﻿imaging transverse sections of maize leaf primordia

Métodos aprimorados para imagens de primórdios de folhas de milho

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>

Autor em destaque: Métodos aprimorados para preparar seções transversais e montagens inteiras desenroladas de primórdios de folhas de milho para fluorescência e imagens confocais  

Métodos aprimorados para preparar seções transversais e montagens inteiras não enroladas de primordia de folhas de milho para fluorescência e imagens confocais

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 Transverse Sections of Maize Leaf Primordia at JoVE.com

Imaging Transverse Sections of Maize Leaf Primordia  

Imagem de seções transversais de Primordia foliar de milho

Comece cortando a muda de milho envelhecida desejada no tecido mesocótilo usando uma pequena faca ou tesoura. Limpe a muda com um pincel. Em seguida, segure o decapador de arame com as mandíbulas voltadas para a calha.Posicione a calha no orifício selecionado e aperte as alças do stripper juntas. Deslize o stripper para longe da calha para aparar a parte superior das folhas. Use uma régua para medir o comprimento da ponta do primórdio até a base da calha.Para iniciar a imagem do primórdio foliar, mantenha a calha firme em uma superfície lisa sob um microscópio estéreo com cerca de 0,8x de aumento. Usando uma lâmina de barbear ou bisturi, faça um corte inicial ligeiramente acima da região alvo para descartar a porção distal da calha. Em seguida, obter seções de cerca de 0,25 a 0,8 milímetros de espessura nos pontos desejados ao longo do comprimento do primórdio.Uma vez feito, monte a seção da folha em uma lâmina de vidro limpa. Aplique algumas gotas de água diretamente na seção usando uma pipeta. E coloque uma tampa por cima.Aplique mais algumas gotas através das bordas da tampa, se necessário. Coloque a lâmina no palco do microscópio de epifluorescência e ajuste as configurações para visualizar o fluoróforo. Usando este método, a amostragem de ressecção casual foi padronizada medindo-se os primórdios antes da secção.Uma tendência foi descoberta mostrando que a borla dois tinha um primórdio mais largo e mais nervuras do que o normal, indicando que o defeito no mutante começou no início do desenvolvimento da folha. Este método também possibilitou o exame sistemático dos padrões de expressão de repórteres de proteínas fluorescentes de resposta hormonal nos primórdios foliares.

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JoVE Journal

Biology

﻿Imaging Transverse Sections 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 ...

Biologia

Imaging Unrolled Whole Mounts of Maize Leaf Primordia

Imagem de montagens inteiras não enroladas de Primordia de folha de milho