Biochemical Analysis Of Blue Light Inhibition Of Hypocotyl Elongation And Induction Of Seed Germination Of Arabidopsis

Cryptochromes are blue light receptors that regulate various photomophogenic responses in plant, including inhibition of hypocotyl elongation, stimulation of cotyledon expansion, and regulation of flowering time. It is not clear about how cryptochromes mediate blue light regulating photomophogenic responses, such as inhibition of hypocotyl elongation. To understand the underling mechanism, the wild-type and some blue light receptor mutants of Arabidopsis were used in this study, and they all are in the col background. In addition, there is no report about regulation of seed germination by blue light, so the effect of blue light on seed germination was also studied in this research. The results of this study were listed as follows:(1) We measured the hypocotyl of one T-DNA insertion mutant scc7-D(suppressors of cry1cry2 7-dominant)which grown under different fluence rates of blue, red, or far-red light. The results showed that scc7-D mutant is hypersensitive to not only blue light but also to red and far-red light, and scc7-D mutant suppressed the long-hypocotyl phenotype of the cry1cry2 parent when grown under blue light, and also suppressed the phenotype of wild-type when grown under red or far red light, but it showed a normally elongated hypocotyl when grown in the dark. Analysis of RT-PCR demonstrated that mRNA expression of GA2ox8 gene flanking the T-DNA insert was significantly increased. It has been shown that GA2ox8 encodes a GA 2β-hydroxidase that catalyzes inactivation of bioactive GA4 or its precursor. The short-hypocotyl phenotype of the scc7-D mutant can be completely reversed by including a bioactive GA (GA3) in the growth medium when grown under blue, red, or far-red light. GA2ox8 overexpressing lines (35S: GFP-GA2ox8-1 and 35S: GFP- GA2ox8-8) also showed a GA-rescuable hypersensitivity to light. We concluded that the short-hypocotyl phenotype of scc7-D mutant under different wavelengths of light was caused by the increased expression of GA2ox8 and reduced accumulation of bioactive GA4.(2) Response of hypocotyl of wild-type, and cry1cry2, cry1, cry2 mutant to bioactive GA3, GA4 or GA biosynthesis inhibitors, pacolobutrazol or ancymidol was tested in this study. The results showed that GA3 or GA4 failed to promote significant hypocotyl elongation in wild-type, and cry1cry2, cry1, cry2 seedlings. However, application of GA biosynthesis inhibitors, pacolobutrazol (10-2μM) or ancymidol (10-1μM) can significantly inhibit hypocotyls elongation of cry1cry2 and cry1, and hypocotyl elongation of cry2 was also inhibited slightly, and cry1cry2, cry1, cry2 seedlings exhibited short hypocotyls phenotype similar to wild-type grown under high fluence rate(20-100μmolm-2 s-1) of blue light . The growth inhibition by the GA inhibitors can be reversed by GA3, such that the long hypocotyl phenotype was restored by GA3 in the presence of GA inhibitor, but long hypocotyl phenotype was not observed in wild-type. These results demonstrated that long hypocotyl phenotype of cry1and cry1cry2 might be due to both elevated level of bioactive GA and enhanced GA transduction. In addition, response of phyB grown under red light and phyA mutants grown under far-red light to GA3, or GA biosynthesis inhibitors, pacolobutrazol, or pacolobutrazol plus GA3 at different concentration was similar to that of cry1 and cry1cry2 grown under blue light. This also demonstrated that long hypocotyl phenotype of phyB and phyA might be also due to both elevated level of bioactive GA and enhanced GA transduction.(3)We cooperated with James Reid's laboratory in Australia to measure GA4 content of wild-type and cry1cry2 mutant using GC-MS method. The results showed that the bioactive GA4 level in wild-type seedlings fall by three folds when it transferred from dark to blue light for 4 h. In contrast, there was no reduction of GA4 in cry1cry2 mutant seedlings. These results demonstrated that cryptochrome-mediated blue light-reduction of bioactive GA4 is responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light(4) Expression of GA2ox genes in wild-type, cry1, cry1cry2 and cry1cry2phyA mutant were analyzed using RT-PCR. Up to eight GA2ox genes in Arabidopsis, and expression of six genes of them were induced variously by blue light treatment except that little transcripts of GA2ox3 and GA2ox5 have been detected in this study. Blue light induced expression of all four GA2ox genes(GA2ox1, GA2ox2, GA2ox6, GA2ox8), especially GA2ox1 and GA2ox8 slightly decreased in cry1 mutant seedlings, but more significantly impaired in the cry1cry2 mutant, and the blue light-induced expression of GA2ox1 was almost completely abolished in the cry1cry2phyA mutant. These results demonstrated that cry1, cry2, and phyA act redundantly to activate expression of most of GA2ox genes to reduce GA4 accumulation. Therefore, cryptochrome-mediated blue light-induction of GA2ox gene expression is at least partially responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light. In addition, red light also variously induced expression of most of members of GA2ox gene family, and phyB and other phytochromes might act redundantly to mediate red light-induced expression of GA2ox genes.(5) There are five GA20ox genes(GA20ox1-5)in Arabidopsis. cry1, cry2, and phyA act redundantly mediating blue light inhibition of GA20ox1-3 genes expression variously. The other two genes can not be detected in this study because of their little expression in Arabidopsis. Expression of GA20ox1 was more significantly inhibited in response to blue light than that of GA20ox2 and GA20ox3. Therefore, cryptochrome-mediated blue light-reduction of GA20ox gene expression is also at least partially responsible for cryptochrome–dependent hypocotyl inhibition in response to blue light. In addition, expression of GA20ox1, GA20ox2and GA20ox3 were also inhibited variously by red light and far-red light treatment. phyB mainly mediated red light-reduced expression of GA20ox3, and phyA mainly mediated far-red light-reduced expression of GA20ox1, and phyB, phyA and other phytochromes might act redundantly to mediate red/far-red light-reduced expression of GA20ox2.(6) Overexpression of GA2ox8 gene suppressed light-induced gene expression of other GA2ox genes, and stimulated light induction of some GA20ox genes expression. Such interactions of expression between members of GA2ox gene family and between GA2ox genes and GA20ox genes may allow a more precise and subtle control of the homeostasis of bioactive GAs in response to light and other environmental fluctuations.(7) GA2ox1 and GA2ox2 genes expression was regulated by clock. The circadian rhythms of its expression peak were in the light (or subjective light) phase and the troughs of the rhythms were in the dark (or subjective dark) phase. The expression of GA2ox4 and GA2ox6 also showed circadian rhythm, but the oscillation was smaller than that of GA2ox1. The other two GA2ox genes, GA2ox7 and GA2ox8, showed a light-dependent diurnal rhythm. The amplitude of GA2ox1 expression is significantly diminished in the cry1cry2 mutant. It demonstrated that cryptochrome alone does play an important function in the regulation of GA2ox1 in seedlings grown in a white-light illuminated photoperiodic condition. These results also showed that circadian rhythmic expression might contribute to the transient light induction of the expression of most of the GA2ox genes to mediate light inhibition of hypocotyl elongation.(8)expression of GA20ox1 and GA20ox3 genes also showed circadian rhythm. Expression of GA20ox2 was regulated by clock when seedlings were grown under white light with long day (16h light/8h dark), but showed a light-dependent diurnal rhythm when seedlings were grown under white light with short day (8h light/16h dark). Cryptochrome mediated white light inhibition of GA20ox1 gene expression. (9) The seed germination of Arabidopsis was induced by blue light, and Cryptochrome mediated blue light induction of early seed germination in which seed germination rate were calculated when seeds were grown under blue light for less than three days. Cryptochrome-mediated blue light induction of seed germination was low fluence rates(0.1-10μmolm-2 s-1)-dependent. Response of wild-type and cry1cry2 mutant seed germination to GA inhibitor pacolobutrazol and ancymidol was investigated. Cry1cry2 mutant was more sensitive to GA inhibitor, and more GA3 was needed to recover inhibition of cry1cry2 by GA inhibitor. We concluded that cryptochrome-mediated increase of GAs synthesis might responsible for cryptochrome–dependent induction of seed germination in response to blue light.