Name: Imaging Unrolled Whole Mounts of Maize Leaf Primordia
Uploaded: 2023-09-22T13:00:00
Duration: 2 min 48 s
Description: Watch this Scientific Journal Video about Imaging Unrolled Whole Mounts of Maize Leaf Primordia at JoVE.com

imaging unrolled whole mounts of maize leaf primordia

Maize Leaf Primordia를 이미징하는 개선된 방법

Grass crops are a major source of food and biofuel for the global population1, and improving leaf anatomy has the potential to increase their productivity2,3. However, our current understanding of how leaf anatomy is regulated in grasses is limited4 and requires the analysis of leaf primordia, as many anatomical and physiological traits of the leaf are predetermined early in development5,6,7. Cellular imaging techniques, such as fluorescence and confocal imaging, are indispensable for studying grass leaf anatomy and cellular traits, but these techniques are difficult to apply to grass leaf primordia because they are deeply ensheathed and rolled within the leaf whorl. We addressed this issue by developing methods for preparing transverse sections and unrolled whole-leaf mounts for fluorescence and confocal analysis of maize leaf primordia, a model system for studying grass leaf anatomy and development2,8.
The maize leaf, like all grass leaves, consists of a strap-like blade with a sheath that wraps around the stem and developing shoot9,10,11,12,13. The leaves develop from the shoot apical meristem (SAM) in a distichous pattern, where each new leaf initiates in the opposite position of the previous leaf, resulting in two ranks of leaves along the vertical axis&#160;(Figure 1A)14 . The developmental stage of each leaf primordium is identified by its position relative to the SAM, with the closest primordium designated as plastochron1 (P1) and the following primordia designated as P2, P3, and so on (Figure 1B,C)2. During development (Figure 1D), the leaf primordium first appears as a crescent-shaped buttress around the base of the SAM (P1), and then grows into a hood-shaped primordium that extends over the meristem (P2)9,10,11. The basal margins of the hood then expand laterally and overlap each other as the tip grows upward, forming a cone-shaped primordium (P3-P5)10. The primordium then rapidly grows in length, and the sheath-blade boundary at the base becomes more prominent with the formation of the ligule, the fringe-like projection on the adaxial side of the leaf (P6/P7). Finally, the leaf unrolls as it emerges from the whorl during steady-state growth, in which the dividing cells are restricted within the small basal region of the blade, forming a gradient with expanding and differentiating cells along the proximal-distal axis (P7/P8)15. The shoot apex of a maize seedling contains multiple primordia at different stages of development, making it an excellent model for studying leaf development8.
Accurate analysis of early leaf development requires staging or the use of standardized criteria to define distinct stages of primordium development in relation to other growth or morphological parameters. Because the leaf primordia are hidden within the grass shoot, investigators typically use parameters such as the age of the plant or the size of the emerging leaves as predictors for the stages and sizes of the leaf primordia9,16. In maize, the chronological age of the plant is determined either by the number of days after planting or germination (DAP/DAG)17,18. The vegetative stage (V stage) is determined by the uppermost leaf with a visible collar, a pale line on the abaxial side between the blade and the sheath that corresponds to the position of the ligule and auricles, a pair of wedge-shaped regions at the base of the blade&#160;(Figure 1A,B)17,19 . Between 20 and 25 DAG, the SAM transitions into an inflorescence meristem and ceases to produce new leaves20. The growth rates of maize leaf primordia can vary depending on the environment and the genotype of the plant. For this reason, plant age and the size of the emerging leaves cannot accurately predict the sizes of leaf primordia; however, using these parameters can help predict the range of primordia stages and sizes for experimental purposes.
Transverse section analysis is a popular method for examining leaf anatomy and development in maize and other grasses because it allows for the sampling of multiple plastochrons in a single section across the shoot21,22,23. This method is also convenient for cellular imaging of fresh samples, as the surrounding leaves serve as a scaffold&#160;that keeps the leaf primordia in place during sectioning and mounting24. However, a disadvantage of this method is that it can be challenging to precisely locate the target plastochron and region within the primordium when sectioning from an intact shoot. Furthermore, because leaf growth varies across plastochrons and along the proximal-distal axis2,5, imprecise sampling could result in an incorrect interpretation of the developmental stage and region of the primordium in a given section. Therefore, developing a method for precise transverse section sampling is critical for ensuring the accuracy and reproducibility of anatomical and developmental analyses of grass leaf primordia.
Whole-leaf mount analysis enables the comprehensive and integrative investigation of tissue and cellular processes that occur at the whole-organ scale, such as proliferative growth25 and vein patterning26,27,28. The method provides a paradermal overview of the leaf, allowing the discovery of distinct processes and patterns that would otherwise be difficult to detect using transverse section analysis24,27. Unlike in Arabidopsis, where there are already established methods for imaging whole-leaf mounts29,30, there is currently no standard method for imaging unrolled whole-leaf mounts in grasses. A previous protocol for unrolling isolated maize leaf primordia involved uncommon materials and was not suitable for cellular imaging31. Advanced imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI), can acquire 3D anatomical information without isolating and unrolling the primordia11,32,33, but they are expensive and require specialized equipment. Developing a technique to overcome the constraints imposed by the rolled and conical morphology of leaf primordia in maize and other grasses would advance investigations into their anatomical and developmental traits.
Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. We used these methods to quantify vein number and map the spatial-temporal hormone distribution in maize leaf primordia with fluorescent proteins (FPs)24. The first method involves removing the upper portion of older leaves from maize seedlings with a wire stripper (Figure 1E). By exposing the tip of the primordium (P5-P7), it becomes possible to determine its length without having to completely remove the older surrounding leaves, thus enabling easy and accurate sectioning. The second method involves unrolling and mounting whole-leaf primordia (P3-P7) with clear, double-sided nano tape (Figure 1F). These methods are suitable for visualizing various FPs24&#160;but need optimization for using fluorescent dyes and clearing reagents. In addition, we outline some procedures for flattening z-stacks, stitching images, and merging channels in ImageJ/FIJI34, which apply to the images produced by the two methods. These methods are useful for routine fluorescence or confocal imaging of maize leaves, but they can also be adapted for other model grass species, such as rice, Setaria, and Brachypodium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65239/65239fig01.jpg" /> 
Figure 1: Organization and morphology of maize leaf primordia and overview of the methods. (A) Schematic representation of a maize seedling. Maize has a distichous phyllotaxy, with the new leaf initiating at the opposite position of the previous leaf. The leaf number indicates the chronological order in which the leaves emerged from germination (i.e., first leaf, L1; second leaf, L2; third leaf, L3; etc.). Each leaf has a distal blade and a basal sheath delineated by a collar that corresponds to the ligule and auricle. The uppermost leaf with the collar visible denotes the vegetative stage. The seedling in this example is at the V2 stage, with the L2 collar (arrowhead) visible. The scissors icon indicates the location at the mesocotyl (me) where the seedling must be cut in order to be collected. (B) Schematic representation of the dissected shoot showing isolated L1 to L4, with the leaf primordia L5 to L9 shown as an enlarged image in (C). The plastochron number indicates the position of the primordium relative to the SAM, with the youngest leaf primordium (P1) closest to the SAM and the older leaf primordia (P2, P3, P4, and so on) successively farther away2. (D) Schematic representation of the morphology of maize leaf primordia from P1 to P5. (E) Schematic overview of the method for transverse section analysis of the maize leaf primordia. (1) Trim the older leaves with a wire stripper. (2) Measure the primordium and section the shoot. (3) Mount the section on a slide for imaging and processing (4, 5). (F) Schematic overview of the method for whole-mount analysis of the maize leaf primordia. (1) Remove the surrounding leaves to extract the primordium. (2) Cut and unroll the primordium flat on the nano tape. (3) Mount the sample for imaging and processing (4, 5). Abbreviations: L = leaf; bl = blade; sh = sheath; co = collar; me = mesocotyl; V = vegetative; P = plastochron; SAM = shoot apical meristem. <a href="https://www.jove.com/files/ftp_upload/65239/65239fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

저자 스포트라이트: 형광 및 컨포칼 이미징을 위한 옥수수 잎 Primordia의 횡절면 및 펼쳐진 전체 마운트를 준비하는 개선된 방법  

형광 및 공초점 이미징을 위해 횡단면을 준비하고 옥수수 잎 원시의 전체 마운트를 펼치기 위한 개선된 방법

Grasses like maize have leaf primordia grow within the shoot, making them difficult to study. We address this problem with improved protocols for preparing maize leaf transverse sections and whole mounts for fluorescence and confocal imaging. The first protocol uses a wire stripper to cut the older leaves, allowing the measurement of the primordium prior to sectioning.The second protocol uses clear, double-sided nano tape to unroll whole leaf primordium. These protocols improve the accuracy of transverse sectioning and enable unrolling of the leaf primordia, which have been very difficult to achieve due to their rolled, conical morphology. These methods will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grasses.Based on these methods, we plan to develop live cell imaging strategies to address questions in grass leaf development.

Watch this Scientific Journal Video about Imaging Unrolled Whole Mounts of Maize Leaf Primordia at JoVE.com

Imaging Unrolled Whole Mounts of Maize Leaf Primordia  

이미징 옥수수 잎 Primordia의 전체 마운트를 펼쳤습니다.

투명한 양면 나노 테이프의 직사각형 조각을 자르고 테이프 상단의 보호 플라스틱 필름을 제거하지 않고 깨끗한 유리 슬라이드의 중앙에 붙임으로써 유리 슬라이드 준비를 시작합니다. 다음으로, 와이어 스트리퍼로 오래된 잎의 윗부분을 제거하여 슈트를 해부합니다. 그런 다음 중풍 장치로 슈트를 잡고 치과 용 탐침으로 주변 잎을 조심스럽게 제거하여 관심있는 원시를 추출하십시오.원시를 장착하려면 테이프에서 보호 필름을 제거하십시오. 노출 된 원시를 테이프에 놓습니다. 면도날을 사용하여 기저부에서 primordium을 자르고, 기초 줄기와 hypocaudal을 버립니다.더 부드럽게 풀려면 바늘 끝을 100% 글리세롤에 담가 내부 또는 인접 잎 표면에 윤활유를 바릅니다. 구부러진 바늘로 치과 용 프로브를 사용하여 primordium을 펼칩니다. 바늘 끝을 표면과 평행하게 놓고 테이프에 부드럽게 눌러 바깥쪽 기저부 가장자리를 펼칩니다.바깥 쪽 여백이 테이프에 붙어 바늘 끝을 잎의 장축과 평행하게 맞 춥니 다. 바늘을 옆으로 부드럽게 밀어 잎을 펼치고 테이프에 평평하게 만듭니다. 펼쳐진 원초에 물 한 방울을 바르십시오.즉시 물방울과 원초에 덮개 슬립을 놓습니다. 커버 슬립의 가장자리를 부드럽게 눌러 테이프에 붙입니다. 완료되면 슬라이드를 에피형광 현미경 스테이지에 올려 형광단을 시각화합니다.이 방법을 따라 옥수수 잎 원시에서 형광 단백질 발현을 시각화하고 분석했습니다. 횡단면 분석을 보완합니다. 전체 잎 마운트 분석은 정맥 형성 동안 공식적인 반응의 조직 및 단계별 패턴을 보여주었습니다.전체 마운트 분석 방법을 통해 옥수수 잎 원시에서 형광 단백질 발현의 정성적 및 정량적 분석이 가능했으며, 이는 압연 형태로 인해 이미지화하기 어려운 옥수수 잎 원시를 검사하는 데 이 방법의 효과를 나타냅니다.

imaging-unrolled-whole-mounts-of-maize-leaf-primordia

Research

JoVE Journal

Biology

Imaging Unrolled Whole Mounts of 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.

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 ...

연구

생물학

﻿Imaging Transverse Sections of Maize Leaf Primordia

Imaging Transverse Sections of Maize Leaf Primordia  

Begin by cutting the desired aged maize seedling at the mesocotyl tissue using a small knife or pair of scissors. Clean the seedling with a paintbrush. Then hold the wire stripper with its jaws facing the chute.Position the chute to the selected hole and squeeze the stripper handles together. Slide the stripper away from the chute to trim the upper portion of the leaves. Use a ruler to measure the length from the primordium's tip to the chute's base.To begin the leaf primordium imaging, hold the chute steady on a smooth surface under a stereo microscope at around 0.8x magnification. Using a razor blade or scalpel, make an initial cut slightly above the target region to discard the distal portion of the chute. Then obtain around 0.25 to 0.8 millimeter thin sections at the desired points along the length of the primordium.Once done, mount the leaf section on a clean glass slide. Apply a few drops of water directly to the section using a pipette. And place a cover slip on top.Apply a few more drops through the edges of the cover slip if needed. Place the slide on the epifluorescence microscope's stage and adjust the settings to visualize the fluorophore. Using this method, chance resection sampling was standardized by measuring the primordia prior to sectioning.A trend was discovered showing that the vanishing tassel two had a wider primordia and more veins than normal, indicating that the defect in the mutant began early in the leaf development. This method also enabled the systematic examination of the expression patterns of hormone response fluorescent protein reporters in the leaf primordia.

옥수수 잎 Primordia의 가로 단면 이미징

Begin the preparation of the glass slide by cutting a rectangular piece of clear, double-sided nano tape and sticking it to the center of a clean glass slide without removing the protective plastic film on the top side of the tape. Next, dissect the chute by removing the upper portion of the older leaves with a wire stripper. Then hold the chute by the mesocaudal and carefully remove the surrounding leaves with a dental probe to extract the primordia of interest.For mounting the primordium, remove the protective film from the tape. Lay the exposed primordium on the tape. Cut the primordium at the base using a razor blade, discarding the basal stem and the hypocaudal.For smoother unrolling, lubricate the inner, or, adaxial leaf surface by dipping the needle tip into 100%glycerol. Unroll the primordium using a dental probe with a bent needle. Position the needle tip parallel to the surface and unfurl the outer basal margin by gently pressing it against the tape.With the outer margin adhering to the tape, align the needle tip parallel to the long axis of the leaf. Gently slide the needle sideways to unroll and flatten the leaf onto the tape. Apply a drop of water to the unrolled primordium.Immediately place a cover slip on the water drop and the primordium. Gently press down the edges of the cover slip so that they adhere to the tape. Once done, place the slide on the epifluorescence microscope stage to visualize the fluorophore.This method was followed to visualize and analyze the fluorescent protein expression in maize leaf primordia. Complementary to the transverse section analysis. The whole leaf mount analysis revealed tissue and stage-specific patterns of formal response during vein formation.Whole mount analysis method enabled qualitative and quantitative analyses of fluorescent protein expression in the maize leaf primordia, indicating the effectiveness of this method in examining the maize leaf primordia which are difficult to image due to their rolled morphology.