我们非常感谢四匿名审稿改善的手稿，以及美国国家科学基金会（批准IOS-0546784）的支持。

1。测量的水合叶的叶片导水率（K 最大 ） <ol><li>收集笋最好在傍晚或晚上木质部的紧张局势低，减少了空气和地方在黑暗的塑料袋充满了湿纸巾。在实验室中，从去年底的每一个拍摄在水中，重新切割至少两个节点。让在纯净水，或其他的解决方案，可以使用用于 K 叶测量拍摄再水合过夜。超纯水被推荐为一个流程解决方案，除非进行调查的一个因素的影响，不同的解决方案。 </li><li>从拍摄部分脱气流程解决方案下的一个新的刀片切叶。超纯水被推荐为一个流程解决方案，除非进行调查的一个因素的影响，不同的解决方案。我们建议使用在一个烧瓶中，用真空泵脱气溶液过夜，但也可以采用其他方法。 </li><li>总结预拉伸封口膜周围的petio乐以确保良好的密封叶柄和油管之间。这一步是特别有用的非圆叶柄，额外的封口膜包装将有助于圆形，叶柄，以适应管内。可以应用于牙科糊剂叶柄之前包装封口膜填补叶柄中的槽。 </li><li>叶片连接根据超纯水硅胶管，以防止空气进入系统。作为叶的上方被抬起的源水，管路中的水是在低于大气压的压力下，并且因此密封件是好的，如果在测量过程中没有空气被抽入油管。另外的叶柄直接进入密封硅胶管，橡胶垫片压缩配件（ 例如 ，（Omnifit除A2227孔适配器; Omnifit接头，剑桥，英国），这个方法是有用的为叶柄特别复杂的形状，或草叶，可卷绕的玻璃杆密封与压缩接头。在可行时，直接进入密封叶柄州市licone管材具有的优点是快速，留下叶柄油管的交界处，是透明的，使气泡更容易看到。管连接叶硬管的量筒上的平衡（±10微克分辨率的），每30秒记录数据到计算机（ 例如，使用“平衡”软件，梅特勒-托利多公司，Greifensee，瑞士）计算流速通过叶。 </li><li>叶近轴面应保持向上串成一大盒风扇的鱼线以上的光源下（&gt; 1000微摩尔•M•S -1光合有效辐射的辐射测量），略高于水平的木框架量筒中的弯液面。叶应放在上面的水的量筒，被驱动，以确保移动到叶​​的水通过蒸发，而不是进入叶压力下由于重力，如将是叶的情况下，如果被放置在气缸中的水的弯液面以下。填充与湿纸巾的小容器被放置的平衡室的内部，以确保在平衡的空气是水饱和的，从而避免了从水源蒸发到大气中的平衡。这种可能性可以检查通过停止流入叶使用四通旋塞阀管路中，并检查，天平上的水筒的质量不会下降。 </li><li>放置一个明确的耐热玻璃容器中充满了水上面的叶子吸收的热量的灯。 </li><li>保持温度在23°C和28°C整个实验过程中，通过添加更多的冷水耐热玻璃容器中，使得热灯不耐热玻璃容器中加热到足以温暖周围的空气下面的叶叶。 </li><li>允许发散上的装置，至少30分钟的叶，从而有足够的时间来适应环境高光照，和的其他onally直到流速稳定，没有向上或向下的趋势，并与至少5-10在30秒的流速的时间间隔的测量的变异系数&lt;5％。它是必不可少的流量达到一个稳定的状态，因为此方法假定一个稳定的Ψ 叶 。以前的研究发现，与这些条件是足够稳定的流动速率（E），Ψ 叶 和 K 叶成立;较长的测量周期后稳定流动测试与测量时间的显示没有关系的 K 叶各7种了广泛范围，叶子电容17,18。稳定一般需要30分钟或者更少，但在一些物种中，可能需要一些物种超过一个小时。 </li></ol> 注 ：当流量是非常低（&lt;8微克-1），可以使用过去五年30秒的时间间隔平均运行稳定。 </p&gt; <ol start="9"><li>丢弃如果流量突然变化，或者由于突发气孔关闭，或明显的，从密封处泄漏或堵塞系统中的颗粒或气泡的测量。 </li><li>当流量稳定后，记录叶片温度与热电偶。 </li><li>从管快速去除叶，轻拍干叶柄和叶放入一个密封的袋子先前已呼出，停止蒸腾。 </li><li>让叶在其袋平衡至少20分钟。 </li><li>平均流量测量的最后10。这将是你的电子测量。 </li><li>当叶是平衡的，衡量一个压力室的最后叶水势（Ψ 最终 ）。 </li><li>与平板扫描仪和图像处理软件，或与叶面积仪测定叶面积。 </li><li> ķ 叶的计算公式为E /-ΔΨ 叶 （其中ΔΨ 叶 =Ψ 最后 </子 - 0 MPa）和叶面积进一步规范。单位是毫摩尔：-1米-2兆帕-1。由于叶片的位置的正上方的带刻度的量筒中的水位，重力的影响，在进入叶的水的压力降低将可忽略不计的，尤其是相对于Ψ 决赛 ，这始终是&gt; -0.1兆帕。 </li><li>若要更正更改在 K 叶诱导的水的粘度的温度依赖性的15,19,20，规范ķ 叶值至25℃，通过使用下面的等式： </li></ol><img alt="式（1）" fo:content-width="3.863in" fo:src="/files/ftp_upload/4179/4179eq1highres.jpg" src="/files/ftp_upload/4179/4179eq1.jpg" /><ol start="18"><li>气孔导度（ 克 ） 为 E除以叶-空气蒸汽压力差，相当于蒸气压力，可确定重新亏损（VPD），确定使用的温度和相对湿度传感器附近的叶。 VPD是使 ​​用下面的式（21）计算</li></ol><img alt="公式2" src="/files/ftp_upload/4179/4179eq2.jpg" fo:content-width="1.67in" fo:src="/files/ftp_upload/4179/4179eq2highres.jpg" /> 其中，RH是相对湿度，VP是饱和蒸汽的空气压力（千帕，温度的函数，根据雅顿降压方程22），和AP的大气压力（千帕）。 2。脱水叶叶片导水率测量<ol><li>收集笋最好在傍晚或晚上木质部的紧张局势低，切在空气中，在黑暗中充满了湿纸巾的塑料袋。在实验室中，从去年底的每一个拍摄在水中，重新切割至少两个节点。让拍摄补充水分过夜，纯净水，或其他解决方案使用的 K LEAF测量。超纯水被推荐为一个流程解决方案，除非进行调查的一个因素的影响，不同的解决方案。 </li><li>剪切分部芽叶成具有至少三个叶下超纯水和脱水一个风扇，用于拍摄的Ψ 叶值的范围内的不同的时间段。 </li><li>一旦枝条脱水，围绕拍摄的每一片叶子放在密封的包装袋（WHIRL-PAK Nasco的，阿特金森堡，WI，USA）已被呼出），然后将湿纸在一个大型密封的袋子，整个拍摄毛巾。 </li><li>让拍摄平衡至少30分钟（强脱水枝条较长的平衡时间可能是必要的）。 </li><li>平衡后，消费的顶部和底部的叶片和措施初始Ψ 叶（=ΨO）用压力室。 </li><li>丢弃在Ψ这两个叶片叶如果该差值是大于0.1兆帕（坚实的拍摄叶糖脱水，此范围可以扩展至0.3MPa）。 </li><li>第三个叶，然后用于确定 K 叶和 G s与EFM，以下步骤1.2 -1.18以上。值得一提的是，获得稳定的状态E的叶子已经严重脱水的叶子，的气孔仍然打开，并产生G S值远高于表皮电导17。 </li><li>为了构建液压脆弱性曲线， 计算 K 叶使用Ψ 最终在步骤1.16，然后情节ķ 叶对无论是最低值，Ψo或Ψ 最后 ， 即 ，在实验过程中所经历的叶片的最强的脱水（=Ψ 最低 ）。同样，G S在叶脱水的下降可以通过绘制G S对Ψ 最低的 。以前的工作SH<html>自己的脱水导致至少部分不可逆的跌幅在 K 叶和 G s 14,16。 </li><li>我们建议获得至少6 K 叶每0.5 MPa的间隔的Ψ 最低值（如果您正在使用的物种是真的容易脱水，此时间间隔可以降低到0.25兆帕）。 </li><li>执行狄克逊对 K 叶值的异常值检验为每个0.5兆帕水潜在的间隔的Ψ 最低的删除异常值从你的弱点曲线23。 </li><li>由于种不同的反应的 K 叶和 G s到脱水14,16，每个物种适应不同的功能，并选择最大似然（AIC值最低;有关详细信息，请参见图14）。我们建议以前在文献中使用的配件至少有四个功能：线性（PG“FO：内容的宽度=”1.4in“佛：SRC =”/ files/ftp_upload/4179/4179eq3highres.jpg“/&gt;），S型（ <img alt="式（4）" src="/files/ftp_upload/4179/4179eq4.jpg" fo:content-width="1.483in" fo:src="/files/ftp_upload/4179/4179eq4highres.jpg"/> ），物流（ <img alt="方程（5）" src="/files/ftp_upload/4179/4179eq5.jpg" fo:content-width="1.7in" fo:src="/files/ftp_upload/4179/4179eq5highres.jpg"/> ）和指数（ <img alt="方程（6）" src="/files/ftp_upload/4179/4179eq6.jpg" fo:content-width="1.583in" fo:src="/files/ftp_upload/4179/4179eq6highres.jpg"/> ）。 </li></ol> 3。代表性的成果叶导水率的下降与脱水变化剧烈跨物种（ 图1）。干旱敏感物种的体验不是更耐旱品种，少负水势下降的情况下测量高光（PAR&gt; 1000微摩尔•米-2 -1）。水潜在的导水率在80％的损失（P 80）梅西奇草本植物向日葵为3兆帕少负Heteromeles钻天柳 ，原产于美国加州丛林。Heteromeles钻天柳线性的响应水势的下降，而向日葵呈非直线下降。 气孔导度（G s）和叶片导水率（K 叶 ）也可以反应显着辐照度（ 图2）。 Raphiolepis表明 ，气孔导度下降了强烈的脱水叶子下测得的高光（PAR&gt; 1000微摩尔•米-2 -1）和在实验室光（PAR &lt;6微摩尔•米-2 -1）。在高光照下，叶片测量与EFM已经脱水的水势，是典型的光线充足的执法机关VES月中旬的一天完整的植物表现出类似的G S测量测孔仪（ 图2中的明星）的完整的植物叶。值得注意的是，对于在最大水化叶片，K 叶的4倍叶片比低辐照度（11比2.8毫摩尔米-2 -1兆帕-1）暴露于高的，因此ķ 叶显示出更强的光响应的水合叶比G S。此外，在高光强，K 叶下降更为强烈的脱水。 P 80 0.93兆帕高比低光照下少负。 <img alt="图1" src="/files/ftp_upload/4179/4179fig1.jpg" /> 图1。的叶片液压漏洞曲线干旱的敏感（顶部）和耐旱（底部）物种（从14,24）。每一个点代表一个罪叶液压电导（K 叶 ）使用蒸发助熔剂法（EFM）上的离体叶GLE测量。竖线代表水势该物种丧失了80％的初始叶导水率（P 80）。最适合用最大似然函数绘制两个物种。 <img alt="式（7）" src="/files/ftp_upload/4179/4179eq7.jpg" fo:content-width="3.293in" fo:src="/files/ftp_upload/4179/4179eq7highres.jpg"/><img alt="图2" src="/files/ftp_upload/4179/4179fig2.jpg" /> 图2。气孔导度（G）和叶片导水率（K 叶 ）下降，叶片水势Raphiolepis指标的响应下测得的高与低光照（16）。 （PAR&gt; 1000，&lt;6毫摩尔•米-2•S-1）。在离体叶片蒸发通量法（EFM），白点和黑点，分别代表叶高和低光照下测得的每一个点代表一个单一的测量。气孔导度曲线的高与低辐照度的方程为： <img alt="式（8）" src="/files/ftp_upload/4179/4179eq8.jpg" fo:content-width="4.727in" fo:src="/files/ftp_upload/4179/4179eq8highres.jpg" />和<img alt="公式9" src="/files/ftp_upload/4179/4179eq9.jpg" fo:content-width="2.26in" fo:src="/files/ftp_upload/4179/4179eq9highres.jpg"/> 。脆弱性曲线的高与低光照方程为： <img alt="式（10）" src="/files/ftp_upload/4179/4179eq10.jpg" fo:content-width="2.7in" fo:src="/files/ftp_upload/4179/4179eq10highres.jpg"/>和<img alt="式（11）" src="/files/ftp_upload/4179/4179eq11.jpg" fo:content-width="1.8in" fo:src="/files/ftp_upload/4179/4179eq11highres.jpg"/> 。在上面板的灰色明星代表均数±站g s和完整的植物叶子上月中旬的一天，一个气孔计测得的叶水势ndard错误。在下部面板上的灰色和黑色竖线代表该物种丧失了80％的初始叶导水率（P 80）分别高和低光照下的水势。

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C. <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 Chemistry and Physics. , (1974).</li><li>Yang, S. D., Tyree, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+resistance+in+Acer+saccharum+shoots+and+its+influence+on+leaf+water+potential+and+transpiration.">Hydraulic resistance in Acer saccharum shoots and its influence on leaf water potential and transpiration.</a> Tree Physiol. 12, 231-242 (1993).</li><li>Pearcy, R. W., Schulze, E. -. D., Zimmermann, R., Pearcy, R. W., Ehleringer, J. R., Mooney, H. A., Rundel, P. 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> Plant Physiological Ecology: Field Methods and Instrumentation. , 137-160 (2000).</li><li>Buck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+equations+for+computing+vapor-pressure+and+enhancement+factor.">New equations for computing vapor-pressure and enhancement factor.</a> Journal of Applied Meteorology. 20, 1527-1532 (1981).</li><li>Sokal, R. R., Rohlf, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biometry. , (1995).</li><li>Scoffoni, C., Rawls, M., McKown, A., Cochard, H., 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=Decline+of+leaf+hydraulic+conductance+with+dehydration:+relationship+to+leaf+size+and+venation+architecture.">Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture.</a> Plant Physiol. 156, 832-843 (2011).</li><li>Sack, L., Tyree, M. T., Holbrook, N. M., Zweiniecki, 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=.">.</a> Vascular Transport in Plants. , (2005).</li><li>Nardini, A., Tyree, M. T., Salleo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylem+cavitation+in+the+leaf+of+Prunus+laurocerasus+and+its+impact+on+leaf+hydraulics.">Xylem cavitation in the leaf of Prunus laurocerasus and its impact on leaf hydraulics.</a> Plant Physiol. 125, 1700-1709 (2001).</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=Stomatal+closure+during+leaf+dehydration,+correlation+with+other+leaf+physiological+traits.">Stomatal closure during leaf dehydration, correlation with other leaf physiological traits.</a> Plant Physiol. 132, 2166-2173 (2003).</li><li>Blackman, C. J., Brodribb, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+measures+of+leaf+capacitance:+insights+into+the+water+transport+pathway+and+hydraulic+conductance+in+leaves.">Two measures of leaf capacitance: insights into the water transport pathway and hydraulic conductance in leaves.</a> Funct. Plant Biol. 38, 118-126 (2011).</li><li>Nardini, A., Raimondo, F., Lo Gullo, M. A., Salleo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leafminers+help+us+understand+leaf+hydraulic+design.">Leafminers help us understand leaf hydraulic design.</a> Plant Cell Environ. 33, 1091-1100 (2010).</li><li>Sheriff, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation+sites+and+distillation+in+leaves.">Evaporation sites and distillation in leaves.</a> Ann. Bot. 41, 1081-1082 (1977).</li><li>Canny, M. J., Huang, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+water+content+and+palisade+cell+size.">Leaf water content and palisade cell size.</a> New Phytol. 170, 75-85 (2006).</li><li>Santiago, L. S., Mulkey, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+test+of+gas+exchange+measurements+on+excised+canopy+branches+of+ten+tropical+tree+species.">A test of gas exchange measurements on excised canopy branches of ten tropical tree species.</a> Photosynthetica. 41, 343-347 (2003).</li><li>Sack, L., Cowan, P. D., Jaikumar, N., 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=The+'hydrology'+of+leaves:+co-ordination+of+structure+and+function+in+temperate+woody+species.">The 'hydrology' of leaves: co-ordination of structure and function in temperate woody species.</a> Plant Cell Environ. 26, 1343-1356 (2003).</li></ol>

没有利益冲突的声明。

蒸发通量这里介绍的方法可以较快的在实验室同时测定气孔导度（30分钟）测定的叶片导水率。 EFM是迄今最密切的方法，该方法如下水在叶片的天然途径，鉴于叶空域和扩散从气孔15中的水蒸发。许多其他的 K 叶测定的实验方法存在25日 ，有三个是特别常见的。 （1）在高压流量的方法（HPFM）的，水被压在高压下通过叶15,26。然而，脆弱性曲线不能用这种方法获得的，因为施加的正压力可能会填充的栓子和补充水分叶肉组织。 （2）在复水动力学方法（RKM），K 叶计算补液，脱水叶之间的类比与CHarging的电容串联一个电阻27,28。这种方法是快速的，但不会重现整个系统的自然蒸腾途径在叶，不容易修改，以适应高光的叶子可以测量。 （3）在真空泵的方法（VPM）叶片被放置在一个腔室，其叶柄连接到一个水源上的平衡，而不同强度的真空被施加到从叶15,26叶驱动水分损失。这种方法可以很费时间。所有这三种方法的测量结果，使用已发现，得到一致的结果，15,28,29，从而在EFM是经常使用的给出其基于蒸腾水分运动，和其相对的速度，并在不同的条件下用于测量ķ 叶及其改性。 所有用于确定ķ 叶片的典型方法涉及的水平的不确定性在量化按urately蒸腾25对应的驱动力。在EFM中，ΔΨ 叶用于计算ķ 叶比真正的驱动力低，因为它是基于散装Ψ 叶后叶是平衡的压力炸弹。此时，堆积Ψ 叶并不一定代表细胞的水势的液压通路，结束在水蒸发后，而是相对较少的细胞低水势水的体积加权平均移动和蒸发，而对于更大量的细胞可以从蒸腾流中分离的高水势。 Ψ 叶的真正动力不同，取决于那里的水蒸发的叶。如果整个叶片的水分蒸发时，大部分Ψ 叶可能是接近的真正动力，如果水蒸发，只有相对少量C英语学习者的气孔附近，大部分Ψ 叶可能大大低估了的真正动力， 即这些细胞的水势实际上可能要低得多，因此，实际ķ 叶将低于与EFM确定。因此，度的 K 叶由EFM确定代表了真实的ķ 叶是敏感水流的途径，这仍然没有得到很好的理解。虽然在EFM提供了相匹配的数据的那些其他用于测量ķ 叶 （见上文）的方法，并且是用于比较使用优异，物种变化水流通路通过叶肉的可能性需要进一步调查。事实上，即使对于给定物种的叶片，这些水通路可能会有所不同在不同条件下， 例如 ，在不同的辐照度和热负荷4,30或不同水分状态（特定组织收缩）31。进一步认识水的运输途径，从而使EFM被用来解决影响整体叶水力输送的途径。 在EFM气孔导度的测量具有允许测定辐照度和脱水反应，并提供匹配G S的响应与 K 叶的优点。此外，后跟补液G S以前叶脱水的效果可以用这种方法进行评估。然而，该方法有两个潜在的缺点。首先，由本克 测量的离体叶片上，而不是一个完整的植物上，和第二，气孔导度的测定是根据气候测量一段距离，而不是从叶片附近的叶片在气孔计室的通风风扇叶，但叶表面与周围空气的平衡。对于某些种类，切除叶子可能会表现出不同的气孔反应比上一个完整的植物叶16,32。然而，对于几个物种G S估计使用的EFM是完整的植物与中期天Ψ 叶 16（ 图2）的叶片的气孔计的测定值上相似。 的EFM可以应用于生理洞察力， 即 ，调查的 K 叶 ，其在结构和解剖学的基础，以及其对环境的响应的动态。此外，该方法可用于用来比较的植物生长在不同的条件下的给定的物种，或年龄的不同，或比较不同物种的植物，适应不同的环境。 通过构建叶脆弱性曲线，可以深入了解进入机制物种的公差干旱。在干旱，已经示出的气蚀和细胞崩溃发生在一些叶木质部<html>物种，并在叶肉细胞收缩在脱水过程中也可能影响水的运动（审查14）。未来的研究将需要确定这些过程的相对重要性在驾驶的 K 叶片气孔关闭的下降。 辐照度也是一个重要的因素影响ķ 叶和高光下的测量的EFM是非常适合的。水通道蛋白的辐射和干旱的综合反应应该获得多大的兴趣。高辐照度导致增加通过活细胞允许更快的水流的水通道蛋白的表达和/或活化，增加K 叶 ，而脱水导致减少的表达和/或de-activatation的水通道蛋白，减少ķ 叶 。 日 K 叶光照和供水的综合影响，需要进一步调查4,10,16,33。 帐篷“&gt;此外，跨物种，K 叶及其脆弱性的供应光水和其他资源3,13,24相关的物种分布的物种从干燥的地区往往表现出更大的阻力液压下降，面临更负面的土壤水势，和更多的负面水势保持了较高的K 叶 ，他们能保持高G S和，继续捕获CO 2进行光合作用13,14。因此，本研究的叶水力特性提供了见解的水平细胞，叶，全厂及其对环境反应，并在所有尺度上，可能会产生许多重要的新发现。

measurement of leaf hydraulic conductance and stomatal conductance and their responses to irradiance and dehydration using the evaporative flux method (efm)

水是一种重要的资源，植物水分的运输系统设置上最大的生长及抗旱性的限制。当植物气孔打开他们的实现了高气孔导度（G）捕获二氧化碳进行光合作用，水失去了的蒸腾1,2。蒸发的水分从空域被替换的细胞壁，反过来取水的叶脉的木质部，依次绘制在根和茎的木质部。通过该系统，由于水是被拉到它经历液压阻力，创建整个系统的张力和低叶水势（Ψ 叶 ）。叶在全厂系统本身是一个重要的瓶颈，占平均30％的植物液压阻力。叶液压电导（是ķ 叶 = 1 /叶液压阻力）的水的流速的比例是整个叶的水的电势梯度，并总结了一个复杂的系统行为：叶柄和几个数量进入维管束鞘的静脉，出口和通过或进入空域蒸发和蒸腾的气孔周围叶肉细胞的水穿过了浓厚的兴趣。K 叶作为重要的生理特征比较物种的，量化的有效性叶片结构和生理水路运输，以及一个关键的变量来研究其结构的变化之间的关系（ 例如 ，在叶叶脉架构）和光合气体交换的影响。此外，K 叶回应强烈的内部和外部的叶3。辐射ķ 叶会显着增加显然是由于变化的表达和活化水通道蛋白，水上运输有关的蛋白质通过膜4， 钾 叶强劲下降博士应，由于气蚀和/或木质部导管和/或渗透损失，由于叶肉和维管束鞘细胞收缩或水通道蛋白失活5-10额外的木质部组织的崩溃。 由于 K 叶可以约束g s和光合速率在 ​​正常浇水条件下，跨物种在干旱，从而限制全株表现，他们可能会决定物种的分布，尤其是旱灾增加的频率和严重程度11-14。 我们提出一个简单的同时测定的方法对离叶的 K 叶和 G s。甲蒸腾叶叶柄油管连接到水源上平衡运行。损失的水从天平上的记录来计算流速通过叶。当达到稳定状态蒸腾（E，毫摩尔•米-2 -1），G <sub&gt; s是通过除以水汽压亏缺确定，和ķ 叶通过除以水使用的压力腔室（K 叶 = E / - Δψ 叶 ，兆帕）15确定的潜在的驱动力。 此方法可用于评估ķ 叶的反应不同的辐照度和脆弱性的 K 叶脱水14,16,17。

Watch this Scientific Journal Video about Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM at 

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

我们描述了一个相对快速（30分钟）和现实的方法同时测量叶片导水率（<em&gt; K</em<sub&gt;叶</sub&gt;）和气孔导度（<em&gt; G</em<sub&gt; S</sub&gt;）蒸腾切除的叶子。该方法可以被修改，以测量的光和脱水反应的<em&gt; K</em<sub&gt;叶</sub&gt;和<em&gt; G</em<sub&gt; S</sub&gt;。

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 ...

measurement-leaf-hydraulic-conductance-stomatal-conductance-their

Research

JoVE Journal

Biology

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

36.9K 次观看.  University of California, Los Angeles. 我们描述了一个相对快速（30分钟）和现实的方法同时测量叶片导水率（ K

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

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

科研

生物学

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  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

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 ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

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, ...

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 ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

在靶标和标记下使用裂解分析植物中的H3K4me3修饰

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...