Ron T. Nagao

Developmentally regulated expression of soybean proline-rich cell wall protein genes.

Previously, we reported the characterization of a developmentally regulated proline-rich cell wall protein (SbPRP1) gene of soybean; the encoded protein is represented by a consensus amino acid repeat structure of Pro-Pro-Val-Tyr-Lys [Hong, J.C., Nagao, R.T., and Key, J.L. (1987). J. Biol. Chem. 262, 8367-8376]. Two other closely related members of this family of proline-rich protein (PRP) genes (SbPRP2 and SbPRP3), which differ from the extensin family of cell wall proteins, have been characterized (J.C. Hong, R.T. Nagao, and J.L. Key, unpublished results). Here we report studies on the regulation of expression of this gene family during soybean development by analyzing various plant organs, including leaves, stems, and roots of etiolated seedlings and light-grown plants, as well as young and mature pods, seed coats, and cotyledons. These organs were tested at different stages of development (young and fully mature tissue). Although a high level of sequence homology is observed at the nucleotide and amino acid sequence level among these three PRP genes/proteins, there are marked differences in the patterns of expression of each gene in different plant organs and organ regions. SbPRP1 is highly expressed and is the predominant form of PRP transcript in the mature hypocotyl, root, and immature seed coat. SbPRP2 is the major form of PRP in the apical hypocotyl and young suspension culture cells. SbPRP3 is the major form of PRP gene expressed in aerial parts; it is highly expressed in leaves, although no expression is detected in the roots.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of soybean proline-rich protein gene expression.

The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes.

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes β-galactosidase, and a polyadenylation 3′-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5′ promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypcotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged.Transgenic plants with a 600 bp promoter construct (−0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a −500 bp promoter had levels of expression similar to the −3.0 kb construct. The −0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 × 10−6 to 5 × 10−5 M 2,4-D and was responsive to as little as 5 × 10−8 M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.

DNA-Binding Study Identifies C-Box and Hybrid C/G-Box or C/A-Box Motifs as High-Affinity Binding Sites for STF1 and LONG HYPOCOTYL5 Proteins

LONG HYPOCOTYL5 (HY5) is a bZIP (basic leucine zipper) transcription factor that activates photomorphogenesis and root development in Arabidopsis (Arabidopsis thaliana). Previously, STF1 (soybean [Glycine max] TGACG-motif binding factor 1), a homologous legume protein with a RING-finger motif and a bZIP domain, was reported in soybean. To investigate the role of STF1, the phenotypes of transgenic Arabidopsis plants overexpressing STF1 and HY5 were compared. In addition, the DNA-binding properties of STF1 and HY5 were extensively studied using random binding site selection and electrophoretic mobility shift assay. Overexpression of STF1 in the hy5 mutant of Arabidopsis restored wild-type photomorphogenic and root development phenotypes of short hypocotyl, accumulation of chlorophyll, and root gravitropism with partial restoration of anthocyanin accumulation. This supports that STF1 is a homolog of HY5 with a role in light and hormone signaling. The DNA-binding properties of STF1 and HY5 are shown to be similar to each other in recognizing many ACGT-containing elements with a consensus sequence motif of 5′-(G/A)(G/A) TGACGT(C/G/A)(A/T/G)-3′. The motif represents a characteristically strong preference for flanking sequence to TGACGT and a larger sequence than the sequences recognized by the G-box binding factor and TGA protein families. The finding of C-box, hybrid C/G-, and C/A-boxes as high-affinity binding sites over the G-box and parameters associated with HY5 recognition define the criteria of HY5/STF1 protein-DNA interaction in the promoter regions. This study helps to predict the precise in vivo binding sites of the HY5 protein from the vast number of putative HY5 genomic binding sites analyzed by chromatin immunoprecipitation on chip.

Isolation and Characterization of Three Soybean Extensin cDNAs

We have characterized three different soybean (Glycine max) mRNAs that encode apoproteins of extensins, a family of cell wall hydroxyproline-rich glycoproteins (HRGPs). These transcripts encoded distinctive Tyr-rich proteins containing characteristic Ser-Pro4 sequences organized in higher-order repetitive units. The first transcript encoded an extensin SbHRGP-1 containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Val-Tyr-Lys, with Val occasionally replaced by Ile or Tyr. The second transcript encoded the SbHRGP-2 protein containing the 16-amino acid repeat Ser-Pro4-Ser-Pro-Ser-Pro4-Tyr-Tyr-Tyr-Lys/His. The third transript encoded the SbHRGP-3 protein containing a variant of 9- or 10-amino acid canonical repeats: Ser-Pro4-Tyr-Lys-Tyr-Pro, Ser-Pro5-Tyr-Lys-Tyr-Pro, and Ser-Pro4-Val-Tyr-Lys-Tyr-Lys, respectively. The dramatic amino acid substitutions in the Tyr-rich blocks (Tyr-X-Tyr-Lys) among these HRGPs indicate that each SbHRGP may have a different function in cell wall architecture.

The transcript abundance of GmGT-2, a new member of the GT-2 family of transcription factors from soybean, is down-regulated by light in a phytochrome-dependent manner

A new member of the GT-2 family of transcription factors, GmGT-2, was isolated from soybean while screening a cDNA library with a protein binding site (D1) in the promoter of Aux28, a member of the Aux/IAA family of auxin-responsive genes. GmGT-2 possesses various primary amino acid sequence characteristics common to all GT-2 factors thus far isolated, including sequence identity in the twin trihelix DNA-binding domains. Recombinant GmGT-2 expressed in Escherichia coli binds oligotetramers of both D1 and various GT-boxes. However, unlike other known members of the GT-2 family, GmGT-2 message levels are down-regulated by light in a phytochrome-dependent manner. Evidence is presented that the expression levels of Aux28 mRNA are also down-regulated by phytochrome. These results and other referenced data implicate the possible convergence of phytochrome and auxin signaling pathways.

Analysis of Multiple Classes of Soybean Heat Shock Genes and Proteins

The influence of high temperature stress (heat shock or HS) and other environmental stress agents on gene expression of soybean seedlings has been extensively studied. The sequence analysis of HS genes has revealed a high degree of conservation among individual members of several heat shock protein (HSP) families and different classes within a family, but some interesting differences have been noted. These studies have also revealed complex patterns of regulation of expression of the HS genes and accumulation of the HSPs. Based primarily upon the deduced amino acid sequence of the HSPs, immunological cross-reactivity, and intracellular localization, the complex group of low molecular weight (LMW) HSP genes have been organized into multiple classes. In soybean several cDNA and genomic clones encoding 20 to 24 kD LMW HSPs have been isolated which represent new classes of the LMW HSP gene super family based on nucleotide/amino acid sequence and cell fractionation analyses. The mRNAs transcribed from these genes are of lower abundance than those for the 15 to 18 kD Class I and II proteins, and these genes occur as small multigene (i.e. three to four) classes or subfamilies. The mRNAs of three of these classes of LMW HSP genes are translated on ER- bound ribosomes and possess hydrophobic leader sequences. The presence of a consensus ER retention sequence on two of these proteins indicates that they probably reside within the ER. The third protein lacks the consensus ER retention signal and presumably is translocated to an as yet unidentified location. The mRNA representing a fourth LMW gene class is translated on unbound cytoplasmic ribosomes, and the predicted protein has a N-terminal sequence with properties similar to that of some proteins which are translocated into mitochondria. Early studies with soybean seedlings indicated that some 22 to 24 kD HSPs are localized in mitochondria. Differential induction by amino analog treatment indicates that genes assigned to the same class based on amino acid similarity and localization can be regulated differently. The possible role of the multiple classes on LMW 15 to 24 kD HSPs in protein protection from denaturation at high temperature (i.e. a chaperone function), based on studies from other laboratories is noted and some of these results will be summarized.

INSTABILITY OF HIGHER PLANT CHROMOSOMAL DNA MOLECULARLY CLONED IN E. COLI VIA RECOMBINANT PLASMIDS

This chapter studies the instability of higher plant chromosomal DNA molecularly cloned in E. coli via recombinant plasmids. High molecular weight DNA from Glycine max (soybean) and Arabidopsis thaliana was highly purified to remove polysaccharide contamination by banding in CsCl-PdI gradients. Restriction fragments were enriched 60-fold for the soybean rDNA genes by preparative agarose gel electrophoresis. Restriction fragments of total DNA from both plants and from fractions enriched for rDNA were inserted into the Hind III and Bam HI sites (tetracycline resistance) of plasmid pBR322 and the Eco RI site (chloramphenicol resistance) of plasmid pBR325. Hundreds of independently isolated transformants containing recombinant plasmids were screened. The vast majority of the plant DNA inserts was less than 10 6 daltons. The size of the input DNA varied from 10 6 to 10 7 daltons with a MW av of 4 × 10 6 daltons. It is shown that the cloned rDNA fragments result from a 60% loss of DNA from within the cloned rDNA fragment.