Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays

Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24-48 h and again from 72-96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs approximately 5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event.     

Cunxi Wang · Kevan P.C. Croft · David F. Hildebrand

 Auxin [α-naphthaleneacetic acid (NAA) or indole-3-acetic acid] can induce the expression of lipoxygenases (LOXs) in cultured immature zygotic embryo cotyledons of soybean [Glycine max. (L.) Merr]. These auxin-induced LOXs are different from those normally expressed in seeds but have the same isoelectric points (pI) as those found in seedlings. The pIs of the two seedling LOXs were determined to be 5.09 and 5.23. One of the auxin-induced LOXs has the same pI (5.09) and molecular mass (94 kDa) as seedling LOX4. The partial amino acid sequences from the purified NAA-induced pI-5.09 LOX are identical to those of LOX4. RNA protection assays showed that NAA induces the expression of LOX4 and LOX5 mRNAs in cultured embryo cotyledons where they are not normally expressed. Soybean genotypes with a polymorphic variant of LOX4 in hypocotyls showed the same variation as NAA-induced LOXs in the embryo cotyledons. These results demonstrate that the NAA-induced pI-5.09 LOX is seedling LOX4 and also suggest that auxin might be directly or indirectly involved in seedling LOX expression during seed germination. Received: 10 January 2000 / Revision received: 16 June 2000 / Accepted: 29 June 2000     

Development and localization of endo-{beta}-mannanase in the embryo of germinating and germinated tomato seeds

The occurrence of endo--mannanase in the embryo of germinating and germinated tomato (Lycopersicon esculentum Mill.) seeds was characterized. The endo--mannanase that developed in the embryo consisted of two isoforms and their molecular masses (41 and 42 kDa) did not correspond to the mass (37-39 kDa) of any isoform present in the endosperm. This indicates that mannanase isoforms present in the embryo are embryo-specific. Specific activities (with locust bean galactomannan as substrate) were also different between the embryonic and the endospermic enzymes. The enzyme was absent from the embryo of seeds imbibed for 2 h. With time after imbibition, mannanase content increased until the radicle had just protruded (day 2). However, the increase was transient and the content rapidly decreased thereafter and fell to an undetectable level on day 4. Tissue prints showed that the activity first appeared at the tip part of the radicle and then at the tip of the cotyledon. Thereafter the activity spread through the embryo tissues from the both tip parts.     

Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae

Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72–96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction. Received: 15 March 1999 / Accepted: 1 May 1999     

Protein Patterns, Characterized by Computer Image Analysis, of Lentil Embryo Axes Germinating Under Salt Stress

Following 16, 40 and 64 h exposure to 0.33 M NaCl given after 8 h water imbibition, lentil seeds showed a gradual decrease of germination upon their transfer to water. These salt related changes were accompanied by modifications in the protein patterns of embryo axes as revealed by two-dimensional electrophoresis separation and by the computer image analysis of protein spots. In comparison with 8 h water imbibed seeds, prominent proteins comprised between the 5.1 – 7.6 pH isoelectric point in the first dimension and 75 – 50 kDa molecular mass in the second dimension showed a significant increase in their abundance as salt exposure increased. On transfer to water to complete germination, the content of many of these proteins decreased at 24h in 2 – 3 cm length embryo axes in comparison with the corresponding embryo axes of seeds continuously imbibed in water for 24 h. Some groups of proteins ranging between 15.5 – 17.3 kDa, already present after 8 h water imbibition, were not detectable after 24 h but were expressed in seeds exposed to NaCl and transferred to water for 24 h. Up- and down-regulated proteins in lentil embryo axes, imbibed under non-lethal salt stress conditions, have been tentatively identified by comparison with the protein map of germinating seeds of the model plant Arabidopsis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Qualitative and quantitative changes in mRNA of castor beans during the initial stages of germination

The increase in extractable RNA during the initial germination stages of castor bean was measured, for both the embryonic axis and the storage endsperm. The extractable rRNA increased between 24 and 72 h after initial imbibition in the embryo and 48–72 h in the endosperm. The mRNA species present over the first 6 days of germination were identified by the products from in vitro translation. The mRNA from dry seeds gave predominantly low molecular weight polypeptide products. Between 0.5 and 1 h of initial imbibition new mRNA species were detectable and the qualitative changes were largely complete by 8 h, some 16 h–40 h before the detectable quantitative changes. Despite the large variations in enzyme activity occurring 48 h–192 h after imbibition, the mRNA species qualitatively varied very little, after this initial change, up to 144 h after imbibition. In the light of this large, early, qualitative change in mRNA, the possible importance of long-lived mRNA in seed germination is discussed.Abbreviations SDS sodium dodecyl sulphate - TMV tobacco mosaic virus - DEP diethyl pyrocarbonate - MDL message-dependent reticulocyte lysate - TCA trichloroacetic acid - MW molecular weight - PAGE polyacrylamide gel electrophoresis     

Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures

 Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397 amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity. Received: 2 September 1999 / Accepted: 30 November 1999     

Actin isoform and α1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch

Summary The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and α_1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of α_1B -adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in α_1B-adrenoceptor or β/γ-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.     

Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Differences in muscle contractile characteristics among bodybuilders, endurance trainers and control subjects

The purpose of this investigation was to compare the myosin heavy chain (MHC) isoform expression of the triceps brachii muscle and isoinertial, isometric and isokinetic strength indices in competitive bodybuilders (CB, n = 5), recreational resistance trainers (RT, n = 5), endurance-trained rowers (ER, n = 5) and control (C, n = 5) subjects. Muscle tissue samples were analysed for MHC isoform content using 6% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The CB possessed significantly smaller (P < 0.05) percentage of MHC type IIb proteins [12.92 (SD 7.08)%] than RT [30.08 (SD 6.58)%] ER [31.20 (SD 2.74)%] and C [38.22 (SD 2.95)%] groups (i.e. CB < RT ≈ ER < C). While the content of MHC type IIa isoforms did not differ significantly between the two resistance-trained groups [CB = 55.76 (SD 5.38)%; RT = 45.72 (SD 7.8)%], CB presented significantly more type IIa MHC isoforms than ER [42.84 (SD 2.98)%] and C [34.72 (SD 1.57)%] subjects (i.e. CB ≈ RT > ER ≈ C). The MHC type I protein content did not differ significantly among RT [24.20 (SD 4.89)%] ER [25.38 (SD 1.67)%] and C [27.06 (SD 1.81)%] groups. The CB [31.32 (SD 2.67)%] presented significantly more type I MHC isoforms only in comparison with RT. However, when changes in the percentage of MHC type I isoforms were converted to effect sizes (ES), it appeared that low statistical power rather than the absence of an effect accounted for the nonsignificant differences between CB and other groups (i.e. CB > RT ≈ ER ≈ C). Significant differences existed in isoinertial strength among the trained athletes (i.e. CB > RT > ER ≈ C), while isometric and isokinetic strength were not significantly different among any of the trained groups. However, the ES transformation of data demonstrated that large differences existed between resistance-trained groups and ER for isometric and isokinetic strength (i.e. CB ≈ RT > ER ≈ C). A statistically significant negative correlation (P < 0.001) was found between MHC type IIb isoforms and isoinertial strength index (r = − 0.68). The MHC type IIa proteins were positively related to all the strength measures considered (r = 0.51 – 0.61; P < 0.001). These data demonstrated different patterns of MHC isoform expression among the different groups of athletes and it is suggested that these differences on occasion may affect the expression of strength. Accepted: 24 September 1996     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Endogenous Rhythmicity in Water Uptake by Seeds

Water uptake (after 4 h imbibition) as a function of the momentof its onset shows rhythms with a period length of approx. 6h. The period length is not affected by a increase in temperatureof 10 °C, suggesting that the oscillation is endogenousin character. Furthermore, heat-killed seeds show no oscillationsin water uptake, indicating that the rhythm described originatesfrom the temperature-sensitive component of imbibition. Phaseolus vulgaris, french bean, imbibition, oscillation in imbibition     

Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

Background  

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform.     

Steroidal regulation of Ihh and Gli1 expression in the rat uterus

Ovarian steroid hormones, progesterone (P4), and estradiol (E2) strictly regulate the endometrial tissue remodeling required for successful embryo implantation. Indian hedgehog (Ihh) is up-regulated by P4 and critically mediates uterine receptivity in the mouse. However, the regulation of Ihh expression during the implantation period still remains unclear. The present study was conducted to elucidate the mechanism of the steroidal regulation in the expression of Ihh and Gli1, the mediator of the Ihh pathway. Ihh mRNA was expressed in the rat uterus on 3.5–5.5 days post-coitus (dpc), while Gli1 expression transiently increased at 3.5 dpc but decreased significantly on 5.5 dpc (P < 0.001). In delayed implantation, the expression of Ihh was induced by the implantation-induced E2 treatment in the primed rat uterus. In contrast, expression of Gli1 was significantly decreased by E2 treatment (P = 0.016). In the case of ICI182.780 (ICI) treatment, Ihh expression was eliminated by ICI, whilst Gli1 expression increased. These results suggest that Ihh expression is maintained at a high level until the initiation of implantation, while the expression of Gli1 is decreased just prior to the initiation of implantation depending on the E2 action. This observation aids in the understanding of the Ihh signaling pathway mediating uterine remodeling for implantation.     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)