Storage protein degradation and proteolytic enzyme activities during germination of water stressed impatiens seeds
Water uptake of impatiens (Impatiens wallerana Hook. f. cv. Super Elfin Coral) seed was measured an an increase in fresh weight every 24 hr during 144 hr of germination. Seeds absorbed most of the water required for germination within the first 3 hr of imbibition, and thus germinated at 60 to 67% moisture on a dry weight basis. Germination started at 48 hr and was complete by 96 hr at 25C. Three distinct groups of storage proteins with estimated molecular weights of 35, 33, and 31 kilodaltons (kd); 26, 23, 21 kd; and two bands less than 14 kd were present in dry seeds. At 48 hr from the start of imbibition, two proteins, 35 and 26 kd, degraded in the radicles. The remainder of the storage proteins degraded by 96 hr. Major depletion of storage proteins coincided with the time of maximum germination. The soluble protein fraction increased and coincided with a decrease in the insoluble fraction during germination under optimum conditions. Under these conditions the activity of leucine aminopeptidase (LAPase) during 144 hr of germination correlated with the major depletion of storage proteins from radicles and cotyledons. Isoelectric focussing showed that only one form of LAPase was present in impatiens seeds. This was determined to be a sulfhydryl protease with peak activity at pH 8. An increase in specific activity indicated synthesis of this enzyme during germination. Water stress of -0.1, -0.2, -0.4, and -0.6 MPa, induced by polyethylene glycol 8000, reduced germination by 13, 49, 91, and 100%, respectively at 96 hr. Under these conditions increases in fresh weight were inhibited by 53, 89, 107, and 106%, respectively. Water potentials that inhibited germination also inhibited degradation of storage proteins and the protein profile remained unchanged. At these levels, the insoluble protein content remained unchanged and the small increase in soluble proteins was not enough to proceed to germination. Threshold concentrations of soluble proteins were required for germination. Water stress caused a decrease in activity of LAPase. The loss of enzyme activity under water stress was due to both inhibition of enzyme synthesis and inactivation of preexisting enzymes. p-Hydroxymercuribenzoate was the most potent inhibitor of LAPase in enzyme assays, but did not inhibit germination. N-Ethylmaleimide (NEM) inhibited the LAPase activity by 70%. Total inhibition of germination by iodoacetamide, NEM, and phenylmethylsulfonyl fluoride indicated involvement of other sulfhydryl proteases or enzymes in germination processes.
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