradually.

We previously described the preparation of a protein fraction that bound to the promoter region of the rRNA gene of Vicia faba. We now describe two additional protein fractions that interact with this DNA element. The three protein fractions have similar binding properties with the exception of their binding stabilities. DNase I footprinting experiments revealed that these protein fractions bind to a region that is located about 60 bp upstream from the site of initiation of transcription. From methylation interference experiments and gel-retardation assays with mutated oligonucleotides, it was evident that the protein fractions required the same residues for the formation of stable DNA-protein(s) complexes. However, the three protein fractions were chromatographically distinguishable and the stability of their binding to DNA was different. Although the functions of these fractions are still unknown, the fact that several protein fractions can bind to a narrow region near the site of initiation of transcription is of considerable interest in terms of the mechanism of transcription of the rRNA gene.

Summary β -Conglycinin, the 7S seed storage protein of soybean ( Glycine max [L.] Merr.), is comprised mainly of three subunits, designated α ' , ex and β . Among these subunits, expression of the β subunit is unique because its accumulation is enhanced under sulfate deficiency in vivo and repressed by exogenously applied L-methionine in cotyledon cultures of soybean. Transgenic Arabidopsis thaliana (L.) Heynh. strains carrying either intact gene encoding the β subunit, or β -glucuronidase fusion were constructed and the effects of sulfur nutritional conditions were analyzed. Expression of the transgene was enhanced under sulfate deficiency and repressed when the siliques of transgenic A. thaliana plants were cultured in the presence of L-methionine. The results indicate that response to sulfur nutritional conditions are conserved in transgenic A. thaliana . By using an A. thaliana mutant, designated mto1-1 , that overaccumulates free methionine, activity of the β subunit gene promoter was shown to be repressed in the seeds. These transgenic and mutant A. thaliana plants will be useful in analyzing the plants' response to nutritional conditions.

Plant growth is directed by the activity of stem cells within meristems. The first meristems are established during early embryogenesis, and this process involves the specification of both stem cells and their organizer cells. One of the earliest events in root meristem initiation is marked by re-specification of the uppermost suspensor cell as hypophysis, the precursor of the organizer. The transcription factor MONOPTEROS (MP) is a key regulator of hypophysis specification, and does so in part by promoting the transport of the plant hormone auxin and by activating the expression of TARGET OF MP (TMO) transcription factors, both of which are required for hypophysis specification. The mechanisms leading to the activation of these genes by MP in a chromatin context are not understood. Here, we show that the PHD-finger proteins OBERON (OBE) and TITANIA (TTA) are essential for MP-dependent embryonic root meristem initiation. TTA1 and TTA2 are functionally redundant and function in the same pathway as OBE1 and OBE2. These PHD-finger proteins interact with each other, and genetic analysis shows that OBE-TTA heterotypic protein complexes promote embryonic root meristem initiation. Furthermore, while MP expression is unaffected by mutations in OBE/TTA genes, expression of MP targets TMO5 and TMO7 is locally lost in obe1 obe2 embryos. PHD-finger proteins have been shown to act in initiation of transcription by interacting with nucleosomes. Indeed, we found that OBE1 binds to chromatin at the TMO7 locus, suggesting a role in its MP-dependent activation. Our data indicate that PHD-finger protein complexes are crucial for the activation of MP-dependent gene expression during embryonic root meristem initiation, and provide a starting point for studying the mechanisms of developmental gene activation within a chromatin context in plants.

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1-1 or abi3-1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.

We have isolated three HSP90-family genes from Arabidopsis: HSP81-1 which is heat-inducible, and HSP81-2 and -3 which are highly expressed under normal growth temperatures. Northern blot analysis and RNase protection analysis, using gene specific probes, showed that HSP81-2 and -3 mRNA were present in all tissues and abundant in roots, floral bud clusters, and flowers at 22 degrees C. A small amount of HSP81-1 mRNA was detected only in roots. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with an HSP81 promoter region fused to a beta-glucuronidase (GUS) gene, confirmed these results. At 22 degrees C, high GUS activity was observed in the root apical meristems, pollen and tapeta in HSP81-2::GUS and HSP81-3::GUS transgenic plants, while only branches of the root in HSP81-1::GUS transgenic plants expressed high GUS activity. After 2 hours of 35 degrees C treatment, extensively high GUS activity was observed in all tissues in HSP81-1::GUS transgenic plants, while elevated but tissue specific expression was observed in HSP81-2 and -3 transgenic plants. Exogenous application of various chemicals such as ABA, GA3, kinetin, IAA, NaCl, and mannitol revealed that 10 mM IAA and 0.1 M NaCl significantly enhanced the accumulation of HSP81-2 and -3 transcripts. Only a slight response to IAA was observed in HSP81-1 mRNA accumulation at 22 degrees C; the increase was possibly caused by a novel pathway other than heat-shock-response pathway.

Pedicel length and orientation (angle) contribute to the diversity of inflorescence architecture, and are important for optimal positioning of the flowers. However, relatively little is known about pedicel development. We previously described the Arabidopsis CORYMBOSA1 (CRM1)/BIG gene, which affects inflorescence architecture by controlling pedicel elongation and orientation. Here, we performed a suppressor screen using the partial loss-of-function allele crm1-13 to identify genes and pathways that affect pedicel development. We identified a hypomorph allele of the meristem identity regulator LEAFY (LFY) as the suppressor. Consistent with this, crm1 pedicels had elevated LFY levels and conditional gain of LFY function produced downward-bending pedicels. Steroid activation of 35S:LFY-GR plants caused a reduction in the cortical cell length in the abaxial domain and additional defects associated with adaxialization. Further analyses of loss of LFY function revealed that LFY is required for reduced cortical cell elongation at the adaxial side of the pedicel base. Defects in conditional LFY gain-of-function pedicels were correlated with decreased BREVIPEDICELLUS (BP) expression, while ASYMMETRIC LEAVES2 (AS2), a transcriptional repressor of BP, and REVOLUTA, a promoter of adaxial cell fate, were highly and ectopically expressed in LFY gain-of-function pedicels. LFY bound to cis-regulatory regions upstream of AS2, and as2 mutations partially suppressed the pedicel length and orientation defects caused by increased LFY activity. These data suggest that LFY activity promotes adaxial cell fate and hence the proper orientation and length of the pedicel partly by directly activating AS2 expression, which suppresses BP expression.

A modified method for studying the initiation of flowering in darkness (dark flowering, DF) in Arabidopsis thaliana has been developed, and the DF process has been examined with the aid of late-flowering mutants. A majority of plants developed floral buds by the use of liquid-shaken cultures in darkness. The late-flowering phenotype in gi and co mutants and early-flowering phenotype in a hy2 mutant disappeared in DF. It was found that wild-type plants grown under DF conditions express light-regulated genes and develop appropriate leaf architecture, as do the light-grown plants, without the apparent differentiation of chloroplasts. The shift experiments from darkness to light revealed the critical duration of growth in darkness for the initiation of DF. These results indicate that the DF process to the initiation of flowering is a mode of development distinct from that in light in Arabidopsis.

We isolated and analyzed mutants of Arabidopsis thaliana, acaulis, with flower stalks that are almost absent or are much reduced in length. The mutations are divided between two loci, acaulisl (acll) and acaulis2 (acl2). The acll-1 mutation has been assigned to linkage group 4 in the vicinity of locus ap2. The acl1-1 mutant showed premature arrest of the inflorescence meristem after the onset of reproductive development, followed by consequent reduction in the number of flower-bearing phytomers and therefore flowers. The apical meristem of the inflorescences was morphologically normal but its radius was about half that of the wild type. The acl1 mutants are also defective in the development of foliage leaves. Both defects could be rescued by growth at a specific temperature (28°C). The length of the cells in acl1-3 mutant was less than that in the wild type but the numbers of cells in leaves and internodes of acl1 mutants were calculated to be the same as those of the wild type. Thus, the defects in inflorescences and leaves were attributed to defects in the process of elongation (maturation) of these cells. Temperature-shift experiments showed that the Acl1+ product was necessary at all developmental stages. A critical stage was shown to exist for recovery from the cessation of development of inflorescence meristems that was caused by the acl1-1 mutation. Grafting experiments showed that the acl1-1 mutation does not affect diffusible substances. An analysis of double mutants carrying both acl1-1 and one of developmental mutations, ap1, clv1, Ify, or tfl1, showed that ACL1 is a new class of gene.

Summary The mutation gigantea (gi) is recessive and belongs to the late-flowering mutations in Arabidopsis thaliana. The late-flowering mutations result in a pronounced delay in flowering due to a prolonged phase of vegetative growth, which is manifested by an increased number of primary foliage leaves in the rosette (i.e. vegetative nodes). To examine the nature of the gi mutation, detailed phenotypic analysis was carried out for three representative mutant alleles. The results indicate that gi mutants have a defect in the promotion of the floral induction process by long-day photoperiods and not in the flowering process per se. Temperature-shift experiments using a partially conditional allele were employed to determine the timing of the functional requirement for the product of the GI locus. The end of the deduced functional period corresponds to the period at which transition of the apical meristem from the vegetative to the reproductive phase occurs. Such timing is in good agreement with the postulated role of the GI locus. These results demonstrate that the GI locus is involved in the promotion of floral initiation (entrance of the meristem into the transitional stage) by long-day photoperiods.

Diverse and precise control is essential for eukaryotic gene expression. This is accomplished through the recruitment of a myriad of proteins to a nascent messenger RNA (mRNA) to mediate modifications, such as capping, splicing, 3′-end processing, and export. Despite being important for every cell, however, the mechanism by which the formation of diverse messenger ribonucleoprotein (mRNP) particles contributes to maintaining intricate systems in the multicellular organism remains incompletely defined. We identified and characterized a mutant gene named erecta mRNA under-expressed (emu) that leads to the defective mRNA accumulation of ERECTA, a developmental regulator in the model plant Arabidopsis thaliana. EMU encodes a protein homologous to a component of the THO complex that is required for the generation of functional mRNPs. Further analysis suggested that EMU is genetically associated with SERRATE, HYPONASTIC LEAVES1, and ARGONAUTE1, which are required for proper RNA maturation or action. Furthermore, mutations in another THO-related gene led to embryonic lethality. These findings support the presence and importance of the THO-related complex in plants as well as yeast and vertebrates.

A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β-glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre-incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat-shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat-shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described.