Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion.
Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion.
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DOI：
10.1111/tpj.12403
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Fry SC
中科院分区：
文献类型：
--
作者：
Chormova D;Messenger DJ;Fry SC
细胞壁果胶结构域鼠李糖半乳糖醛酸-II(RG-II)通过硼酸二酯桥进行交联，影响细胞壁的膨胀、厚度和孔隙率。以前，人们对这种交联体的机制或亚细胞位置知之甚少。用聚丙烯酰胺凝胶电泳法(PAGE)分离单体和二聚体(含硼)RG-II，我们证实了Pb2+在体外促进了H3BO3依赖的二聚反应。高达50 mM的H3BO3浓度并不能阻止交联。在体内实验中，我们成功地培养了‘Paul’s Scarlet‘月季(Rosa sp.)无硼培养液中的细胞：其壁上结合的果胶含有单体RG-II结构域，但没有可检测到的二聚体。因此，含有RG-II结构域的果胶可以被固定在壁上而不是通过硼桥。在3.3μm中重新加入H3BO3可在24小时内逐渐出现RG-II二聚体，但没有检测到现有单体的损失，这表明只有新合成的RG-II才能发生硼桥联。与此一致的是，其多糖生物合成机制受到损害(碳饥饿、呼吸抑制、厌氧、冷冻或煮沸)的玫瑰培养物失去了产生RG-II二聚体的能力。我们得出结论，RG-II通常在合成或分泌过程中变得与硼桥联，但不是在分泌后。支持这一结论的是，当加入ROSA培养时，外源[~3H]RG-II既没有在介质中二聚化，也没有与现有的壁相关RG-II结构域交联。总之，在培养的ROSA细胞中，RG-II结构域有一个短暂的机会窗口，可以在原生质内或分泌过程中进行硼桥接，但分泌到质外体是一个无法返回的点，超过这个点就不容易发生额外的硼桥接。
The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb2+ promotes H3BO3-dependent dimerisation in vitro. H3BO3 concentrations as high as 50 mm did not prevent cross-linking. For in-vivo experiments, we successfully cultured ‘Paul's Scarlet’ rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3BO3 to 3.3 μm triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [3H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur.
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