Meng Xiaoying, Hua Zhihua, Wang Ning, Fields Allison M, Dowd Peter E, Kao Teh-hui
Intercollege Graduate Degree Program in Plant Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Sex Plant Reprod. 2009 Dec;22(4):263-75. doi: 10.1007/s00497-009-0114-3. Epub 2009 Sep 16.
The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature "S-RNase", the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S(2)-RNase, S(3)-RNase and non-glycosylated S(3)-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin-myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.
基于S-RNase的自交不亲和性（SI）的特异性由两个S位点基因控制，即雌蕊S-RNase基因和花粉S位点F-box基因。S-RNase在传递细胞中合成；其信号肽在分泌到传递通道的过程中被切除；而本研究的对象——成熟的“S-RNase”，通过一种尚不清楚的机制被生长中的花粉管吸收。吸收后，S-RNase在非自身（可亲和）和自身（不亲和）花粉管的液泡区室中被隔离，随后不亲和花粉管中该区室的破坏与SI反应的开始相关。然而，含有S-RNase的区室如何在不亲和花粉管中被特异性破坏尚不清楚。在这里，我们通过在转基因植物的自身（不亲和）和非自身（可亲和）花粉中直接表达融合有绿色荧光蛋白（GFP）的矮牵牛S(2)-RNase、S(3)-RNase和非糖基化S(3)-RNase，绕过了S-RNase的吸收步骤。我们发现，这些异位表达的S-RNase均未影响其自身或非自身花粉/花粉管的活力或SI行为。基于体外萌发花粉管的GFP荧光，所有这些S-RNase都在自身和非自身花粉管中被隔离。此外，含有S-RNase的区室在活花粉管中是动态的，其移动依赖于基于肌动蛋白-肌球蛋白的分子马达系统。所有这些结果表明，糖基化对于花粉管中表达的S-RNase的隔离不是必需的，并且花粉的细胞质是SI中S-RNase细胞毒性作用的位点。
