Popper Zoë A, Fry Stephen C
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK.
Planta. 2008 Mar;227(4):781-94. doi: 10.1007/s00425-007-0656-2. Epub 2007 Nov 7.
We tested two hypotheses for the mechanism by which xyloglucan-pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using alpha-(1-->5)-L-[(3)H]arabino- or beta-(1-->4)-D-[(3)H]galacto-oligosaccharides as model acceptor substrates. The (3)H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [(3)H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [(3)H]Xyloglucan-pectin complexes were detectable <4 min after [(3)H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan-pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [(3)H]xyloglucan that was anionic remained almost constant at approximately 50% for > or =6 days, showing that the xyloglucan-pectin bond was stable in vivo. Some [(3)H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only approximately 30% of the sloughed [(3)H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that approximately 50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan-pectin bonds are stable and the pectic moiety aids wall-assembly.
我们针对拟南芥细胞培养物中木葡聚糖 - 果胶共价键形成的机制测试了两种假说。假说1提出了以木葡聚糖作为供体底物、鼠李半乳糖醛酸聚糖-I（RG-I）侧链作为受体的异源转糖基化作用。我们使用α-(1→5)-L-[(3)H]阿拉伯糖基或β-(1→4)-D-[(3)H]半乳糖基寡糖作为模型受体底物，寻找催化此反应的酶活性。将(3)H-寡糖（添加或不添加木葡聚糖）提供给活的拟南芥细胞培养物、透化细胞、无细胞提取物或四种纯的木葡聚糖内转糖基酶（XTH）。未发生异源转糖基化作用。因此，我们无法支持假说1。假说2提出一些木葡聚糖是作为RG-I上的侧链从头合成的。为了对此进行测试，我们用[(3)H]阿拉伯糖对拟南芥细胞培养物进行脉冲标记，并监测阴离子型（与果胶结合的）木葡聚糖的放射性标记情况，通过离子交换色谱法将其与游离木葡聚糖分离。在添加[(3)H]阿拉伯糖后不到4分钟就可检测到[(3)H]木葡聚糖 - 果胶复合物，这比多糖分泌的转运时间短，表明木葡聚糖 - 果胶键是在原生质体内形成的。此后，与细胞壁结合的[(3)H]木葡聚糖中阴离子型的比例在≥6天的时间内几乎保持恒定在约50%，表明木葡聚糖 - 果胶键在体内是稳定的。一些[(3)H]木葡聚糖迅速脱落到培养基中而不是与细胞壁结合。脱落的[(3)H]木葡聚糖中只有约30%是阴离子型 的，这表明与果胶结合促进了木葡聚糖整合到细胞壁中。我们得出结论，培养的拟南芥细胞中约50%的木葡聚糖是在果胶引物上合成的，然后分泌到质外体中，在那里木葡聚糖 - 果胶键是稳定的，并且果胶部分有助于细胞壁组装。
