?假彩色图像可以显示源自拟南芥表皮的单个细胞。已经标记了荧光的细胞被设计用来追踪葡萄糖的分布。来自Carnegie植物分子生物学系的科学家首次在完好无损的活体植物组织中实时观察糖的利用。在全新的成像技术的协助下，该研究小组已经观察到植物根部维持极低浓度的糖，比先前估计的要低100,000倍。这项新技术将可以开辟在植物中研究糖代谢方面的研究，从而为在提高食品和生物燃料产量方面的工程改造提供更好的保障。
??在来自Carnegie研究机构成员Wolf Frommer领导下，研究人员通过遗传改造来设计一种能编码荧光标签来监测在模式植物拟南芥根叶中的葡萄糖浓度。这项技术史无前例地可以避免时间和空间的局限，而直接追踪活体原状的植物组织中葡萄糖。该研究发表在九月份国际顶尖杂志《植物细胞》上。 该研究小组同时也发明了一种针对蔗糖的荧光共振能量转移传感器。这个研究工作将发表在九月份这期《生物化学》杂志上。
??Frommer认为：“迄今为止，我们已经掌握一些线索，是关于在多细胞植物中单个细胞中糖的数量。我们通常将植物的根和叶碾成粉末，然后平均所有细胞的成份，但是如果在单个细胞糖水平升高和降低，我们将从平均水平上是看不出来的。还有单个细胞在亚细胞水平上糖分布存在差异，所以在特定时间段几乎不可能知道在亚细胞结构上的糖水平”。
??Frommer同时还强调：“时间分辨率是另一个瓶颈，我们间隔时间段采集组织样品，但如果糖含量在波动，我们可能就错过最佳的时间点。我们最近发明的技术可以避免在单细胞水平实时监测糖流量时面临的所有瓶颈，以及可以解决有关亚细胞水平分辨率的问题”。
??Frommer和他的同事已经使用拟似于荧光共振能量转移(FRET)传感器样的成像标签，来追踪动物细胞中糖和神经递质。最近，FRET传感器的课题组使用该技术研究谷氨酸盐（一种重要的哺乳动物神经递质）。Frommer已经在培养的哺乳动物细胞内追踪葡萄糖，但是到现在为止，在植物组织上证明还是有问题的，主要是来自植物病毒防疫机制以及在一些植物组织中高背景荧光的干扰。
??为了克服这些障碍，Frommer课题组对此传感器做了经典的改善，将它们插入到没有防卫基因的拟南芥突变体上，此时的荧光标签可以避免先前的弊端发挥更好的功效。
??Frommer解释：“这可能不是理想的，因为只能用在防卫突变体的植株上。最优的应该是这些传感器能在任何野生型植株的遗传背景下发挥功效。现在我们开始找到关于植物处理糖途径的重要障碍，我们将继续改善此传感器以保证其发挥功效”。
??Frommer谈到：“这项技术的中心就在于巧妙而简单。通过计算机的模拟设计，我们可能设计FRET标签来活灵活现地追踪活细胞中任何小分子。象这样的成像技术在有关代谢方面的研究有着广泛的应用前景，同时也将帮助我们回答压在植物学家心头一些最紧迫的问题，例如在糖分布过程中单个基因发挥什么样的功能。依次这项技术将有助于我们改造植物从而提高产量”。
英文原文：
Sugar metabolism tracked in living plant tissues, in real time
This false-color image shows a cell from the epidermis of an Arabidopsis thaliana plant marked with fluorescent imaging sensors designed to detect the sugar glucose. (Click image for full caption and credit info.)
Scientists at Carnegie’s Department of Plant Biology have made the first real-time observations of sugars in the cells of intact and living plant tissues. With the help of groundbreaking imaging techniques, the group has determined that plants maintain extremely low levels of sugar in their roots—as much as 100,000 times lower than previous estimates. The new technology will enable new studies of sugar metabolism in plants, which will inform the effort to engineer higher crop yields for food and biofuel production.
Led by Carnegie staff member Wolf Frommer, the researchers designed genetically-encoded fluorescent tags to monitor glucose, an important sugar, in leaf and root tissues of the model plant Arabidopsis thaliana. The technique has allowed the researchers to track glucose over time and space at unprecedented detail, in living and undisturbed plant tissues. The work appears in the September issue of the journal Plant Cell*. The group has also developed a FRET sensor for sucrose, a major transport sugar in plants. This work will appear in the September issue of the Journal of Biological Chemistry**.
“Until now, we have had few clues regarding how much sugar is in an individual cell in a multicellular plant,” Frommer said. “We normally grind up a leaf or a root and average the information for all cells, but if sugar levels rise in one cell and drop in another, we would see no change in this average.” Also, because the cell can distribute sugar among subcellular organelles, it is nearly impossible to know how much sugar is in any cell compartment at a given time.
“Time resolution is another problem,” Frommer added. “We can sample tissue at intervals, but if the sugar changes in waves, we might miss the right time point. Our new technology addresses all of these problems by measuring sugar flux in real time in individual cells, with subcellular resolution.”
Frommer and his colleagues have used similar imaging tags, called fluorescent resonance energy transfer (FRET) sensors, to track sugars and neurotransmitters in animal cells. Most recently, the group used FRET sensors to study glutamate, an important mammalian neurotransmitter. Frommer has tracked glucose in cultured mammalian cells, but until now, plant tissues had proven problematic because of interference from the plants’ virus defense mechanisms, as well as high background fluorescence in some plants.
To surmount these issues, Frommer’s team dramatically improved the sensors, while inserting them in mutant Arabidopsis plants with disabled defense genes. The fluorescent tags worked well where they had failed before.
“It may not be ideal to use defense-mutant plants—the ideal would be for the sensors to work in any wild-type genetic background,” Frommer explained. “But proving that the sensors can work in plants is an important first step. Now we can begin addressing important questions about the way plants manage sugar distribution while we continue to improve the sensors.”
In preliminary experiments, Frommer’s group compared fluctuations in glucose levels in root tissue and leaf epidermis—the topmost layer that absorbs sunlight—and found that the plant maintained glucose at higher levels in leaf tissue than in roots. In fact, the researchers found that root cells contain sugar at concentrations at least 100,000 times lower than previous estimates.
FRET sensors are encoded by genes that, in theory, can be engineered into any cell line or organism. They are made of two fluorescent proteins that produce different colors of light—one cyan and one yellow—connected by a third protein that resembles a hinged clam shell. The two fluorescent proteins are derived from jellyfish, and the third from a bacterium; the shape of the clam shell protein determines which sugar or other molecule the sensor can detect. When a target molecule such as glucose or sucrose binds to the third protein, the hinge opens, changing the distance and orientation of the fluorescent proteins. This physical change affects the energy transfer between the cyan and yellow markers.
When the researchers hit the tags with light of a specific wavelength, the cyan tag starts to fluoresce. If the yellow tag is close enough, the cyan tag will transfer its energy to the yellow tag, causing it to resonate and fluoresce as well. This energy transfer affects how much cyan and yellow fluorescence can be seen, and by calculating this ratio, researchers can accurately track molecules such as glucose and sucrose in both time and space.
“The strength of this technology lies in its elegant simplicity; with the power of computational design, we can potentially design FRET tags to detect virtually any small molecule in living cells,” Frommer said. “Imaging techniques like this are the next frontier in the study of metabolism, and will help to answer some of the most pressing questions on plant biologists’ minds, such as the role of individual genes in the distribution of sugars. This in turn can help us engineer plants to produce more biomass.”
