?假彩色圖像可以顯示源自擬南芥表皮的單個細胞。已經(jīng)標(biāo)記了熒光的細胞被設(shè)計用來追蹤葡萄糖的分布,。來自Carnegie植物分子生物學(xué)系的科學(xué)家首次在完好無損的活體植物組織中實時觀察糖的利用,。在全新的成像技術(shù)的協(xié)助下,，該研究小組已經(jīng)觀察到植物根部維持極低濃度的糖，比先前估計的要低100,000倍,。這項新技術(shù)將可以開辟在植物中研究糖代謝方面的研究,，從而為在提高食品和生物燃料產(chǎn)量方面的工程改造提供更好的保障。
??在來自Carnegie研究機構(gòu)成員Wolf Frommer領(lǐng)導(dǎo)下,，研究人員通過遺傳改造來設(shè)計一種能編碼熒光標(biāo)簽來監(jiān)測在模式植物擬南芥根葉中的葡萄糖濃度,。這項技術(shù)史無前例地可以避免時間和空間的局限,，而直接追蹤活體原狀的植物組織中葡萄糖。該研究發(fā)表在九月份國際頂尖雜志《植物細胞》上,。 該研究小組同時也發(fā)明了一種針對蔗糖的熒光共振能量轉(zhuǎn)移傳感器,。這個研究工作將發(fā)表在九月份這期《生物化學(xué)》雜志上。
??Frommer認為：“迄今為止,，我們已經(jīng)掌握一些線索,，是關(guān)于在多細胞植物中單個細胞中糖的數(shù)量。我們通常將植物的根和葉碾成粉末,，然后平均所有細胞的成份,，但是如果在單個細胞糖水平升高和降低，我們將從平均水平上是看不出來的,。還有單個細胞在亞細胞水平上糖分布存在差異,，所以在特定時間段幾乎不可能知道在亞細胞結(jié)構(gòu)上的糖水平”。
??Frommer同時還強調(diào)：“時間分辨率是另一個瓶頸,，我們間隔時間段采集組織樣品,，但如果糖含量在波動，我們可能就錯過最佳的時間點,。我們最近發(fā)明的技術(shù)可以避免在單細胞水平實時監(jiān)測糖流量時面臨的所有瓶頸,，以及可以解決有關(guān)亞細胞水平分辨率的問題”。
??Frommer和他的同事已經(jīng)使用擬似于熒光共振能量轉(zhuǎn)移(FRET)傳感器樣的成像標(biāo)簽,，來追蹤動物細胞中糖和神經(jīng)遞質(zhì),。最近，F(xiàn)RET傳感器的課題組使用該技術(shù)研究谷氨酸鹽（一種重要的哺乳動物神經(jīng)遞質(zhì)）,。Frommer已經(jīng)在培養(yǎng)的哺乳動物細胞內(nèi)追蹤葡萄糖,，但是到現(xiàn)在為止，在植物組織上證明還是有問題的,，主要是來自植物病毒防疫機制以及在一些植物組織中高背景熒光的干擾,。
??為了克服這些障礙，F(xiàn)rommer課題組對此傳感器做了經(jīng)典的改善,，將它們插入到?jīng)]有防衛(wèi)基因的擬南芥突變體上,，此時的熒光標(biāo)簽可以避免先前的弊端發(fā)揮更好的功效。
??Frommer解釋：“這可能不是理想的,，因為只能用在防衛(wèi)突變體的植株上,。最優(yōu)的應(yīng)該是這些傳感器能在任何野生型植株的遺傳背景下發(fā)揮功效。現(xiàn)在我們開始找到關(guān)于植物處理糖途徑的重要障礙,，我們將繼續(xù)改善此傳感器以保證其發(fā)揮功效”,。
??Frommer談到：“這項技術(shù)的中心就在于巧妙而簡單。通過計算機的模擬設(shè)計，我們可能設(shè)計FRET標(biāo)簽來活靈活現(xiàn)地追蹤活細胞中任何小分子,。象這樣的成像技術(shù)在有關(guān)代謝方面的研究有著廣泛的應(yīng)用前景,，同時也將幫助我們回答壓在植物學(xué)家心頭一些最緊迫的問題,，例如在糖分布過程中單個基因發(fā)揮什么樣的功能,。依次這項技術(shù)將有助于我們改造植物從而提高產(chǎn)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.”
