We are indebted to Xuefei Yang, Sabine Both, Lin Chen and Kaitian Wang for coordinating the fieldwork and establishing the BEF-China experiment. We are also grateful to the whole BEF-China research group for their general support. BEF-China is mainly funded by the German Research Foundation (DFG FOR 891/1 and 2) and funding for this particular project was provided by the German Research Foundation to H.B. (DFG BR 1698/9-2). We are also thankful for the travel grants and summer schools financed by the Sino-German Centre for Research Promotion in Beijing (GZ 524, 592, 698, 699 and 785). In addition we would like to thank David Eichenberg, Michael Staab, Katja Grotius, Silvana Tornack, Lin Chen, and Shouren Zhang for their support in the field and in the lab.

1.气孔导度<ol><li>气孔导度的测量 注：利用一个简单类型的稳态气孔计（十边形SC1）的作者。该气孔计的设计，具有体积小，直观的手动操作，可靠性高的优点。当测量在外地气孔导度，确保以最小化测量个体之间的距离为一个重复测量周期是最有效的可能。 </li><li>选择根据一个重现的图案不同物种和个体的叶（相同高度，相同的曝光，在植物中相同的位置，如果可能的话只从相同的节点，并且仅来自一个类别;太阳或阴影叶等）。 <ol><li>只有度量叶子在健康的，未受损和完全发育的条件。标记上的个人（例如，使用扎带彩色带），叶子以确保重复测量是在相同的叶进行。 注：测量电气孔导度对此语句应只在叶面上，同时避免了中脉和强大的叶脉。 </li><li>开始在清晨测量日出服用九时五十五重复测量点前气孔导度值显示在中午明显下降。 注：测量每日课程将提供良好的数据分析VPD和气孔导度之间的关系。 </li></ol></li><li> VPD测量<ol><li>与每个克 S-测量，记录温度和相对湿度优选与便携式记录仪直接测量在相同的叶的位置的条件。为了计算蒸汽压赤字利用八月，罗氏马格努斯公式15。 </li></ol></li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/52738/52738eq1.jpg" width="300" /> （E S）=饱和水蒸汽压力[百帕] <ol start="4"><li> G型的S - VPD响应<ol><li>现在积种明智的全部G 对阵 VPD数据，单个叶片所有的日常课程合并为每个物种一一分析。提取由搜索最大值从气孔导度数据中观察到的最大值。要缩放模型种明智的可比性，通过观察该物种（G S / G SMAX）的最大值除以观测值。 </li><li>每个物种，倒退克S的 logits（克S /克SMAX）到VPD和VPD的使用广义线性模型用二项式误差分布（a，b和c表示回归参数 ​​）的二次项： </li></ol></li></ol><img alt="公式（2）" src="/files/ftp_upload/52738/52738eq2.jpg" width="300" /><img alt="图1" src="/files/ftp_upload/52738/52738fig1highres.jpg" wIDþ=“700”/&gt; 图中绘制的值与拟合模型克 S- 1例- VPD气孔导度绘制成蒸气压赤字供种枫香功能。空点表示的观测值。 （A）最大气孔导度，VPD的最大气孔导度和平均气孔导度从绝对因而不可伸缩的气孔导度 （G s）的数据中提取。 （二）缩放气孔导度数据（G S / G SMAX）绘制提取相关参数（显示为实心点），20。重新打印许可20。 <a href="https://www.jove.com/files/ftp_upload/52738/52738fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52738/52738fig2highres.jpg" wi嗞嗞=“400”/&gt; 图2.所有的拟合模型的所有物种。模型图的气孔导度数据VPD回归的所有物种。常绿树种用红线20由黑色线条，落叶树种表示。经许可后20重新打印。 注意：使用logits而不是直接回归克 S-到VPD，通向该模型的最大值不超过克SMAX的点和克 S-接近0在高温VPD。 <ol start="5"><li>提取气孔调节参数的每一个物种<ol><li>计算绝对建模克SMAX值（MaxFit在图1B）。要做到这一点，从计算设定1.4.2的 ​​一阶导数为零，这给VPD gsMaxFit = -b / 2a中的VPD最大气孔导度。将VPD gsMaxFit到1.4.2的 ​​公式，提高到E，获得MaxFit的力量。计算并购每个品种的电导测量的EAN（ 见图1A）。 注：使用统计软件R（http://www.r-project.org）。 </li><li>计算的相对值，从比例模型（克S /克SMAX），提取气孔导度和VPD值以下两点：（1）气孔导度和VPD在模型的最大（MaxFit）和（2）VPD在曲线拐点的第二个点（参见图1B）。这个值乘由G 和S-MAX获得绝对克 S-值这些点。 图 2所示为单一车型分析了所有39种的完整覆盖。 </li></ol></li></ol> 2.测量气孔性状<ol><li>采取样品优选从完全相同的树叶已用于气孔导度的测量结果。如果这是不可能的，应用施加到选择叶片相同的选择过程对于气孔导度的测量，优选在相同的个人。 </li><li>涂上薄薄一层无色，快速烘干指甲油（测试不同品牌，有些人比其他人更适合）到一个新的样本。如果样本不能被立即处理，将它们存储在70％的酒精。后指甲油干燥后出来，轻轻地从叶剥离的印象，并继续进行微观分析，与正常的叶样品。 注意：在叶子以高密度毛状体的情况下施加氢氧化钠或1的前一步骤：乙酸和氧气的过氧化1溶液可以导致更好的结果。 </li><li>光学显微镜分析<ol><li>一个相机连接到能够40倍之间的放大倍率为400X的光学显微镜。后相机连接到显微镜，匹配采取光学放大倍数和图像的分辨率， 例如，用一个尺度的帮助下的图像。 </li><li>采用开源​​的我像ImageJ的16法师处理软件分析这些照片。 <ol><li>绘制一个形状与来自图像的图像分析工具形状工具在一个地区，没有污垢，拇指版画，受损区域，或大叶脉。计数气孔在这方面并在总共至少50000微米2％的样品。 </li><li>测量气孔保卫细胞长度和孔长。计算每平方毫米的气孔的数量。计算气孔指数作为气孔密度的比率为表皮每mm 2的细胞数。 </li></ol></li></ol></li></ol>叶脉性状3.评估注：有关叶脉特征的评估，修改后的袋＆Scoffoni 17的协议受聘。 <ol><li>样品制备 注：为了优化叶脉的知名度，叶先漂白，然后用番红和孔雀石绿染色。 <ol><li>漂白LEAV上课，让他们至少72小时，在50％的溶液消色剂（试验不同的品牌，又有些人可能会更好地工作）。另外，使用5％NaOH溶液，或10％的KOH溶液或25％的H 2 O 2的溶液中。 </li><li>加热溶液到30℃或从步骤3.1.1结合不同的解决方案更好的效果。水冲洗几次之后。适应漂白过程的具体种类，这取决于它们的叶特性。 注：叶片较厚，可能需要的解决方案浴或更积极的解决方案更长的时间。更薄，更嫩叶可在不到72小时的漂白到令人满意的程度。 </li><li>着色叶子将它们放置在100％的乙醇。它们上色2 - 1％番红溶液30分钟。以提高着色，只有几秒钟添加用1％孔雀绿溶液另外的处理。适应的协议，以每种时间和强度而言，以达到最佳的效果。冲洗SE水veral次之后。如果叶子是深染，在乙醇或消一段时间可能会有所帮助。 </li></ol></li><li>样品分析<ol><li>扫描叶子背光扫描仪在约 1200 dpi的分辨率。匹配带到图片， 例如的分辨率的扫描 ，带刻度的帮助下，以确保该像素长度可以追溯到绝对长度扫描的叶的措施。 </li><li>测量叶面积，周长，长度和宽度。计算几个指标， 比如，长/宽和周长2 /面积。剪下1 1厘米的矩形出来的画面的中间。测量的第一和第二顺序静脉直径（不包括主要的中央静脉）。测量的第1阶的静脉在这个样方（静脉密度）的长度。 </li></ol></li></ol>其他叶性状4.评估注：驴子典型的叶片性状，如具体的叶面积（SLA），叶干物质含量（LDMC），叶面积，元素的内容，叶习惯，叶pinnation，叶型化合物，叶缘类型等等以下建立的方案18,19。 <ol><li>观察叶片性状<ol><li>评估叶pinnation，叶型化合物，叶缘型和外蜜腺18,19通过观察在该领域中存在。 </li></ol></li><li>分析叶片性状<ol><li>收集新鲜叶样品，优选作为用于其它测量，比叶面积（SLA），叶面积，叶干物质含量（LDMC）18,19的测定相同的个体。 48小时后，在干燥炉中在80℃下，测量元素的含量和比率，优选在相同的叶子。 </li></ol></li></ol>

<ol>
	<li>Schulze, E. -. D., Kelliher, F., K&#246;rner, C., Lloyd, J., Leuning, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+maximum+stomatal+conductance,+ecosystem+surface+conductance,+carbon+assimilation+rate,+and+plant+nitrogen+nutrition:+a+global+ecology+scaling+exercise.">Relationships among maximum stomatal conductance, ecosystem surface conductance, carbon assimilation rate, and plant nitrogen nutrition: a global ecology scaling exercise.</a> Annual Review of Ecology and Systematics. 25 (1), 629-662 (1994).</li><li>Chaturvedi, R. K., Raghubanshi, A. S., Singh, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+tree+seedlings+in+a+tropical+dry+forest+in+relation+to+soil+moisture+and+leaf+traits.">Growth of tree seedlings in a tropical dry forest in relation to soil moisture and leaf traits.</a> Journal of Plant Ecology. 6 (2), 158-170 (2013).</li><li>Santiago, L. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+evolution+of+leaf+shape+and+physiology+in+the+woody+Sonchus.+alliance+(Asteraceae:+Sonchinae)+in+Macaronesia.">Correlated evolution of leaf shape and physiology in the woody Sonchus. alliance (Asteraceae: Sonchinae) in Macaronesia.</a> International Journal of Plant Sciences. 170 (1), 83-92 (2009).</li><li>Aasamaa, K., Sober, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+conductance+and+stomatal+sensitivity+in+six+deciduous+tree+species.">Hydraulic conductance and stomatal sensitivity in six deciduous tree species.</a> Biologia Plantarum. 44 (1), 65-73 (2001).</li><li>Waring, R. H., Chen, J., Gao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-water+relations+at+multiple+scales:+integration+from+observations,+modeling+and+remote+sensing.">Plant-water relations at multiple scales: integration from observations, modeling and remote sensing.</a> Journal of Plant Ecology-UK. 4, 1-2 (2011).</li><li>Oren, R., Sperry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+and+synthesis+of+intra-+and+interspecific+variation+in+stomatal+sensitivity+to+vapour+pressure+deficit.">Survey and synthesis of intra- and interspecific variation in stomatal sensitivity to vapour pressure deficit.</a> Plant, Cell and Environment. 22 (12), 1515-1526 (1999).</li><li>Leuning, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+appraisal+of+a+combined+stomatal-photosynthesis+model+for+C3+plants.">A critical appraisal of a combined stomatal-photosynthesis model for C3 plants.</a> Plant, Cell and Environment. 18 (4), 339-355 (1995).</li><li>Patan&#232;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+area+index,+leaf+transpiration+and+stomatal+conductance+as+affected+by+soil+water+deficit+and+vpd+in+processing+tomato+in+semi+arid+mediterranean+climate.">Leaf area index, leaf transpiration and stomatal conductance as affected by soil water deficit and vpd in processing tomato in semi arid mediterranean climate.</a> Journal of Agronomy and Crop Science. 197 (3), 165-176 (2011).</li><li>Jarvis, A. J., Davies, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coupled+response+of+stomatal+conductance+to+photosynthesis+and+transpiration.">The coupled response of stomatal conductance to photosynthesis and transpiration.</a> Journal of Experimental Botany. 49 (Special Issue), 399-406 (1998).</li><li>Roelfsema, M. R. G., Hedrich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+the+light+of+stomatal+opening:+new+insights+into+&#8220;the+Watergate.&#8221;.">In the light of stomatal opening: new insights into &#8220;the Watergate.&#8221;.</a> New Phytologist. 167 (3), 665-691 (2005).</li><li>Wong, S. C., Cowan, I. R., Farquhar, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+conductance+correlates+with+photosynthetic+capacity.">Stomatal conductance correlates with photosynthetic capacity.</a> Nature. 282 (5737), 424-426 (1979).</li><li>Sobrado, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+conductance+and+water+potential+differences+inside+leaves+of+tropical+evergreen+and+deciduous+species.">Hydraulic conductance and water potential differences inside leaves of tropical evergreen and deciduous species.</a> Biologia Plantarum. 40 (4), 633-637 (1998).</li><li>Zhang, Y. -. J., Meinzer, F. C., Qi, J. -. H., Goldstein, G., Cao, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midday+stomatal+conductance+is+more+related+to+stem+rather+than+leaf+water+status+in+subtropical+deciduous+and+evergreen+broadleaf+trees.">Midday stomatal conductance is more related to stem rather than leaf water status in subtropical deciduous and evergreen broadleaf trees.</a> Plant, Cell and Environment. 36 (1), 149-158 (2013).</li><li>Aerts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+of+being+evergreen.">The advantages of being evergreen.</a> Trends in Ecology, &amp; Evolution. 10 (10), 402-407 (1995).</li><li>Murray, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+computation+of+saturation+vapor+pressure.">On the computation of saturation vapor pressure.</a> Journal of Applied Meteorology. 6 (1), 203-204 (1967).</li><li>Scoffoni, C., Sack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+leaf+vein+traits.">Quantifying leaf vein traits.</a> PrometheusWiki. , (2011).</li><li>Cornelissen, J. H. C., Lavorel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+handbook+of+protocols+for+standardised+and+easy+measurement+of+plant+functional+traits+worldwide.">A handbook of protocols for standardised and easy measurement of plant functional traits worldwide.</a> Australian Journal of Botany. 51 (4), 335-380 (2003).</li><li>Perez-Harguindeguy, N., D&#237;az, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+handbook+for+standardised+measurement+of+plant+functional+traits+worldwide.">New handbook for standardised measurement of plant functional traits worldwide.</a> Australian Journal of Botany. 61 (3), 167-234 (2013).</li><li>Kr&#246;ber, W., Bruelheide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transpiration+and+stomatal+control:+A+cross-species+study+of+leaf+traits+in+39+evergreen+and+deciduous+broadleaved+subtropical+tree+species.">Transpiration and stomatal control: A cross-species study of leaf traits in 39 evergreen and deciduous broadleaved subtropical tree species.</a> Trees. 28 (3), 901-914 (2014).</li><li>Larcher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiological plant ecology. , 514 (2003).</li><li>Bunce, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+transpiration+control+stomatal+responses+to+water+vapour+pressure+deficit.">Does transpiration control stomatal responses to water vapour pressure deficit.</a> Plant, Cell and Environment. 20 (1), 131-135 (1997).</li><li>Bunce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+leaf+hydraulics+limit+stomatal+conductance+at+high+water+vapour+pressure+deficits.">How do leaf hydraulics limit stomatal conductance at high water vapour pressure deficits.</a> Plant, Cell and Environment. 29 (8), 1644-1650 (2006).</li><li>Poorter, L., Bongers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+traits+are+good+predictors+of+plant+performance+across+53+rain+forest+species.">Leaf traits are good predictors of plant performance across 53 rain forest species.</a> Ecological Sciences of America. 87 (7), 1733-1743 (2006).</li><li>Juhrbandt, J., Leuschner, C., H&#246;lscher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+maximal+stomatal+conductance+and+leaf+traits+in+eight+Southeast+Asian+early+successional+tree+species.">The relationship between maximal stomatal conductance and leaf traits in eight Southeast Asian early successional tree species.</a> Forest Ecology and Management. 202 (1-3), 245-256 (2004).</li><li>Brodribb, T. J., Holbrook, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+physiology+does+not+predict+leaf+habit;+examples+from+tropical+dry+forest.">Leaf physiology does not predict leaf habit; examples from tropical dry forest.</a> Trees. 19 (3), 290-295 (2005).</li><li>Kr&#246;ber, W., Zhang, S., Ehmig, M., Bruelheide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylem+hydraulic+structure+and+the+Leaf+Economics+Spectrum+-+A+cross-species+study+of+39+evergreen+and+deciduous+broadleaved+subtropical+tree+species.">Xylem hydraulic structure and the Leaf Economics Spectrum - A cross-species study of 39 evergreen and deciduous broadleaved subtropical tree species.</a> PLOS ONE. 9 (11), e109211 (2014).</li><li>Kr&#246;ber, W., Heklau, H., Bruelheide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+morphology+of+40+evergreen+and+deciduous+broadleaved+subtropical+tree+species+and+relationships+to+functional+ecophysiological+traits.">Leaf morphology of 40 evergreen and deciduous broadleaved subtropical tree species and relationships to functional ecophysiological traits.</a> Plant Biology. 17 (2), 373-383 (2015).</li></ol>

The authors have nothing to disclose.

气孔调节通过本文介绍的方法提取的参数强调气孔性状，如气孔密度，气孔指数的重要性。这些新的关系证明联从生理模型参数的形态，解剖和化学叶片性状20的潜力。与其它方法相比，本方法带有捕获在哪些气孔导被下调到一半的建模克的最大的一个独特的和明确的VPD值的优点。 在协议中所概述的所有步骤中最关键的是气孔导度的测量结果。由于气孔导度的环境气候条件下的多因素调控对克 S-强大的影响力。气孔导度测量在相对 ​​湿度高和低光照强度可能是不可靠21-23。对于形态和ANATomical性状，该协议应当始终适于包括在研究中的目标物种。特别是在静脉密度分析，漂白和叶的染色的持续时间应该改变，这取决于叶片结构和韧性。该方法的潜在限制包括物种，其中气孔导度的测量是不可能的或复杂的，容易出现由于非凡的叶子形式的错误。这可能包括针叶树和草很窄叶片。 我们的结果部分地确认气孔导度参数和叶经济学谱（LES），它对应于几个其他研究的叶性状之间的链路的第一个假设。例如，Poorter和Bongers（2006）24日报道 克 S-并由LES， 如代表性状之间的密切联系，与增加叶片寿命克 S-下降。因此，舒尔茨等人（1994）1 demonst叶氮含量和G SMAX之间的额定明确的联系。同样，Juhrbandt 等人 （2004）25发现摹SMAX和叶面积和叶片含氮量之间显著的关系。 我们的第二个明显的区别就叶习惯的假设都没有得到证实。高变化中的常绿和落叶习惯测量参数和特征表明，叶的习惯不是LES的一个很好的描述。布罗德里布和霍尔布鲁克（2005）26探讨了叶习性和叶片生理策略并不一定必然连接，因为广泛的性状变化是在所有类型的叶习惯普遍。 该方法可以扩展到性状等于叶，例如，有关木质部液压性状的植物的器官，如特定木质部水力传导率和显微镜木材性状27的生理特性。同样，其他类型为来源于显微镜，如栅栏组织结构和epicuticular蜡层结构叶片性状可列入28。 总之，这项研究证实了LES和气孔调节之间的密切联系。此外，这里介绍的方法，揭示了那些不相关的LES气孔调节模式方面。尤其是比叶性状，如气孔的大小，密度及指数，以及静脉长度未来值得关注的功能性植物的研究。

以超前的功能的理解植物叶片，最近的许多研究都试图关系形态，解剖和化学叶性状的生理反应，如叶片气孔导度（G S）1-4。此外，叶性状，气孔导度的强烈影响的环境条件，如光合活性光子通量密度，空气温度和VPD 5。各种方式已经提出了模型的gs -这主要是基于GS的上VPD 6线性回归VPD曲线6-8。与此相反，在本研究中提出的模型倒退相对气孔导度（即，克S与最大气孔导克SMAX的比值）上VPD，并通过添加VPD作为二次回归术语占非线性的logits。 相比其他车型，新车型可以用于获得描述参数在其中g S的下缺水下调的VPD。类似地，获得了VPD在哪些克S是最大的。因此生理参数可以预期紧密相连碳同化9,10所反映的LES这些模型参数和关键叶片性状养分和资源分配之间的密切联系，应该预计3,11。结果，有也应该是气孔调节与LES性状战略之间的紧密关系。这样的关系，预计特别是用于叶片习惯（常绿对落叶），为叶习惯上都相关的LES和与水分利用效率12,13。常绿树种一般增长较慢，但在环境较差的营养物质14更有效。因此，叶的习惯应该翻译成不同的气孔调节模式，以较为保守的用水战略比落叶树种。 比较aring大集阔叶树种在花园里常见的情况，以下假设进行了测试：1）给G S模型的参数- VPD模型连接到相关的叶经济学频谱叶片性状。 2）常绿植物具有较低的平均克 S-和G SMAX比落叶树种值。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SC 1 Porometer</td>
			<td>Decagon</td>
			<td>NA</td>
			<td>Any other porometer is suitable</td>
		</tr>
		<tr>
			<td>Cable ties to mark leaves</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plastic sample bags</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Paper sample bags</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Hygrometer</td>
			<td>Trotec</td>
			<td>NA</td>
			<td>Any other is suitable</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Axioskop 2 plus</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Any other is suitable</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>5% NaOH</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>10% KOH</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>25% H2O2</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Malachite green</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Safranine</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

relating stomatal conductance to leaf functional traits

叶功能性状是重要的，因为它们反映生理功能，如蒸腾和碳同化。尤其是，叶形态特征有总结植物战略的水分利用效率，增长方式的转变和养分利用方面的潜力。叶经济学谱（LES）是在功能性植物生态学一个公认的框架，并反映增加比叶面积（SLA），叶氮，磷和阳离子含量，降低叶干物质含量（LDMC）和碳氮比率的梯度（ CN）。在LES描述了不同的策略，从那个短命叶，每叶重较高的光合能力，长寿命的叶子与低质量为基础的碳同化率。但是，不包括在LES的性状可以提供关于该物种的生理学的其他信息，如那些与气孔控制。协议提出了广泛的叶函数的对最终的性状，包括LES的特质，同时也特质，独立于LES的。特别是，一种新的方法介绍，涉及气孔导蒸汽压赤字植物的监管行为。气孔调节由此产生的参数就可以比LES和其他植物功能性状。研究结果表明，LES的功能叶性状也有效预测的气孔调节的参数。例如，叶碳浓度呈正相关，蒸气压亏缺（VPD）的拐点和电导VPD曲线的最大值。但是，不包括在LES的性状添加的信息在解释气孔控制的参数：VPD在电导VPD曲线的拐点较低的物种具有更高气孔密度和较高的气孔指数。总体而言，气孔和静脉性状更强大的预测解释气孔T法令在LES使用汉特质。

气孔导度和气孔调节许多参数，发现可能与形态学，解剖学和化学叶片性状。在下文中，重点将是链接到VPD的拐点，这降低与气孔密度（P = 0.04）和气孔指数（P = 0.03），并随叶片的碳含量（P = 0.02， 参见图3）。研究结果表明，在拐点下降VPD有气孔密度和气孔指数的下降。与此相反，气孔导度没有参数表现出明显的关系，叶的习惯。在两组叶习惯的高差异表明，不同的监管机制小组常绿落叶习惯中同时存在。 <img alt="图3" src="/files/ftp_upload/52738/52738fig3highres.jpg" width="700" /> 图3.结果图为日水汽压亏缺（VPD）的气孔导度调节和叶性状模式之间E连接在克 S-的拐点- VPD曲线（VpdPoi）为（A）气孔密度，（B）气孔指数函数， （ 三）叶碳含量20。重新打印许可20。 <a href="https://www.jove.com/files/ftp_upload/52738/52738fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Relating Stomatal Conductance to Leaf Functional Traits at JoVE.com

Relating Stomatal Conductance to Leaf Functional Traits

揭开怎样的生理和形态链接可以提高设备的机械运作有了更深的认识离开。我们提出这两个程序从气孔导度的测量和相关性与传统的功能叶片性状得到气孔调节的参数。

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

relating-stomatal-conductance-to-leaf-functional-traits

Research

JoVE Journal

Biology

19.0K 次观看.  Martin-Luther-University Halle-Wittenberg. 揭开怎样的生理和形态链接可以提高设备的机械运作有了更深的认识离开。我们提出这两个程序从气孔导度的测量和相关性与传统的功能叶片性状得到气孔调节的参数。 

视频: Relating Stomatal Conductance to Leaf Functional Traits

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

科研

生物学

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

心脏压力容量环分析小鼠使用电导导管

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

在实验室条件下收集和长期维护切叶蚁（Atta）

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

通过表皮表型评分 鉴定 参与气孔发育的基因

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

用于映射植物根系架构特征的简单协议

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Methods Article

与气孔导度，以叶功能性状