探討硝酸鹽調控開花的路徑

Abstract

硝酸鹽不僅是植物的主要氮源，更是調節開花的重要信號分子。硝酸鹽轉運蛋白NRT1.13是一個硝酸鹽感應蛋白，可在低硝酸鹽條件下促進開花。此外，在酵母菌雙雜合試驗中，它與硝酸鹽信號相關的蛋白：磷酸激酶CIPK8、CIPK23和轉錄因子NLP2、NLP7產生交互作用。為了探討硝酸鹽如何及何時參與開花，我們在正常以及低硝酸鹽條件下觀察野生型、nrt1.13、cipk8、cipk23、nlp2和nlp7的開花表型以及幼年期至成年期的轉換時間。結果顯示在低硝酸鹽條件下，野生型在藉由延長幼年期而延遲開花，nrt1.13和nlp7則是透過延長成年期來影響開花。cipk8和nlp2雖然提早開花但他們的開花時間不受硝酸鹽所調控，而cipk23的開花時間則與野生型無異。 此外，為了了解硝酸鹽所調控的開花路徑，我們在nrt1.13，野生型和nlp7中分析已知開花相關基因，例如FLC，FT和SPL3的表現情形。結果顯示，FLC 在nrt1.13葉片及莖頂分生組織SAM的表現量均增加。值得注意的是，在nrt1.13葉片中，FLC受低硝酸鹽的誘導更為顯著。這表示NRT1.13可以在葉片中監測硝酸鹽含量，並通過抑制葉片中的FLC來促進開花。 在低硝酸鹽環境下的野生型葉片中，FLC表現量上升，而FT表現量下降。然而，FT的反應早於FLC。這表示除了NRT1.13所調控的路徑外，低硝酸鹽也透過其他未知的路徑抑制FT並延遲開花。 在低硝酸鹽條件下的nlp7中，我們並沒有找到在葉片中被調控的基因，但在莖頂分生組織SAM中，我們發現SPL3表現量上升的速度變慢，此現象合乎nlp7只在低硝酸鹽條件下延後開花的表現型。這表示NLP7可能透過加速表現莖頂分生組織SAM中SPL3表現而促進開花，進而對低硝酸鹽環境做出適當反應。 在本篇研究中，我們發現硝酸鹽透過NRT1.13和NLP7調節開花，在此之外仍有其他未知途徑參與其中，這也反映出硝酸鹽調控的開花機制是相當複雜的。

Nitrate is not only a major N source of plants but also a vital signaling molecule regulating flowering. NRT1.13 is a putative nitrate sensor and promotes flowering at low nitrate. Yeast-two hybrid assays showed that NRT1.13 interacts with CIPK8, CIPK23, NLP2 and NLP7, the regulators in nitrate signaling pathway. To verify how and when nitrate participates in flowering control, the flowering phenotype and vegetative phase change of wild type, nrt1.13, cipk8, cipk23, nlp2 and nlp7 were examined under normal or low nitrate conditions. The results show that wild type delays flowering at low nitrate through prolonging juvenile stage. Compared to wild type, nrt1.13 and nlp7 delay flowering especially at low nitrate through prolonging adult stage. However, the flowering of cipk8 and nlp2 are earlier but nitrate-independent, while flowering of cipk23 is similar to wild type. In addition, we monitored temporal and spatial expression changes of floral-related genes in nrt1.13, wild type and nlp7 to uncover the nitrate-regulatory pathways. The results show that compared to wild type, FLC expression increased in nrt1.13 in both leaf and SAM. It is worth noting that in leaf, the increase at low nitrate was more obvious in nrt1.13. This suggests that NRT1.13 could monitor low nitrate then promotes flowering through repressing FLC expression in leaf. In wild type, FLC expression was up-regulated and FT expression was down-regulated in leaf under low nitrate condition; however, the major difference of FT expression was previous to that of FLC expression. This implied that apart from NRT1.13-dependent pathway, low nitrate postpones flowering through other unidentified pathways, which repressing FT expression in leaf. In nlp7, expression of the genes examined show no differences in leaf. In SAM, the ascending rate of SPL3 became slow under low nitrate condition, corresponding to the nitrate-dependently late flowering phenotype. This suggests that NLP7 responds to low nitrate then promotes flowering through accelerates SPL3 expression in SAM. In this study, we found that nitrate regulates flowering through NRT1.13-dependent and NLP7-dependent pathways; behind these, there are still other unidentified pathways. This demonstrates that nitrate regulates flowering through multiple mechanism.