Calcium, Reactive Oxygen Species, and Pollen Tube Growth

[00:87517]近年来，对花粉管生长的研究取得了长足的进展，越来越多的尖端技术被用于研究这一问题。一个重要的结论是花粉管尖端是一个高度动态的结构。伸长不是稳定的，而是振荡的，许多重要的生理变量也是振荡的，包括胞质Ca2+、胞质pH、净Ca2+内流、净H+内流和净K+内流。这些变量相对于生长振荡的相位关系是复杂的，振荡是如何建立和维持的，以及连接变量的反馈机制是什么，都不清楚。建议直接检测囊泡分泌并确定其相对于伸长的时间。用电学方法测量分泌物的尝试被证明是无效的，所以现在采用光学方法。通过这些方法，可以精确测量囊泡分泌的时间。胞质内Ca2+振荡所需的大部分Ca2+必须来自细胞内储存，最可能的来源是分泌囊泡本身。建议分离囊泡并直接测定其吸收和释放Ca2+的能力。这些事件的生理控制也将被确定。为了表征花粉管中可能存在的Ca2+通道，将对花粉粒原生质体进行全细胞夹持。工作模型假定存在膨胀压力振荡，这似乎是生长振荡的基础。计算机视觉的超高分辨率光学测量将用于确定花粉管直径是否因膨胀振荡而振荡。如果是这样，我们就可以根据其他变量确定这些振荡的相位。最后，将使用发光探针pholasin直接观察生长尖端ROS的产生，在ROS存在下，pholasin的发光量增加了几个数量级。

0087517RobinsonThe study of tip growth in general and pollen tube growth in particular has been an area of considerable progress in recent years and more and more sophisticated techniques have been brought to bear on the problem. One important conclusion that has emerged is that the pollen tube tip is a highly dynamic structure. Elongation is not steady, but oscillatory, and a number of important physiological variables are also oscillatory, including cytosolic Ca2+, cytosolic pH, net Ca2+ influx, net H+ influx, and net K+ influx. The phase relationships of these variables with respect to growth oscillations are complex, and it is not at all clear how the oscillations are established and maintained, and what are the feedback mechanisms that connect the variables.It is proposed to directly detect vesicle secretion and determine its timing relative to elongation. Attempts to use electrical methods for measuring secretion have proved fruitless, so optical methods are now employed. By these means, accurate measurements of the timing of vesicle secretion will be made.A substantial fraction of the Ca2+ that is required for the oscillations in cytosolic Ca2+ must come from intracellular stores, and the most likely sources are the secretory vesicles themselves. It is proposed to isolate the vesicles and directly determine their ability to take up and release Ca2+. The physiological controls of these events will also be determined.In order to characterize putative Ca2+ channels in pollen tubes, whole cell clamping of pollen grain protoplasts will be done. The working model posits the existence of turgor pressure oscillations, which appear to underlie the growth oscillations. Ultra-high resolution optical measurement by computer vision will be used to determine if the diameter of pollen tubes oscillates due to turgor oscillations. If so, we will determine the phase of these oscillations with respect to other variables.Finally, the production of ROS at the growing tip will be directly observed, using a luminescent probe, pholasin, which increases its light emission by several orders of magnitude in the presence of ROS.