The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

Isolation of a cDNA clone for pea (Pisum sativum) seed lipoxygenase.

A lipoxygenase cDNA clone, pCD45, was identified in a Pisum sativum L. (pea) seed mRNA cDNA library by hybrid-release/translation followed by immunoprecipitation with antiserum raised against lipoxygenase from Glycine max L. (soya bean). pCD45 hybrid-selected an mRNA encoding the larger of the two polypeptides of Mr approximately 95 000 that were immunoprecipitated from cell-free translation products of pea seed poly(A)-containing RNA by the G. max anti-lipoxygenase. 'Northern'-blot analysis showed the mRNA that hybridized to pCD45 to be approximately 3000 nucleotides in length. Three to five copies of the lipoxygenase gene corresponding to pCD45 were estimated to be present per haploid Pisum genome; hybridization of the cDNA insert from pCD45 to G. max DNA was also detected.     

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.     

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.     

Cloning, expression, and sequence conservation of pathogenesis-related gene transcripts of potato

Treatment of potato tuber disks with arachidonic acid elicits the accumulation of several mRNAs. cDNA clones corresponding to two of these mRNAs were isolated and characterized. Nucleotide sequence analysis reveals that both clones (pSTH-2 and pSTH-21) contain an open-reading frame coding for a 155-amino acid polypeptide. The polypeptides encoded by the two clones differ by only six amino acids and show a high degree of similarity with PR protein sequences from pea (approximately 42%) and parsley (approximately 37%). mRNAs corresponding to the two potato cDNA clones also accumulate in Solanum chacoense and in tomato following elicitor treatment. Maximum accumulation of the mRNAs corresponding to the two cDNA clones is reached 24 hr after elicitor treatment of the tuber disks. pSTH-2-related mRNAs also accumulate in tubers after wounding or treatment with eicosapentaenoic acid and are detected in potato and tomato leaves treated with a Phytophthora infestans mycelium homogenate. The presence of these conserved genes in species from three plant families and the similarity of their induction pattern suggest an important function during the plant defense response.     

The Auxins IAA and 4-Cl-IAA Differentially Modify Gibberellin Action via Ethylene Response in Developing Pea Fruit

This study explores the unique growth-regulatory roles of two naturally occurring auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA), and their interactions with gibberellin (GA) during early pea (Pisum sativum L.) fruit development. We have previously shown that 4-Cl-IAA can replace the seed requirement in pea pericarp growth (length and fresh weight), whereas IAA had no effect or was inhibitory. When applied simultaneously, gibberellin (GA₃ or GA₁) and 4-Cl-IAA had a synergistic effect on pericarp growth. In the present study, we found that simultaneous application of IAA and GA₃ to deseeded pericarps inhibited GA₃-stimulated growth. The inhibitory effect of IAA on GA-stimulated growth was mimicked by treatment with ethephon (ethylene releasing agent), and the inhibitory effects of IAA and ethylene on GA-mediated growth were reversed by silver thiosulfate (STS), an ethylene action inhibitor. Although pretreatment with STS could retard senescence of IAA-treated pericarps, STS pretreatment did not lead to IAA-induced pericarp growth. Although 4-Cl-IAA stimulated growth whereas IAA was ineffective, both auxins induced similar levels of ethylene evolution. However, only 4-Cl-IAA-stimulated growth was insensitive to the effects of ethylene. Gibberellin treatment did not influence the amount of ethylene released from pericarps in the presence or absence of either auxin. We propose a growth regulatory role for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. Additionally, ethylene (IAA-induced or IAA-independent) may inhibit GA responses under physiological conditions that limit fruit growth.     

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Regulation of root formation by auxin-ethylene interaction in pea stem cuttings

The possibility was investigated that the inhibition of rooting in pea ( Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.     

Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea. Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea. Of the two GS polypeptides in leaves (44 and 38 kd), the 44-kd GS polypeptide is predominant and is localized to the chloroplast stroma. In roots, the predominant GS polypeptide is 38 kd. Upon Rhizobium infection of roots, three 37-kd GS polypeptides increase in abundance in the nodules relative to uninfected roots. cDNA clones encoding three different GS mRNAs have been characterized. Hybrid-select translation has identified three different GS primary translation products (49, 38 and 37 kd). Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38- and 37-kd GS primary translation products. A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49-kd putative precursor to the mature chloroplast GS polypeptide. cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.     

Dexamethasone and estrogen regulate Xenopus laevis albumin mRNA levels

The regulation of albumin mRNA levels by steroid hormones was examined in a primary Xenopus liver culture system. In the absence of exogenous steroid hormone, albumin mRNA levels declined rapidly in culture. Dexamethasone is required for preservation of albumin mRNA levels in culture and effects a greater than 10 fold induction of albumin mRNA in cultures maintained in steroid hormone-free medium. Estrogen can override the effect of dexamethasone and elicits a decline of greater than 80% in albumin mRNA levels. Our cDNA clones of the mRNAs encoding the two 74,000 dalton and one 72,000 dalton cellular albumins allowed us to show that all three albumin mRNAs were down regulated by estrogen.