Hetero-trans-b-glucanase (HTG), a unique cell-wall remodelling enzyme from Equisetum: action and potential to enhance mechanical properties of cereals

背景和目的我们最近发现了一种独特的酶（HTG或异反式-β-葡聚糖酶），仅在一组非开花植物中发现，即木贼。开花植物缺乏HTG，即使它们的细胞壁含有链状分子，至少在试管中，HTG可以切割和重新连接。我们现在的目标是发现（a）HTG对马尾植物有什么好处，（B）HTG可以实现的“切割和重新连接”反应的全部范围，（c）当HTG从马尾人工转移到作物植物时会发生什么。我们预测，马尾酶将赋予开花作物，例如小麦，以迄今为止只有马尾才能获得的方式加强其茎的能力。这种作物可以获得更好的抗倒伏性（风暴损害）。值得注意的是，马尾HTG是已知的唯一一种来自任何生物的酶，可以“切割和重新连接”纤维素分子，纤维素是植物细胞壁的主要成分。它可以将纤维素链嫁接到另一种称为木葡聚糖的细胞壁构建材料上。HTG还可以将第三种这样的材料（MLG或混合键葡聚糖）的链接枝到木葡聚糖上。因此，HTG可以产生纤维素-木葡聚糖键和MLG-木葡聚糖键。由此产生的“混合”聚合物被认为可以加强马尾。我们将发现HTG产生的确切时间和地点，以及这种连接的形成，在马尾。这可能会给HTG的自然作用提供线索。我们还将发现HTG在试管中与多种植物细胞壁多糖混合时可以催化哪些新反应。这可以提供新的“混合”聚合物，当按比例放大时，其可以是具有商业价值的新材料。为了进一步我们的HTG的基本知识，我们还将调查哪些酶的氨基酸是重要的，其能力（在试管中）重新配置纤维素和MLG。该项目的一个主要部分涉及人工引入马尾的HTG活动到开花植物，包括双子叶植物和谷物，并测量的后果。我们的工业合作伙伴（拜耳作物科学）将在小麦方面开展这项工作。我们预测，以这种方式遗传转化的任何作物植物将能够在其细胞壁中产生纤维素-木葡聚糖键，并且谷物（与双子叶植物不同，其具有MLG以及纤维素和木葡聚糖）将另外能够产生MLG-木葡聚糖键。我们将通过实验来检验这些预测。我们还将测试HTG赋予的开花植物是否更强壮，以及它们是否改变了形状或大小。我们将通过测量使植物茎弯曲或折断所需的力来量化植物的机械强度。植物细胞壁分子结构的任何变化都可能影响其生长和强度，因为细胞壁在决定这些特征方面发挥着关键作用。项目的受益者具有较强的茎的谷物品种通常遭受较少的倒伏，但这种强化通常是通过植物生长较厚的茎以较低的谷物产量为代价来实现的。在不显著增加茎生物量的情况下，给予谷物HTG可在细胞壁中形成新的聚合物间键，并赋予类似的强化，从而不损害收获。以这种方式改变谷物将使植物育种者和农民以及公众受益，因为随着风暴和暴雨变得更加频繁，气候变化会提高谷物生产的可靠性。此外，随着对HTG重新配置生物质材料，特别是纤维素（世界上最丰富的有机物质）的能力的了解不断增加，生物技术专家有了通过无污染的“绿色”工艺创造新材料（例如，用于专业论文和医疗应用）的新机会。

BACKGROUND AND PURPOSE We recently discovered a unique enzyme (HTG or hetero-trans-b-glucanase), found only in a group of non-flowering plants, the horsetails. Flowering plants lack HTG even though their cell walls contain the chain-like molecules which, at least in the test-tube, HTG can cut and re-join. We now aim to discover (a) what good HTG does horsetail plants, (b) the full range of 'cutting and re-joining' reactions that HTG can achieve, (c) what happens when HTG from horsetails is artificially transferred to crop plants. We predict that the horsetail enzyme will endow flowering crops, e.g. wheat, with the ability to strengthen their stems in a manner hitherto only available to horsetails. Such crops may acquire improved resistance to lodging (storm damage). OBJECTIVES AND EXPECTED OUTCOMESRemarkably, horsetail HTG is the only known enzyme from any living thing that can 'cut and re-join' molecules of cellulose, the major constituent of plant cell walls. It can graft a cellulose chain onto a chain of a different cell-wall building material called xyloglucan. HTG can also graft chains of a third such material (MLG or mixed-linkage glucan) onto xyloglucan. HTG can thus create cellulose-to-xyloglucan and MLG-to-xyloglucan linkages. The resulting 'hybrid' polymers are thought to strengthen horsetails. We will discover exactly when and where HTG is produced, and such linkages are formed, in horsetails. This will potentially give clues about HTG's natural roles. We will also discover what new reactions HTG can catalyse when mixed in the test-tube with diverse plant cell-wall polysaccharides. This may afford new 'hybrid' polymers, which when scaled up may be commercially valuable new materials. To further our fundamental knowledge of HTG, we will also investigate which of the enzyme's amino acids are important for its ability (in the test-tube) to re-configure cellulose and MLG. A major part of this project involves artificially introducing the horsetail's HTG activities into flowering plants, including both dicotyledons and cereals, and measuring the consequences. Our industrial collaborators (Bayer CropScience) will do this work in the case of wheat. We predict that any crop plants genetically transformed in this way will be able to create cellulose-to-xyloglucan linkages in their cell walls, and that cereals (which, unlike dicots, possess MLG as well as cellulose and xyloglucan) will in addition be able to make MLG-to-xyloglucan linkages. We will test these predictions experimentally. We will also test whether the HTG-endowed flowering plants are stronger, and whether they have an altered shape or size. We will quantify the plants' mechanical strength by measuring the force required to bend or break their stems. Any changes to the molecular architecture a plant's cell walls are likely to affect its growth and strength because of the pivotal roles that cell walls play in dictating these features. BENEFICIARIES OF THE PROJECTCereal varieties with stronger stems often suffer less lodging, but such strengthening is usually achieved by the plant growing thicker stems at the expense of lower grain yield. Artificially giving cereals HTG may form novel inter-polymer linkages in the cell wall and confer similar strengthening without significant increases in stem biomass and thus without compromising the harvest. Modifying cereals in this way would benefit plant breeders and farmers, as well as the general public, by improving the reliability of grain production in a changing climate as storms and heavy rains become more frequent. In addition, increasing knowledge of HTG's ability to reconfigure biomass materials, especially cellulose (the world's most abundant organic substance), offers biotechnologists novel opportunities to create new materials (e.g. for specialist papers and medical applications) via non-polluting 'green' processes.