Bologa Karin L, Fernie Alisdair R, Leisse Andrea, Loureiro Marcello Ehlers, Geigenberger Peter
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany.
Plant Physiol. 2003 Aug;132(4):2058-72. doi: 10.1104/pp.103.022236.
Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-beta-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of (14)C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.
植物拥有两条用于蔗糖（Suc）降解的替代生化途径。一条途径是通过转化酶水解，随后经己糖激酶和果糖激酶磷酸化；另一条途径是植物特有的，涉及由蔗糖合酶（SuSy）催化的依赖UDP的蔗糖裂解。在本研究中，我们直接测试了通过异位过表达转化酶或蔗糖磷酸化酶绕过内源性SuSy途径是否会影响生长中的块茎内部的氧气水平，以及这是否是其淀粉含量降低的原因。（a）转基因块茎内的氧气张力低于野生型块茎。在块茎组织的前10毫米内氧气张力降低，且这种梯度在转基因块茎中更陡。（b）过表达转化酶的块茎中甘油醛-3-磷酸脱氢酶、乳酸脱氢酶和乙醇脱氢酶的活性更高，（c）乳酸水平也更高。（d）与野生型背景相比，在过表达转化酶的背景下，低氧敏感的Adh1-β-葡萄糖醛酸酶报告基因构建体的表达诱导更强。（e）完整的转基因块茎的ATP与ADP比值低于野生型。当转基因块茎切片在21%（v/v）氧气中孵育时，ATP与ADP比值恢复到野生型水平。（f）淀粉含量从块茎的外围向中心降低。在转基因品系中这种降低更为明显，导致淀粉含量降低，尤其是在块茎中心附近。（g）更详细地分析了代谢通量（基于（14）C-葡萄糖的重新分布）和ATP与ADP比值，将在不同外部氧气张力（0%、1%、4%、8%、12%和21%[v/v]）下孵育的切片与完整块茎进行比较。表达蔗糖磷酸化酶的品系的切片在高氧条件下具有与野生型相似的ATP与ADP比值，淀粉合成速度也与野生型一样快，但在完整块茎内部典型的低氧张力下，其ATP与ADP比值较低，淀粉合成速率也低于野生型。（h）在野生型块茎切片中，低于环境的氧气浓度导致特定SuSy基因的mRNA水平选择性增加，而编码液泡和质外体转化酶的基因的mRNA水平降低。（i）这些结果表明，在生长中的块茎中，抑制转化酶并通过SuSy提供的能量成本较低的途径动员蔗糖很重要，因为它能保存氧气，并使内部氧气张力维持在比其他情况更高的水平。
