Joe L. Key

Comparative analysis of physical stress responses in soybean seedlings using cloned heat shock cDNAs.

SummarySoybean seedlings were subjected to a wide range of physical (abiotic) or environmental stresses. Cloned cDNAs to heat shock (hs)-induced mRNAs were used to assess whether these diverse stresses induced the accumulation of poly(A)RNAs in common with those induced by hs. Northern blot hybridization analyses indicated that a wide range of stress agents lead to the accumulation of detectable levels of several of the hs-induced poly(A)RNAs; the relative concentration of those RNAs ‘induced’ by the wide range of stress agents (e.g. water stress, salt stress, anaerobiosis, high concentrations of hormones, etc.), was generally in the order of 100-fold lower than that induced by hs. There are two notable exceptions to that pattern of response to the stress agents. First, arsenite treatment resulted in accumulation of the ‘hs poly(A)RNAs’ to levels similar to those induced by hs. Cadmium also induced a somewhat normal spectrum of the ‘hs poly(A)RNAs’, but generally lower levels accumulated than in hs- and arsenite0treated tissues. Second, one set of poly(A)RNAs which are present at low and variable levels in control (non-stressed tissue) tissue, and which are increased some 5- to 10-fold by hs, increased in relative concentration in response to a wide range of the stress agents similarly to the response to hs. The physiological significance of the accumulation of this set of poly(A)RNAs (which translate into four electrophoretically different 27 kd proteins) is not known, but they certainly seem to serve as a monitor (or barometer) of physiological stress conditions. Cadmium treatment results in the accumulation of those same poly(A)RNAs and an additional band of higher molecular weight poly(A)RNA homologous to the same hs cDNA clone (clone pCE 54). Ethylene seems to have no obvious causal relationship to the hs response, even though hs-treated seedlings display some symptoms similar to those exhibited by ethylene-treated seedlings.

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)

Auxin‐regulated gene expression in higher plants

The naturally occurring auxin, indole‐3‐acetic acid, is thought to regulate normal growth and developmental processes in higher plants, including cell extension, cell division, and cell differentiation. Applied auxins, including synthetic and natural auxins, can dramatically alter normal growth and developmental patterns, and applied auxins have been shown to modify expression of genes transcribed by all three classes of nuclear RNA polymerase (i.e., RNA polymerase I, II, and III). The regulation of specific genes by auxin may occur over long (e.g., several hours to days) or short (e.g., minutes) time spans after hormone application. The more interesting responses to applied auxins are the short‐term responses since these are more likely primary responses to the hormone. Several experimental approaches have been used to demonstrate that applied auxins rapidly alter gene expression, and these include in vivo labeling of polypeptides, in vitro translation of mRNAs, and cDNA cloning of auxin‐regulated mRNAs ...

Sequence organization of the soybean genome.

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.

Molecular and cellular biology of the heat-shock response

Publisher Summary This chapter discusses the three major aspects of the heat-shock (HS) response including a comparative analysis of HS gene families and their expression, regulation of HS gene transcription, and functions of the heat-shock proteins (HSPs). This major protein family is also referred to as HSP70, reflecting the molecular weight of the major Drosophila HSP. This heat-inducible protein is conserved throughout evolution, and close related proteins are identified in a wide range of organisms including prokaryotes. In all eukaryotic organisms that are carefully examined, the HSP70 genes consist of a family of close related genes. The complexity and number of genes comprising the HSP70 family differ among species. Despite the number of organisms that are investigated, it is difficult to make generalizations about the composition of groups of HSPs from various species. Biochemical and genetic studies indicate that some HSPs or their cognates are present in organisms at normal growth temperatures and perform essential roles in normal cell function. The identification of some of these functions is valuable in providing clues to the function of HSPs at high temperatures.

Regulatable endogenous production of cytokinins up to toxic levels in transgenic plants and plant tissues

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.

Structure and expression of two auxin-inducible genes from Arabidopsis.

Two genes from Arabidopsis thaliana related to the auxin-inducible Aux28 and Aux22 genes of soybean have been isolated. These genes belong to a small multi-gene family and are similar to the soybean Aux gene family in the sequence of the predicted proteins, intron/exon locations, and auxin-enhanced expression of their transcripts. Application of auxin to 8-day old Arabidopsis plants, 4-day old etiolated seedlings, and suspension culture cells all resulted in enhanced Aux transcript levels. Comparison of the promoter sequences from the soybean and Arabidopsis genes yielded no significant sequence conservation; however, three regions of near sequence identity are present between the two Arabidopsis Aux genes.

Isolation and characterization of three families of auxin down-regulated cDNA clones

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a λZap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907–8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be upregulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.

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.

Development of a heat shock inducible expression cassette for plants: Characterization of parameters for its use in transient expression assays

A heat-inducible expression cassette has been constructed to study the conditional expression of sense or antisense orientations of any sequence of interest in transgenic plants or plant tissues. The construct includes the promoter and all but 5 bases of the mRNA leader from the soybeanGmhsp17.5-E gene, the polylinker from pUC18 (modified to remove the ATG), and a fragment that contains the polyadenylation signal and site from the nopaline synthase gene. Analysis of transient expression of a construct containing the β-glucuronidase (GUS) coding sequence cloned in the cassette and introduced intoNicotiana plumbaginifolia protoplasts by electroporation shows that the promoter has high expression at heat shock temperatures. This construct is expressed at a roughly 80-fold higher level per unit time than a cauliflower mosaic virus 35S gene promoter-GUS construction. The heat shock promoter is regulated positively by supercoiling in this transient assay system. The level of expression of HS-GUS constructions with the polyadenylation sites from either the nopaline synthase gene or theGmhsp17.5-E gene was similar. Constructs with a perfect fusion at the 5′ end had higher levels of expression than those with the corresponding nonperfect transcriptional fusion.