Spermidine-induced Early Germination Of Lettuce Seed Is Probably Relevant To The Nitric Oxide Production

Spermine (Spm), spermidine (Spd) and their diamine obligate precursor putrescine (Put) are micro molecular aliphatic polycations that are ubiquitous in all plant cells. They have been proposed to be closely associated with plant development, morphogenesis and respond to diverse stresses. Nitric oxide (NO) is a reactive oxygen molecule. As a plant cell signaling molecule, NO participates in a variety of plant growth, development regulation. Both polyamines and NO play important role in seed germination and seedling development, and it have been proved that polyamines can induce NO production in plant tissues. However, there are no evidences that polyamines are relevant to the NO production in the process of seed germination and seedling development, and it has not reported that how polyamines induce NO production. Given these, in this study we choose a normal breed of lettuce (Lactuca sativa) seed (Guasihong) as the experimental material treated with different exogenous matters to investigate the roles and interactions of polyamines and NO in the process of lettuce seed germination and seedling development. The results are as follows:Treatments with exogenous polyamines in low concentrations promoted early germination of lettuce seed, of which 0.5 mM Spd was most effective, followed by Spm and Put. The detection of time course of seed germination showed that 0.5 mM Put, Spd and Spm imbibitions could significantly promote the early germination of lettuce seed, but this promotive effect disappeared after 48 h. Consistent with this, treatment with 1.0 mM Cyclohexylamine (CHA, spermidine synthase inhibitor) had a strong inhibitory effect on the lettuce seed germination. The effect of polyamines imbibitions on the lettuce seedling development showed that 0.5 mM Spd could significantly promote the growth of root as the length and fresh weight increased, whereas Put had no obvious promotive effect on root development and Spm did not work. Differing from the root development, Put had a clear role in promoting the plant height and fresh weight of lettuce seedling stems and leaves, and treatments with Spd and Spm had no effect. It showed that treatments with CHA had little role in the plant height and the fresh weight of seedling stems and leaves, but had certain promotive effect on the root development. These results suggest that polyamines are involved in the lettuce seed germination and seedling development.Treatment with exogenous NO donor sodium nitroprusside (SNP) markedly promoted seed germination in a dose-dependent way with optimal concentration at 100μM, whereas 200μM SNP failed to increase germination. The time-course study showed that the promotive effect of 100μM SNP on the early germination of lettuce seed was obvious in 36 h after treatment, and this effect disappeared in 48 h, showing a similar effect to the exogenous Spd treatment. Treatment with SNP could promoted the development of the lettuce seedling lateral roots, and had inhibitory effect on the length of taproots, plant height, fresh weight of seedling stems and leaves to some extent.Application of the NO scavenger cPTIO to lettuce seed soaked with Spd or SNP could significantly reduce the Spd- or SNP-induced increment of seed germination. These findings suggest that Spd and SNP probably has a similar role in the process of promoting the early germination of lettuce seed, and it also indicates that the mechanism of the seed early germination may be relevant to the NO production.According to the principle that NOx, produced by NO and O2 reaction, can react with sulfanilic acid (SA) in the presence of N-naphthyl ethylenediamine hydrochloride (NEDD) at pH7.4, causing certain changes of the optical density at 496 nm, we investigated the A496 changes in extracting solutions after Spd and SNP soaking. The results showed that the A496 increased in both Spd and SNP treatments, while decreased after treatment with exogenous NO scavenger cPTIO. The NO fluorescence detected with DAQ, a NO-sensitive fluorescent probe, showed that both Spd and SNP treatments enforced NO fluorescence which mainly occurred near the embryonic part, and that 0.1 mM cPTIO could suppress Spd- or SNP-induced increment of NO fluorescence in seeds concomitantly with the inhibitory effect on the seed germination promoted by Spd and SNP.Nitric oxide synthase (NOS) and nitrate reductase are probably involved in the synthesis of NO in plant cells. Treatments with NOS inhibitor L-NAME and nitrate reductase inhibitor sodium tungstate (T-Na) showed that T-Na intensively inhibit the early germination of lettuce seed promoted by Spd, but there was no germination difference between Spd + T-Na and T-Na treatments. The effect of exogenous SNP on recovering T-Na-induced germination inhibition was not obvious while treated with Spd + T-Na. L-NAME inhibited the seed germination significantly but SNP could markedly recover this inhibition effect (p﹤0.05). The NR activity and enzyme-spectrum analyzing results showed that Spd less than 0.5 mM treatment had a significant role in promoting the activity of NR in lettuce seed, and the enzyme-spectrum broadened likewise.In conclusion, we believed that the polyamines especially spermidine might be related to the NO production in the process of promoting the early germination of lettuce seed, but the mechanism of Spd-induced NO formation needed more and further study.