McCarthy Avery, Chung Michelle, Ivanov Alexander G, Krol Marianna, Inman Michael, Maxwell Denis P, Hüner Norman P A
Department of Biology and the Biotron Centre for Experimental Climate Change Research, University of Western Ontario, London, ON, N6A 5B7 Canada,.
J Plant Physiol. 2016 Jul 20;199:40-51. doi: 10.1016/j.jplph.2016.05.008. Epub 2016 May 19.
An established cell suspension culture of Arabidopsis thaliana var. Landsberg erecta was grown in liquid media containing 0-15%(w/v) sucrose. Exponential growth rates of about 0.40d were maintained between 1.5-6%(w/v) sucrose, which decreased to about 0.30d between 6 and 15%(w/v) sucrose. Despite the presence of external sucrose, cells maintained a stay-green phenotype at 0-15% (w/v) sucrose. Sucrose stimulated transcript levels of genes involved in the chlorophyll biosynthetic pathway (ChlH, ChlI2, DVR). Although most of the genes associated with photosystem II and photosystem I reaction centers and light harvesting complexes as well as genes associated with the cytochrome b6f and the ATP synthase complexes were downregulated or remained unaffected by high sucrose, immunoblotting indicated that protein levels of PsaA, Lhcb2 and Rubisco per gram fresh weight changed minimallyon a Chl basis as a function of external sucrose concentration. The green cell culture was photosynthetically competent based on light-dependent, CO-saturated rates of O evolution as well as Fv/Fm and P700 oxidation. Similar to Arabidopsis WT seedlings, the suspension cells etiolated in the dark and but remained green in the light. However, the exponential growth rate of the cell suspension cultures in the dark (0.45±0.07d) was comparable to that in the light (0.42±0.02d). High external sucrose levels induced feedback inhibition of photosynthesis as indicated by the increase in excitation pressure measured as a function of external sucrose concentration. Regardless, the cell suspension culture still maintained a stay-green phenotype in the light at sucrose concentrations from 0 to 15%(w/v) due, in part, to a stimulation of photoprotection through nonphotochemical quenching. The stay-green, sugar-insensitive phenotype of the cell suspension contrasted with the sugar-dependent, non-green phenotype of Arabidopsis Landsberg erecta WT seedlings grown at comparable external sucrose concentrations. It appears that the commonly used Arabidopsis thaliana var. Landsberg erecta cell suspension culture has undergone significant genetic change since its original generation in 1993. We suggest that this genetic alteration has inhibited the sucrose sensing/signaling pathway coupled with a stimulation of chlorophyll an accumulation in the light with minimal effects on the composition and function of its photosynthetic apparatus. Therefore, caution must be exercised in the interpretation of physiological and biochemical data obtained from experimental use of this culture in any comparison with wild-type Arabidopsis seedlings.
将拟南芥Landsberg erecta变种的已建立细胞悬浮培养物在含有0 - 15%（w/v）蔗糖的液体培养基中培养。在1.5 - 6%（w/v）蔗糖之间维持约0.40 d的指数生长速率，在6 - 15%（w/v）蔗糖之间降至约0.30 d。尽管存在外部蔗糖，但细胞在0 - 15%（w/v）蔗糖下维持绿持型表型。蔗糖刺激了参与叶绿素生物合成途径的基因（ChlH、ChlI2、DVR）的转录水平。尽管与光系统II和光系统I反应中心以及光捕获复合体相关的大多数基因以及与细胞色素b6f和ATP合酶复合体相关的基因在高蔗糖条件下被下调或未受影响，但免疫印迹表明，以叶绿素为基础，每克鲜重的PsaA、Lhcb2和Rubisco蛋白水平随外部蔗糖浓度的变化最小。基于光依赖的、CO饱和的O2释放速率以及Fv/Fm和P700氧化，绿色细胞培养物具有光合活性。与拟南芥野生型幼苗相似，悬浮细胞在黑暗中黄化，但在光照下保持绿色。然而，细胞悬浮培养物在黑暗中的指数生长速率（0.45±0.07 d）与在光照下的指数生长速率（0.42±0.02 d）相当。如以外部蔗糖浓度为函数测量的激发压力增加所示，高外部蔗糖水平诱导光合作用的反馈抑制。尽管如此，细胞悬浮培养物在0至15%（w/v）蔗糖浓度下在光照下仍维持绿持型表型，部分原因是通过非光化学猝灭对光保护的刺激。细胞悬浮培养物的绿持型、糖不敏感表型与在相当外部蔗糖浓度下生长的拟南芥Landsberg erecta野生型幼苗的糖依赖型、非绿色表型形成对比。自1993年最初传代以来，常用的拟南芥Landsberg erecta变种细胞悬浮培养物似乎经历了显著的遗传变化。我们认为这种遗传改变抑制了蔗糖感知/信号传导途径，同时在光照下刺激了叶绿素的积累，对其光合装置的组成和功能影响最小。因此，在将从该培养物的实验使用中获得的生理和生化数据与野生型拟南芥幼苗进行任何比较时，必须谨慎解释。
