Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner.

MAIN CONCLUSION: By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.

Differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls in response to various stimuli.

Ethylene can be produced by a variety of developmental and environmental factors such as ripening, the plant hormone auxin, and mechanical wounding via a biosynthetic pathway including AdoMet synthase, ACC synthase, and ACC oxidase steps. ACC synthase and ACC oxidase are known to be encoded by multigene families, and are believed to be differentially expressed in response to various stimuli. In mung bean, ACC synthase is encoded by 7 genes, ACS1, ACS2 ACS3, ACS4, ACS5, ACS6, and ACS7, and ACC oxidase by 2 genes, ACO1 and ACO2. In this study, was have investigated differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls under various conditions by the semiquantitative RT-PCR method. Primers which can specifically bind and amplify each cDNAs of ACS1, ACS2, ACS3, ACS4, ACS5, ACS6, ACS7, and ACO1, and ACO2 were designed and used to monitor the responses to various stimuli. Transcripts of ACO1 and ACO2 were accumulated constitutively in the hypocotyl segments even without andy treatment. After cold treatment on intact plant, transcripts of ACS5, ACS6, and ACS7 were accumulated in the hypocotyl segments. We also found the excision of hypocotyl segments and incubation in a buffer solution, a typical way of chemical treatments to hypocotyl segments, lowered the level of ACO2 transcripts with little change of the level of ACO1 transcripts. In response to incubation with IAA (0.1 mM) of excised hypocotyl segments, transcripts of ACS1, ACS6, and ACS7 were accumulated and the level of ACO2 transcripts was increased. Transcripts of ACS1, ACS2, ACS3, ACS5, ACS6 and ACS7 were induced by incubation with OGA (50 micrograms/ml), while the transcripts of ACS4 were accumulated and the level of ACO2 transcripts was increased by incubation with 1 mM LiCl. Our results strongly suggest that all seven ACC synthase genes and two ACC oxidase genes must be active and each gene is differentially regulated by a different subset of the inducing factors.

Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock.

1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P < 0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS-rats. 3. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10-9- 10-6 M). Pretreatment with aminoguanidine (15 mg kg- 1, i.v.,at 20 min prior to LPS) significantly prevented this LPS-induced hyporeactivity to NA ex vivo.4. Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 +/- 0.2 pmol mg-1 min-1 (control, n = 4) to 4.8 +/- 0.3 pmol mg-1 min-1 (P<0.05, n = 6). In LPS-rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44+/- 5% (n = 6, P <0.05).Moreover, when added in vitro to lung homogenates obtained from LPS-rats, aminoguanidine and N omega-nitro-L-arginine methyl ester (L-NAME; 10-8 to 10-3 M) caused a concentration-dependent inhibition of iNOS activity (n = 3-6, IC50: 30 +/- 12 and 11 +/- 6pEM, respectively P>0.05). In contrast,aminoguanidine was a less potent inhibitor than L-NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3-6, IC50 is 140 +/- 10 and 0.6 +/- 0.1 I1M, respectively, P<0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L-NAME(maximal inhibition at 90 min and 30 min, respectively).5. Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg-1, i.p.)resulted in a survival rate of only 8% at 24 h (n = 12). However, therapeutic application of aminoguanidine (15 mg kg-1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75%(n = 8), whereas L-NAME (3 mg kg-1, i.p. at 2 h and 6 h after LPS) did not affect the survival rate(11%, n=9).6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine,or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock.