This research was supported by the National Nature Science Foundation of China (No.31671577), the Science and Technology Innovation Special Construction Funded Program of Beijing Academy of Agriculture and Forestry Sciences(KJCX20180423), the Research Development Program of China (2016YFD0300605-01), the Beijing Natural Science Foundation (5174033), the Beijing Postdoctoral Research Foundation (2016 ZZ-66), and the Beijing Academy of Agricultural and Forestry Sciences Grant (KJCX20170404), (JNKYT201604).

1. Sample Preparation Protocol
<ol>
	<li>For the sampling, collect the stem, leaf, and root from fresh maize plants and divide them into three types of sample groups (each group with four replications). Then, cut them into small segments using a surgical blade in the following manner: (1) cut a segment of the middle stem internode 1 - 1.5 cm in length; (2) cut a segment of the maximum width of the leaf 0.5 - 3 cm in length along the vertical direction with the main vein; (3) cut a segment of the crown root 0.5 cm in length.</li>
	<li>For the FAA fixation, soak the sample segments in an FAA solution (90:5:5 v/v/v, 70% ethanol:100% formaldehyde:100% acetic acid) for at least 3 d.</li>
	<li>Perform the dehydration procedure in six sequential ethanol gradients (i.e., 30%, 50%, 70%, 85%, 95% and 100%) and set the processing time of each ethanol gradient as 30 min.</li>
	<li>Place the plant materials in the corresponding sample baskets manufactured using a 3-D printer; then, quickly transfer the sample baskets to the sample cell of a CO2 critical-point drying system. Set the drying parameters as follows: 
	(1)&#160;CO2 in: fast speed. Holder fillers: 100%. 
	(2)&#160;Charge: CO2 charge delay 120 s into the cycle. Exchange speed: 5. Cycle number: 12. 
	&#8203;(3)&#160;Gas out: heat, fast. Speed: slow, 50%.
	<ol>
		<li>According to the morphological differences of maize root, stem, and leaf, design and print sample baskets using a 3-D printer (e.g., Figure 1).</li>
	</ol>
	</li>
	<li>Place the dried plant materials (maize root, stem, or leaf) in a 50-mL centrifuge tube with 2 g of solid iodine to dye the plant materials with volatile iodine vapor and, then, place the tubes in a lightproof room for 4 - 5 h.</li>
</ol>
2. Micro-CT Scanning Protocol
<ol>
	<li>To scan into the raw CT data, set the CT scanning parameters as follows: 40 kV/250 &#181;A (for stem and leaf) or 34 kV/210 &#181;A (for root). Set the corresponding scanning ranges for the different sizes and volumes of the plant materials used, and adjust the imaging pixel sizes as follows: 2.0 &#181;m (for the maize root), 6.77 &#181;m (for the maize stem), and 10.0 &#181;m (for the maize leaf).</li>
	<li>To reconstruct slice images, convert the raw CT data into CT slice images with a 2K resolution (2,000 x 2,000 pixels) using an image reconstruction software. More details are provided in the NRecon User Manual (http://bruker-microct.com/next/NReconUserGuide.pdf).</li>
</ol>
3. Image Analysis Protocol for a Single CT Image of a Maize Stem or Leaf
NOTE: Use automatic imaging software for vascular bundles to conduct the phenotyping analysis of vascular bundles within the CT slice images of the maize stem and leaf (Figure 2). The software usage steps are described as follows.
<ol>
	<li>Appoint the organ type to initialize different algorithm pipelines. Click the Method parameters button and select maize stem or maize leaf in the first drop-down box.</li>
	<li>To import the images, click the Data management button, set the work directory, and import automatically all slice images in this directory. Select single or multi-slice images into the image pipelines.</li>
	<li>Determine the actual pixel size of the image. Click the Method parameters button and enter the actual pixel size of the image in the edit item of the pixel size.</li>
	<li>For the phenotyping computation, click the Phenotyping computation button to automatically extract phenotypic traits of the vascular bundles for all selected slice images.</li>
	<li>Click the Statistic analysis button to output the results as a TXT or CSV file format.</li>
</ol>
4. Image Analysis Protocol for CT Image Series of a Maize Root
NOTE: The CT image series of maize roots are utilized to extract the 3-D structures of metaxylem vessels using image-processing software. The main steps are as follows.
<ol>
	<li>Import the reconstructed images of maize roots (in BMP file format) and determine the accurate spacing parameters (the size of a voxel [i.e., x, y, z]). Use the recursive Gaussian tool to smoothen these images to improve the image quality.</li>
	<li>Conduct 3-D segmentation of metaxylem vessels by adjusting the threshold parameters; this generates a uniform color label for each connected metaxylem vessel.</li>
	<li>Improve and identify the metaxylem vessels interactively using morphology, bitwise, and flood-fill operations.</li>
	<li>Conduct volume visualization and surface reconstruction of the vessels. Use the mask statistics tool to count and measure the phenotypic traits of a vessel in the 2-D and 3-D levels.</li>
</ol>

<ol>
	<li>Lucas, W. J., 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=The+plant+vascular+system:+evolution,+development+and+functions.">The plant vascular system: evolution, development and functions.</a> Journal of Integrative Plant Biology. 55, 294-388 (2013).</li><li>Gou, L., 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=Effect+of+population+density+on+stalk+lodging+resistant+mechanism+and+agronomic+characteristics+of+maize.">Effect of population density on stalk lodging resistant mechanism and agronomic characteristics of maize.</a> Acta Agronomica Sinia. 33, 1688-1695 (2007).</li><li>Hu, H., 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=QTL+mapping+of+stalk+bending+strength+in+a+recombinant+inbred+line+maize+population.">QTL mapping of stalk bending strength in a recombinant inbred line maize population.</a> Theoretical and Applied Genetics. 126, 2257-2266 (2013).</li><li>Wilson, J. R., Mertens, D. R., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolates+of+cell+types+from+sorghum+stems:+Digestion,+cell+wall+and+anatomical+characteristics.">Isolates of cell types from sorghum stems: Digestion, cell wall and anatomical characteristics.</a> Journal of the Science of Food and Agriculture. 63, 407-417 (1993).</li><li>Hatfield, R., Wilson, J., Mertens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+cell+walls+isolated+from+cell+types+of+grain+sorghum+stems.">Composition of cell walls isolated from cell types of grain sorghum stems.</a> Journal of the Science of Food and Agriculture. 79, 891-899 (1999).</li><li>Du, J., 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=Micron-scale+phenotyping+quantification+and+three-dimensional+microstructure+reconstruction+of+vascular+bundles+within+maize+stems+based+on+micro-CT+scanning.">Micron-scale phenotyping quantification and three-dimensional microstructure reconstruction of vascular bundles within maize stems based on micro-CT scanning.</a> Functional Plant Biology. 44 (1), 10-22 (2016).</li><li>Pan, X., 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=Reconstruction+of+Maize+Roots+and+Quantitative+Analysis+of+Metaxylem+Vessels+based+on+X-ray+Micro-Computed+Tomography.">Reconstruction of Maize Roots and Quantitative Analysis of Metaxylem Vessels based on X-ray Micro-Computed Tomography.</a> Canadian Journal of Plant Science. 98 (2), 457-466 (2018).</li><li>McElrone, A. J., Choat, B., Parkinson, D. Y., MacDowell, A. A., Brodersen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+resolution+computed+tomography+to+visualize+the+three+dimensional+structure+and+function+of+plant+vasculature.">Using high resolution computed tomography to visualize the three dimensional structure and function of plant vasculature.</a> Journal of Visualized Experiments. (74), e50162 (2013).</li><li>Cloetens, P., Mache, R., Schlenker, M., Lerbs-Mache, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phase+tomography+of+Arabidopsis+seeds+reveals+intercellular+void+network.">Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 103, 14626-14630 (2006).</li><li>Dorca-Fornell, C., 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=Increased+leaf+mesophyll+porosity+following+transient+retinoblastoma-related+protein+silencing+is+revealed+by+microcomputed+tomography+imaging+and+leads+to+a+system-level+physiological+response+to+the+altered+cell+division+pattern.">Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern.</a> Plant Journal. 76 (6), 914-929 (2013).</li><li>Verboven, P., 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=Void+space+inside+the+developing+seed+of+Brassica+napus+and+the+modelling+of+its+function.">Void space inside the developing seed of Brassica napus and the modelling of its function.</a> New Phytologist. 199, 936-947 (2013).</li><li>Brodersen, C. R., Roark, L. C., Pittermann, 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+physiological+implications+of+primary+xylem+organization+in+two+ferns.">The physiological implications of primary xylem organization in two ferns.</a> Plant, Cell &amp; Environment. 35, 1898-1911 (2012).</li><li>Choat, B., Brodersen, C. R., McElrone, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrotron+X-ray+microtomography+of+xylem+embolism+in+Sequoia+sempervirens+saplings+during+cycles+of+drought+and+recovery.">Synchrotron X-ray microtomography of xylem embolism in Sequoia sempervirens saplings during cycles of drought and recovery.</a> New Phytologist. 205, 1095-1105 (2015).</li><li>Torres-Ruiz, J. M., 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=Direct+x-ray+microtomography+observation+confirms+the+induction+of+embolism+upon+xylem+cutting+under+tension.">Direct x-ray microtomography observation confirms the induction of embolism upon xylem cutting under tension.</a> Plant Physiology. 167, 40-43 (2015).</li><li>Staedler, Y. M., Masson, D., Sch&#246;nenberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+tissues+in+3D+via.+x-ray+tomography:+simple+contrasting+methods+allow+high+resolution+imaging.">Plant tissues in 3D via. x-ray tomography: simple contrasting methods allow high resolution imaging.</a> PLoS One. 8, 75295 (2013).</li><li>Zhang, Y., Legay, S., Barri&#232;re, Y., M&#233;chin, V., Legland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+quantification+of+stained+maize+stem+section+describes+lignin+spatial+distribution+within+the+whole+stem.">Color quantification of stained maize stem section describes lignin spatial distribution within the whole stem.</a> Journal of the Science of Food and Agriculture. 61, 3186-3192 (2013).</li><li>Legland, D., Devaux, M. F., Guillon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+mapping+of+maize+bundle+intensity+at+the+stem+scale+using+spatial+normalisation+of+replicated+images.">Statistical mapping of maize bundle intensity at the stem scale using spatial normalisation of replicated images.</a> PLoS One. 9 (3), 90673 (2014).</li><li>Heckwolf, S., Heckwolf, M., Kaeppler, S. M., de Leon, N., Spalding, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+analysis+of+anatomical+traits+in+stem+transections+of+maize+and+other+grasses.">Image analysis of anatomical traits in stem transections of maize and other grasses.</a> Plant Methods. 11, 26 (2015).</li><li>Wu, H., Jaeger, M., Wang, M., Li, B., Zhang, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+distribution+of+vessels,+passage+cells+and+lateral+roots+along+the+root+axis+of+winter+wheat+(Triticum+aestivum).">Three-dimensional distribution of vessels, passage cells and lateral roots along the root axis of winter wheat (Triticum aestivum).</a> Annals of Botany. 107, 843-853 (2011).</li><li>Chopin, J., Laga, H., Huang, C. Y., Heuer, S., Miklavcic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RootAnalyzer:+A+Cross-Section+Image+Analysis+Tool+for+Automated+Characterization+of+Root+Cells+and+Tissues.">RootAnalyzer: A Cross-Section Image Analysis Tool for Automated Characterization of Root Cells and Tissues.</a> PLoS One. 10, 0137655 (2015).</li><li>Passot, 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=Characterization+of+pearl+millet+root+architecture+and+anatomy+reveals+three+types+of+lateral+roots.">Characterization of pearl millet root architecture and anatomy reveals three types of lateral roots.</a> Frontiers in Plant Science. 7, 829 (2016).</li></ol>

The authors have nothing to disclose.

With the successful application of CT technology in the fields of biomedicine and materials science, this technology has been gradually introduced into the fields of botany and agriculture, promoting researches in plant life sciences as a promising technical tool. In the late 1990s, CT technology was first used to study the morphological structures and development of plant root systems. In the past decade, synchrotron HRCT has become a powerful, nondestructive tool for plant biologists, and has been successfully used to ...

The maize vascular system runs through the entire plant, from the root and stem to the leaves, which forms the key transportation paths for delivering water, mineral nutrients, and organic substances1. Another important function of the vascular system is to provide mechanical support for the maize plant. For example, the morphology, number, and distribution of vascular bundles in roots and stems are closely related to the lodging resistance of maize plants2,3. At present, studies on the anatomical structure of vascular bundles mainly utilize microscopic and ultramicroscopic techniques to display the anatomical structures of a certain part of the stem, leaf, or root, and then measure and count these structures of interest by manual investigation. Undoubtedly, manual measurement of various microscopic structures in large-scale microimages is a very tedious and inefficient work and severely limits the precision of microphenotypic traits, owing to its subjectivity and inconsistency4,5.
Maize has no secondary growth, and the cell content essentially consists of water in the primary meristem. Without any pretreatment, fresh samples of maize tissues can be directly scanned using a micro-CT device; however, the scanning results are probably poor and rough. The main reasons are summarized as follows: (1) low attenuation densities of plant tissues, resulting in a low contrast of atomic number and high noise in images; (2) fresh plant materials are prone to dehydrate and shrink during the normal scanning environment, as reported by Du6. The abovementioned problems have become the main constraints for the development and application of microphenotyping technology for maize, wheat, rice, and other monocotyledons. Here, we introduce the &#39;sample preparation protocol&#39; to pretreat the samples of maize stem, leaf, and root. This protocol avoids the dehydration and deformation of plant materials during the CT scanning; thus, it is beneficial to increase the preservation time of plant samples with nondeformation. Moreover, the dyeing step based on solid iodine also enhances the contrast of plant materials; thus, it makes significant improvements in the imaging quality of micro-CT. Furthermore, we developed image processing software, named VesselParser, to process the CT images of maize stems and leaves. This software integrates a set of image-processing pipelines to perform high-throughput and automatic phenotyping analysis for 2-D CT images of different plant tissues. Vascular bundles in the entire cross-section of the maize stem and leaf are detected, extracted, and identified using an automatic image-processing method. As a result, we obtain 31 microscopic phenotypes of the maize stem and 33 microscopic phenotypes of the maize leaf. For the CT image series of the maize root, we developed an image-processing scheme to acquire 3-D phenotypic traits of metaxylem vessels. This scheme is superior in efficiency of image acquisition and reconstruction compared with traditional methods.
These results indicate that the image processing pipelines considering the imaging characteristics of ordinary X-ray micro-CT provide an effective method for the microscopic phenotyping of vascular bundles; this extremely widens the applications of CT techniques in plant science and improves the automatic phenotyping of plant materials at cellular resolution6,7.

<table><tbody><tr><td>Skyscan 1172 X-ray computed tomography system</td><td>Bruker Corporation, Belgium</td><td>NA</td><td>For CT scanning</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; critical point drying system (Leica CPD300)</td><td>Leica Corporation, Germany</td><td>NA</td><td>For sample drying</td></tr><tr><td>Ethanol</td><td>Any</td><td>NA</td><td>For FAA fixation</td></tr><tr><td>Formaldehyde</td><td>Any</td><td>NA</td><td>For FAA fixation</td></tr><tr><td>Acetic acid</td><td>Any</td><td>NA</td><td>For FAA fixation</td></tr><tr><td>Surgical blade</td><td>Any</td><td>NA</td><td>For cutting the sample sgements</td></tr><tr><td>3D printer</td><td>Makerbot replicator 2, MakerBot Industries, USA</td><td>NA</td><td>For printing the sample baskets of maize root, stem, and leaf</td></tr><tr><td>Centrifuge tube</td><td>Corning, USA</td><td>NA</td><td>Place the root, stem, or leaf materials</td></tr><tr><td>Solid iodine</td><td>Any</td><td>NA</td><td>For sample dyeing</td></tr><tr><td>SkyScan Nrecon software</td><td>SkyScan NRecon, Version: 1.6.9.4, Bruker Corporation, Belgium</td><td>NA</td><td>For image reconstruction</td></tr><tr><td>VesselParser software</td><td>VesselParser, Version: 3.0, National Engineering Research Center for Information Technology in Agriculture (NERCITA), Beijing, China</td><td>NA</td><td>Image analysis protocol for single CT image of maize stem or leaf</td></tr><tr><td>ScanIP</td><td>ScanIP, Version: 7.0; Simpleware, Exeter, UK</td><td>NA</td><td>3D image processing software</td></tr><tr><td>Latex gloves</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Tweezers</td><td>Any</td><td>NA</td><td></td></tr></tbody></table>

micron-scale phenotyping techniques of maize vascular bundles based on x-ray microcomputed tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

The sample preparation protocol suitable for ordinary micro-CT scanning not only prevents the deformation of plant tissues but also enhances the X-ray absorption contrast. Pretreated plant materials are scanned using a micro-CT system into high-quality slice images, and the highest resolution can reach 2 &#181;m/pixel. Figure 4 shows the scanned micro-CT images of stem, leaf, and root, and the image contrast has a significant improvement compared with the res...

Watch this Scientific Journal Video about Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography at JoVE.com

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

micron-scale-phenotyping-techniques-maize-vascular-bundles-based-on-x

Research

JoVE Journal

Biology

8.7K Views.  Beijing Academy of Agriculture and Forestry Sciences. We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Video: Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochemistry

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Environment

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Bioengineering

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for&#160;euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

Micro-CT is a non-destructive tool that can analyze plant structures in three dimensions. The present protocol describes the sample preparation to leverage micro-CT to analyze parasitic plant structure and function. Different species are used to highlight the advantages of this method when coupled with specific preparations.

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Improved Methods for Imaging Maize Leaf Primordia

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

Author Spotlight: Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging  

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

Potato Resilience Analysis Under Climate-Related Stresses

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Microscopical Analysis for Enhancing Photosynthetic Efficiency in C4 Crops

Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The effectiveness of C4 photosynthesis and intrinsic water use efficiency are directly linked to the share of mesophyll and bundle leaf cells,&#160;size and density of bundle sheaths, and size, density, and cell wall thickness of bundle sheath cells. Rapid microscopical analysis of these traits can be performed on free-hand and semi-thin sections using conventional light microscopy, providing valuable information about photosynthetic efficiency in C4 crops by means of identifying and examining specific cell types. Additionally, errors in freehand and semi-thin section preparation that affect anatomical measurements and cell type diagnoses are shown, as well as how to avoid these errors. This microscopical approach offers an efficient means of gathering insights into photosynthetic acclimation to environmental variation and aids in the rapid screening of crops for future climates.

Author Spotlight: Innovative Approaches to Understanding Plant Structure-Function Relationships for Climate-Resilient Crops

Assessing the anatomical differences between C3 and C4 leaf cross sections helps understand photosynthesis efficiency. This paper describes free-hand and semi-thin leaf cross sections preparation and examination, along with the caveats in the preparation for the crop species Triticum aestivum and Zea mays.

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The ...

Imaging and Analysis for Quantifying Maize (Zea mays) Abiotic Stress Phenotypes

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic stresses. However, traditional methods of measuring plants can be time-consuming, inaccurate, and destructive, particularly when assessing traits such as biomass and height on an individual basis. Image-based analysis offers an opportunity to reduce labor, improve accuracy and precision, and enable repeated measurements of the same plant over multiple time points. While high-throughput phenotyping facilities provide a solution, they are often prohibitively expensive and limited in availability worldwide. A method is described for capturing plant images using low-cost single-board computers and digital cameras within a photo studio setup, accompanied by an image analysis pipeline based on the free and open-source package PlantCV. This method offers a fast and accurate protocol for image capture, labeling, and transfer, along with best practices for ensuring high-quality image acquisition. Through image analysis, more than 10 phenotypes were quantified for all images in the experiment simultaneously, including traits such as plant size, height, and color. These methods were used to characterize the response of the Zea mays (maize) inbred variety B73 to heat, drought, and combined heat and drought abiotic stress conditions.

A method is presented for low-cost imaging of plants to measure phenotypes, along with best practices for image capture and an image analysis pipeline for quantifying plant traits. These methods were applied to measure maize (Zea mays) phenotypes under heat, drought, and combined abiotic stress conditions.

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

Summary

Explore More Videos

Chapters in this video

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

Assessing Structural Traits in Triticum aestivum and Zea mays for C₃ and C₄ Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

Imaging and Analysis for Quantifying Maize (Zea mays) Abiotic Stress Phenotypes