Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase
Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase
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功能性nia基因数量减少的烟草突变体通过改变转录的昼夜调节、翻译后修饰和硝酸还原酶的周转来补偿
作者：
W. Scheible;A. González;R. Morcuende;M. Lauerer;M. Geiger;J. Glaab;A. Gojon;E. Schulze;M. Stitt
尽管硝酸还原酶（NR，EC www.example.com）被认为控制硝酸盐同化的速率，但具有野生型（WT）NR活性（NRA）的40 - 45%的突变体与WT生长一样快。研究了烟草（Nicotiana tabacumL.）CV. Gatersleben）突变体具有一个或两个而不是四个功能基因补偿。(i)突变体叶片中的niatranscript较高。然而，昼夜节律保留在突变体中，在夜晚结束时达到最大值，并在光周期期间强烈下降。(ii)WT叶片硝酸还原酶蛋白和硝酸还原酶活性在光照3 - 4 h后达到最大值，然后在光周期的后半部分和夜间的前半部分下降50 - 60%。光照3-4h后，突变体叶片中NR蛋白和NRA含量减少40-60%，但NR含量在光周期内没有下降。在光周期结束时，WT和突变体含有相似水平的NR蛋白和NRA。(iii)变暗导致WT和突变体中NR的快速失活。然而，在突变体中，这种失活在1 - 3小时黑暗后逆转。Calyculin A阻止了这种逆转。当在测定中包括镁以区分NR的活性和非活性形式时，突变体在夜间比WT含有多50%的活性。[15 N]-硝酸盐转化为有机化合物在叶中的第一个6小时的夜晚是60%的速度比在WT的突变体。(iv)WT植物在增强的二氧化碳中的生长阻止了光周期的第二部分期间NRA的下降，并导致NR在黑暗中的重新激活。(v)NR在光照下的稳定性增加和暗失活的逆转与叶片中谷氨酰胺水平的降低相关。在离体叶片中加入谷氨酰胺后，即使在光照下，也能加速NR的分解，导致NR失活。(vi)还研究了根的日变化。野生型中，光照4h后，nitransscript含量达到最大值，随后逐渐下降。根中NRA转录量的变化幅度小于叶，且无昼夜变化。在突变体中，niatranscript水平高，通过光周期和第一部分的夜晚。NRA在白天低50%，但在夜间上升到几乎与WT一样高的活动。[15 N]-硝酸盐同化率的突变体的根类似于在WT在第一个6小时的夜晚。(vii)还比较了具有非常低的NRA的Nia30（145）转化体和硝酸盐缺乏的WT植物中的昼夜变化。两组植物具有相似的低生长率。硝酸还原酶在Nia30（145）的叶或根中没有表现出昼夜节律，叶含有非常低的谷氨酰胺，NR在黑暗中不发光。每天用0.2mM硝酸盐浇灌硝酸盐缺乏的WT植物。浇水后，叶片中的总蛋白、NR蛋白和NRA含量出现一个小的高峰，随后谷氨酰胺和其它氨基酸含量出现短暂的增加。夜间谷氨酰胺低，NR不升高。在根中，有一个非常显着的增加硝酸盐，niatranscript和NRA 2 - 3小时后，每天浇水0.2 mM硝酸盐。（viii）得出结论，WT植物具有过量的硝酸盐同化能力。它们每天只在很短的时间内利用这种潜在的能力，然后根据条件下调硝酸盐同化反应，以积累硝酸盐同化产物或耗尽外部硝酸盐。具有较低硝酸盐同化能力的基因型通过增加NR和NR的表达来补偿。
Although nitrate reductase (NR, EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40–45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacumL. cv. Gatersleben) mutants with one or two instead of four functionalniagenes compensate. (i) Theniatranscript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3–4 h light in WT leaves, and then decreased by 50–60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40–60% less NR protein and NRA after 3–4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1–3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount ofniatranscript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants,niatranscript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak ofniatranscript, NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate,niatranscript and NRA 2–3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and …
DOI：
10.1016/0006-291x(89)92189-x
发表时间：
1989-03-31
影响因子：
3.1
作者：
ISHIHARA, H;MARTIN, BL;HARTSHORNE, DJ
通讯作者：
HARTSHORNE, DJ
DOI：
10.1073/pnas.89.5.1861
发表时间：
1992-03-01
影响因子：
11.1
作者：
CHENG, CL;ACEDO, GN;CONKLING, MA
通讯作者：
CONKLING, MA