In vivo and in vitro Activity of 1-aminocyclopropane-1-carboxylic Acid Oxidase in Germinating Seeds of China Aster (Callistephus chinensis Nees)

The activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; EC 1.4.3.3) in germinating seeds of Callistephus chinensis was studied. For maximum recovery of ACO activity in vitro, the presence of 10% (w/v) insoluble polyvinylpolypyrrolidone (PVPP) and 30% of glycerol in the extraction medium was necessary. The optimum pH for this activity was 7.0. Ethylene production by whole achenes or enzymatic extract increased due to increasing 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations. Saturation level of ACC for in vivo ACO activity was 10−1 M and Vmax was 10.89 nL C2H4·mg protein−1·h−1. For in vitro ACO activity, the saturation level of ACC was 10−3 M and Vmax was 2.299 nL C2H4·mg protein−1·h−1. Both, in vivo and in vitro ACO activities did not follow Michaelis-Menten kinetics. The Hill coefficients (h) were estimated on the basis of non-linear estimation. Their values were 0.63 for in vivo ACO activity and 1.73 for in vitro ACO activity. The experimental data show that ACO from C. chinensis seeds is an oligomeric enzyme with at least two active sites. During seed germination, in vitro ACO activity was detectable after 12 hours of imbibition, while in vivo ACC conversion to ethylene was observed after 24 h, i.e. – after radicle protrusion. The activity of ACO in C. chinensis seeds is associated with germination sensu stricto, and might be a good marker of this process.

Physiological and biochemical changes induced in sunflower seeds by osmopriming and subsequent drying, storage and aging

Sunflower (Helianthus annuus L.) seeds show more germination at high temperatures (25–30°C) than at temperatures below 20°C. Osmopriming with polyethylene glycol-6000 for 3–5 days at 15°C strongly increases germination at suboptimal temperatures. This stimulatory effect of priming persists after seed redrying and during subsequent storage at 20°C (55% RH) for at least 14 weeks. However, primed seeds deteriorate faster than untreated seeds during accelerated aging (45°C, 100% RH). The longer the priming treatment, the higher is the amount of germination but at the same time the higher is the sensitivity of seeds to accelerated aging. Priming enhances the respiratory activity of seeds transferred onto water and their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. These effects remain after drying the seeds and are maintained in part during dry storage, whereas they disappear during accelerated aging. These results suggest that ACC-dependent ethylene production might be a good indicator of seed vigour; it increases with duration of priming and decreases very early during aging, well before significant loss of seed viability. Decrease in ACC conversion to ethylene indicates that aging is probably associated with membrane deterioration since in vivo ACC oxidase activity depends on membrane properties. However, no increase in electrolyte leakage is observed during aging.

Antagonism by Clopyralid of Picloram-Induced Ethylene Biosynthesis in Rapeseed Plants

The effect of clopyralid pretreatment (500 g/ha) on picloram-induced ethylene, ACC (1-aminocyclopropane-l-carboxylic acid), and MACC [l-(malonylamino)-cyclopropane-1-carboxylic acid] was measured in rapeseed plants that were treated with 50 or 100 g/ha of picloram. In contrast to plants that did not receive a clopyralid pretreatment, ethylene biosynthesis was significantly reduced in plants pretreated with clopyralid prior to picloram. Picloram- induced levels of ACC also were significantly reduced in plants receiving pretreatment with clopyralid. In contrast, there was no difference between the levels of MACC in plants that were and were not pretreated with clopyralid. Therefore, the mechanism by which clopyralid pretreatment interferes with picloram-induced synthesis of both ACC and ethylene may be manifested through the blocking of de novo synthesis of ACC synthase normally stimulated by picloram. The lack of significant difference in MACC levels between plants that were and were not pretreated with clopyralid precludes the stimulation of enhanced ACC conversion to MACC as an exclusive mechanism of clopyralid’s antidoting activity. It is likely that the rate of picloram-induced ACC synthesis by plants receiving pretreatment is within their capacity to convert ACC to MACC, thereby limiting the substrate available for conversion to ethylene. In contrast, it appears that the extent of ACC synthesis by plants receiving no pretreatment supersedes their capacity for conversion to MACC. thereby resulting in greatly enhanced rates of ethylene evolution and subsequent development of injury symptoms.

Antagonism by Clopyralid of Picloram-Induced Ethylene Biosynthesis in Rapeseed Plants

The effect of clopyralid pretreatment (500 g/ha) on picloram-induced ethylene, ACC (1-aminocyclopropane-1-carboxylic acid), and MACC [1-(malonylamino)-cyclopropane-1-carboxylic acid] was measured in rapeseed plants that were treated with 50 or 100 g/ha of picloram. In contrast to plants that did not receive a clopyralid pretreatment, ethylene biosynthesis was significantly reduced in plants pretreated with clopyralid prior to picloram. Piclo­ram-induced levels of ACC also were significantly reduced in plants receiving pretreatment with clopyralid. In contrast, there was no difference between the levels of MACC in plants that were and were not pretreated with clopyralid. Therefore, the mechanism by which clopyralid pretreatment interferes with picloram-induced synthesis of both ACC and ethylene may be manifested through the blocking of de novo synthesis of ACC synthase normally stimulated by picloram. The lack of significant difference in MACC levels between plants that were and were not pretreated with clopyralid precludes the stimulation of enhanced ACC conversion to MACC as an exclusive mechanism of clopyralid’s antidoting activity. It is likely that the rate of picloram-induced ACC synthesis by plants receiving pretreatment is within their capacity to convert ACC to MACC, thereby limiting the substrate available for conversion to ethylene. In contrast, it appears that the extent of ACC synthesis by plants receiving no pretreatment su­persedes their capacity for conversion to MACC, thereby resulting in greatly enhanced rates of ethylene evolution and subsequent development of injury symptoms.