Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase
Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase
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DOI：
10.1073/pnas.1200352109
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发表时间：
2012-03-13
影响因子：
11.1
通讯作者：
DeBolt, Seth
中科院分区：
文献类型：
--
作者：
Harris, Darby M.;Corbin, Kendall;DeBolt, Seth
植物中纤维素生物合成的潜在机制是复杂的，并且仍然知之甚少。一个中心问题涉及微纤维结构的机制，以及这是如何连接到纤维素合成酶（CESA）的催化聚合作用。此外，目前还不清楚纤维素微纤维结构的修饰是否可以通过遗传实现，这在生物基经济中可能是变革性的。为了在植物中探索这些过程，我们开发了一个化学遗传工具箱，其中包含拟南芥中CESA1（A903V）和CESA3（T942I）的C-末端跨膜结构域区域中的药理学抑制剂和相应的抗性赋予点突变。使用C-13固态核磁共振光谱和X-射线衍射，我们表明，纤维素微纤维显示减少的宽度和一个额外的纤维素C4峰指示的结晶度是中间的表面和内部的野生型葡聚糖，表明在微纤维形成过程中的葡聚糖链协会的差异。与较低微原纤维结晶度的测量一致，来自突变CESA 1（A903V）和CESA 3（T942I）的纤维素提取物显示出比野生型更高的糖化效率。使用活细胞成像来跟踪荧光标记的CESA，我们发现这些突变体在质膜中显示出增加的CESA速度，这表明聚合速率增加。总的来说，这些数据表明CESA 1（A903V）和CESA 3（T942I）在结晶度方面具有改性的微原纤维结构，并表明在植物中，如在细菌中，结晶在生物药理学上限制聚合。
The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using C-13 solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.