Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, P.O. Box 1106, 06504, New Haven, CT, USA.
Planta. 1989 Aug;179(1):67-72. doi: 10.1007/BF00395772.
Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 μmol quanta·m(-2)·s(-1)) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO2. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO2), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed (14)CO2 at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of (14)CO2 or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.
设计了程序，用于从缺乏丝氨酸：乙醛酸氨基转移酶（SGAT；EC 2.6.1.45）活性的 Nicotiana sylvestris Speg. et Comes 的 sat 突变体原生质体细胞培养物中进行异养培养和自养建立。增加光子通量率（黑暗，40，80 μmol 量子·m(-2)·s(-1)) 可提高空气中和 1% CO2 中自养（无蔗糖）野生型（WT）培养物的生长速率。在抑制光呼吸的条件下（1% CO2），突变体培养物对光表现出相似的反应，并保持 WT 叶绿素水平的 65%。然而，在正常空气中，sat 培养物会发生严重的光呼吸毒性，生长速度非常缓慢，叶绿素迅速降至 WT 的 1%以下。低水平的蔗糖（0.3%）可完全逆转空气中突变细胞的光呼吸毒性。在空气中，突变体培养物保持 WT 叶绿素水平的 75%，显示出对光的生长刺激，并以与 WT 相同的速率固定（14）CO2。自养 sat 培养物在空气中积累丝氨酸，水平几乎是 WT 培养物的九倍。在空气中的混合营养（0.3%蔗糖）sat 培养物中，丝氨酸积累到相似的水平，但对（14）CO2 的固定或生长没有有害影响，表明高浓度的丝氨酸无毒，并且 sat 突变的毒性可能源于卡尔文循环中间体的消耗。自养 sat 培养物用于选择实验，旨在鉴定恢复在空气中生长能力的自发回复。从 678000 个 sat 菌落的群体中，回收了 23 个植物，它们在空气中持续生长是由于重新获得了 SGAT 活性。其中 22 个的 SGAT 水平在 WT 的 25%至 50%之间，但有一个的 SGAT 活性低于 WT 的 10%，最终表现出典型的 sat 突变体症状。讨论了自养 sat 培养物在选择减少核酮糖-1,5-二磷酸羧化酶/加氧酶（EC 4.1.1.39）加氧酶活性的叶绿体突变中的应用。
