Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.     

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.     

Direct embryo formation in leaves of Camillia japonica L.

Summary The culture conditions for direct embryo formation in leaves of Camellia japonica L. were established. An auxin treatment followed by incubation during 11 days in darkness on diluted Murashige and Skoog modified basal medium induced direct morphogenesis. The number of subcultures, subculture interval and leaf age affected in vitro leaf response. The results showed that the cells from a cultured leaf respond differently to the same culture conditions by forming embryos, roots, and non-morphogenic as well as organogenic callus. Direct embryo formation occurred only in the marginal leaf regions. Direct root formation only occurred in a well-defined region of the midrib whereas callus was preferentially formed on the leaf basis. The results suggest the existence of differences in morphogenic competence according to leaf regions. Plantlet regeneration was successfully achieved from somatic embryos and from leaf basisderived callus, via shoot bud induction.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Studies of the gene expression in a somatic hybrid of dihaploid Solanum tuberosum and diploid S. brevidens using two-dimensional protein electrophoresis

Two-dimensional protein electrophoretic patterns of leaf, stem and microtuber were compared between a somatic hybrid (Solanum tuberosum + S. brevidens) and parental plants. Polypeptide spots observed in leaf of the somatic hybrid (BT-1) were similar to those of S. brevidens. In the stem of BT-1, the spots characteristic for each parental plants were also observed. Three specific spots (W, X, Y) found in BT-1 were identical to those of S. tuberosum, however their appearance in S. brevidens depended on the culture conditions (observed at 16h daylength regime, but not in the dark with high sucrose concentration). Potato tuber storage protein patatin was observed in small amounts in the microtubers of BT-1. The data indicated that gene expression unique to each parental plants also existed in the somatic hybrid.Abbreviations BAP 6-benzylaminopurine - 2,4-D (2,4-dichlorophenoxy) acetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

A pleiotropic mutation results in cross-resistance to auxin, abscisic acid and paclobutrazol

Summary Mutant lines of Nicotiana plumbaginifolia resistant to the synthetic auxins 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) were isolated as germinating seedlings on selective medium. In each case, resistance was conferred by a single recessive nuclear mutation at one of 3 loci designated iba1, iba2 and iba3. Labelling studies with ¹⁴C NAA suggest that resistance was not due to changes in the uptake or metabolism of NAA. Plants homozygous for the iba1 mutation exhibit a syndrome of atypical germination and growth suggestive of a defect in the biosynthesis, metabolism or localization of abscisic acid. Wild-type seeds treated with gibberellin exhibit the same syndrome, including resistance to NAA and IBA. On the basis of these observations, we propose that auxin toxicity in seeds may be mediated by a block in gibberellin biosynthesis.Abbreviations ABA abscisic acid - GA gibberellic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - p-cell protoplast-derived cell - 2,4-D 2,4-dichlorophenoxyacetic acid     

Purification and some properties of chalcone synthase from a carrot suspension culture induced for anthocyanin synthesis and preparation of its specific antiserum

Chalcone synthase was purified to homogeneity by polyacrylamide gel electrophoresis from cell suspension cultures of carrot in which anthocyanin synthesis was induced by transferring the cells from a medium containing 2,4-dichlorophenoxy-acetic acid (2,4-D) to one lacking it. A molecular weight of 80,000-85,000 for the enzyme was determined by gel filtration and disc-gel polyacrylamide electrophoresis, and one of about 40,600 for the subunit by SDS slab-gel electrophoresis. The primary reaction product was chalcone and the pH optimum of the reaction was 8.0. The Km values for 4-coumaroyl-CoA and malonyl-CoA were 5.7 microM and 18 microM, respectively. These properties of carrot chalcone synthase were discussed in comparison to those of that from cell cultures of parsley reported previously. Antiserum against chalcone synthase from carrot was obtained from mice bred under specific pathogen free conditions. Crossreactivity was examined by Western-blotting, and the high specificity of the antiserum against chalcone synthase was demonstrated.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

Synchronization of somatic embryogenesis from carrot cells at high frequency as a basis for the mass production of embryos

Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.(1) Small spherical single cells from suspension cultures obtained by sieving and density gradient centrifugation in Percoll solutions differentiated to embryogenic cell clusters at high frequency when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (0.05 micromolar), zeatin (1 micromolar) and mannitol (0.2 molar). (2) Embryogenic cell clusters from suspension cultures obtained by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed for a short time synchronously differentiated to embryos, especially globular embryos at high frequency, when they were cultured in a medium containing zeatin (0.1 micromolar) but no auxin. (3) Embryogenic cell clusters obtained by above method are cultured at cell densities of 2×10³ cell clusters ml^-1. Globular embryos which were sieved from embryos induced synchronously differentiated to torpedo-shaped embryos at high frequency when they were cultured at densities below 150 globular embryos ml^-1.Using these systems, the whole process of embryogenesis from single cells to whole plants could be synchronously induced at high frequency.Abbreviations ABA abscissic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin A₃ - IAA indoleacetic acid - NAA naphthylacetic acid     

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4‐dichlorophenoxyacetic acid‐mediated alteration of actin in Arabidopsis roots

2,4‐Dichlorophenoxyacetic acid (2,4‐D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole‐3‐acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4‐D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4‐D‐specific mutants suggested that 2,4‐D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4‐D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4‐D but not IAA altered the actin structure in long‐term and short‐term assays. Analysis of the 2,4‐D‐specific mutant aar1‐1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4‐D‐induced depolymerization of actin. The ubiquitin proteasome mutants tir1‐1 and axr1‐12, which show enhanced resistance to 2,4‐D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4‐D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4‐D on the organization of actin filaments. Roots of the double mutant aar1‐1 tir1‐1 also showed enhanced resistance to 2,4‐D‐induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4‐D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF^TIR1 ubiquitin proteasome components.     

OsARF16, a transcription factor,is required for auxin and phosphate starvation response in rice (Oryza sativa L.)

Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (?Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and ?Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and ?Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and ?Pi treatments. In ?Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation‐induced genes (PSIs) were less induced by ?Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under ?Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and ?Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice.