Published: September 22nd, 2023
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 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.
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 (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 (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 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 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.
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. Please click here to view a larger version of this figure.
1. Staging maize leaf development and designing the experiment
Figure 2: Sampling scheme for transverse section analysis of maize leaf primordia. (A, left) Proximal shoot of a 7 DAG maize seedling, showing an exposed fourth leaf (L4) at P7. The broken lines indicate the seven sampling points along the primordium, from 0.5 mm to 10 mm. (A, right) Schematic representation of the leaf primordia, with the projected size and position of each plastochron: P7 (white); P6 (magenta); P5 (blue); P4 (green); and P3 until the SAM (yellow). (B-H) Fluorescence images of transverse sections representing the sampling points shown in A from 10 mm (B) to 0.5 mm (H). The primordia are pseudocolored according to the plastochron color scheme in (A). The sections were imaged with an epifluorescence microscope using a longpass-emission UV filter for autofluorescence. Scale bar = 200 µm (B-H). This figure has been modified and reproduced with permission from Robil and McSteen24. Abbreviations: DAG = days after germination; P = plastochron; SAM = shoot apical meristem; † = leaf sheath or pre-ligule proper; * = shoot apical meristem; ** = stem. Please click here to view a larger version of this figure.
2. Imaging transverse sections of maize leaf primordia
Table 1: Illumination and image acquisition settings used for fluorescence and confocal imaging of selected maize FP reporters. Abbreviations: FP = fluorescent protein; TRITC = tetramethylrhodamine; FITC = fluorescein isothiocyanate; WLL = white light laser; Ar-ion = argon ion laser; HyD = hybrid detector; AU = Airy unit; Hz = Hertz, scan line per second. Please click here to download this Table.
3. Imaging unrolled whole mounts of maize leaf primordia
Table 2: Troubleshooting common problems in imaging transverse sections and whole mounts of maize leaf primordia. Please click here to download this Table.
Figure 3: Suboptimal transverse section and whole-mount preparation of maize leaf primordia. (A-D) Representative confocal images of transverse sections of leaf primordia with a plasma membrane marker, PIP2-1-CFP (A), and a plasma membrane-binding fluorescent dye, FM 4-64 (B), with corresponding bright-field images (C,D). When compared to PIP2-1-CFP, FM 4-64 displays suboptimal visualization of cell outlines. (E-M) Representative fluorescence images of whole mounts of leaf primordia showing the presence of tearing (E-G), bruised surfaces† (H), air bubbles* (I-K), rolled back margins (L), and photobleached regions‡ (M). The leaf primordia express DII-Venus (E-G), GAR2-YFP (H-J), mDII-Venus (K), PIN1a-YFP (L), and DR5-RFP (M). Scale bar = 200 µm (A-D); 500 µm (E-M). Figure 3A has been modified and reproduced with permission from Robil and McSteen24, while Figure 5B-M are unpublished data from the authors. Abbreviations: P = plastochron; YFP = yellow fluorescent protein; RFP = red fluorescent protein; CFP = cyan fluorescent protein; PIP2-1-CFP = pZmPIP2-1::ZmPIP2-1:CFP; DII-Venus = pZmUbi:DII:YFP-NLS; GAR2-YFP = pZmGAR2::ZmGAR2:YFP; mDII-Venus = pZmUbi:mDII:YFP-NLS; PIN1a-YFP = pZmPIN1a::ZmPIN1a:YFP; DR5-RFP = DR5rev::mRFPer; BF = bright-field. Please click here to view a larger version of this figure.
4. Processing the images using ImageJ/FIJI
Transverse section analysis of maize leaf primordia
We used protocol section 2 to quantify vein number and characterize hormone response patterns in transverse sections of maize leaf primordia with FPs (Figure 4)24. To assess the role of the plant hormone auxin in leaf growth and vein formation, we quantified the number of veins in the leaf primordia of an auxin-deficient maize mutant, vanishing tassel254. In early plastochrons, developing veins exhibit distinct cellularization in the median cell layer of the maize leaf primordium21,22. However, identifying and counting veins using conventional histological techniques22 can be labor-intensive and time-consuming. Thus, to quantify the veins, we utilized the maize auxin efflux protein marker pZmPIN1a::ZmPIN1a:YFP41 (PIN1a-YFP hereafter), which marks developing veins and procambial strands (PCSs; Figure 4A). Using protocol section 2, we were able to standardize the transverse section sampling by measuring the primordia prior to sectioning (Figure 4B,C). We discovered a trend in which vt2 has a wider primordium and more veins than normal (Figure 4D,E)24, which is consistent with data from fully expanded leaves55, indicating that the defect in vt2 began early in leaf development. Using protocol section 2, we were also able to systematically examine the expression patterns of hormone response FP reporters in the leaf primordia (see examples of the images in Figure 4F-I). Through a standardized transverse section sampling scheme, we mapped the distribution of auxin, cytokinin (CK), and gibberellic acid (GA) responses across different plastochrons and regions of leaf primordia, and discovered novel response patterns that we hypothesize have implications for leaf growth and vein formation24. Therefore, these representative results demonstrate the utility of protocol section 2 for transverse section analysis of maize leaf primordia.
Figure 4: Representative results for transverse section analysis of maize leaf primordia. (A-E) Quantification of vein number and primordium width in leaf primordia of normal and vanishing tassel2 with the auxin efflux protein marker PIN1a-YFP. (A) Representative fluorescence images of transverse sections of P5 to P7 expressing PIN1a-YFP in developing veins and procambial strands. The transverse sections were imaged with an epifluorescence microscope using a FITC filter (495-519 nm excitation). The number of veins and the primordium width were quantified using the multi-point and freehand line tools in FIJI/ImageJ, respectively. (B) A maize seedling with the upper leaf whorls removed with a wire stripper to expose the tip of the fourth leaf (L4). The bracket spans the projected primordium length, with the dashed line indicating the mid-length. (C) Schematic diagram of varying primordium shapes from P5 to P7, illustrating how vein number and primordium width at the mid-length (horizontal dashed line) can vary depending on the developmental stage of the primordium. (D,E) Measurement of primordium width (D) and vein number (E) at the mid-length section of the L4 of normal and vt2. The trendline represents 10 mm rolling averages of measurements ± standard error of the mean (SEM). (F-I) Representative confocal images of transverse sections of leaf primordia expressing combinations of PIN1a-YFP, auxin response reporter, DR5-RFP, cytokinin response reporter, TCS-Tomato, gibberellic acid-responsive marker, GAR2-YFP, and mDII-Venus, a mutated version of the auxin signaling input reporter DII-Venus. The FP channels are superimposed on the bright-field channel in each image. Scale bar = 200 µm (A); 10 mm (B); 100 µm (F-I). This figure has been modified and reproduced with permission from Robil and McSteen24. Abbreviations: DAG = days after germination; P = plastochron; vt2 = vanishing tassel2; PCS = procambial strand; YFP = yellow fluorescent protein; FITC = fluorescein isothiocyanate; RFP = red fluorescent protein; PIN1a-YFP = pZmPIN1a::ZmPIN1a:YFP; DR5-RFP = DR5rev::mRFPer; TCS-Tomato = TCSv2::NLS-tdTomato; GAR2-YFP = pZmGAR2::ZmGAR2:YFP; mDII-Venus = pZmUbi:mDII:YFP-NLS. Please click here to view a larger version of this figure.
Whole-mount analysis of maize leaf primordia
We followed protocol section 3 to visualize and analyze FP expression in whole-leaf mounts of maize leaf primordia (Figure 5). By visualizing vein patterns with PIN1a-YFP, we found that the formation of veins occurs in the entire primordium during early plastochrons, but this process becomes restricted in the proximal regions later in development (Figure 5A)24. Complementary to the transverse section analyses, the whole-leaf mount analyses have revealed tissue- and stage-specific patterns of hormone response during vein formation24. One example is the expression pattern of the CK response reporter TCSv2::NLS-tdTomato37 (TCS-Tomato), relative to PIN1a-YFP expression (Figure 5B)24. By following protocol section 3, we were able to perform both qualitative and quantitative analyses of FP expression in the leaf primordia (Figure 5D-F; unpublished data). We examined the expression patterns of an auxin response reporter, DR5rev::mRFPer41 (DR5-RFP), and a GA-responsive marker, pZmGAR2::ZmGAR2:YFP39 (GAR2-YFP), in whole-leaf mounts of single and double mutants of vt2 and dwarf plant3 (d3), a GA-deficient mutant56 (Figure 5D). We also compared the relative levels of cell proliferation between normal and vt2 leaf primordia using pZmCyclin-D2B::ZmCyclin-D2B:YFP42 (Cyclin-D2B-YFP), which is a marker for the G1/S transition in the cell cycle57 (Figure 5E,F). While there was no significant difference between normal and vt2, Cyclin-D2B-YFP expression was consistent with the known cell proliferation profile of early plastochrons31. We conclude that protocol section 3 is an effective method for analyzing whole mounts of maize leaf primordia, which are difficult to image due to their rolled morphology.
Figure 5: Representative results for whole-mount analysis of maize leaf primordia. (A) Representative fluorescence images of leaf primordia of 7 DAG maize seedlings showing developing veins and procambial strands, as marked by the auxin efflux protein marker PIN1a-YFP. P3-P6 primordia were excised from the base, unrolled, and flattened with the adaxial side up. Insets show close-ups of P3 and P4. In P5 and P6, dashed lines demarcate the distal end of the proliferative zones, where the majority of procambial strands are still developing and extending. (B,C) Representative confocal images showing the expression of PIN1a-YFP and cytokinin response reporter, TCS-Tomato, in the proximal marginal region of a P5 primordium. (D) Representative fluorescence images of P4 primordia showing the expression of auxin response reporter, DR5-RFP, and gibberellic acid-responsive marker, GAR2-YFP in normal and single and double mutants of vanishing tassel2 and dwarf plant3. (E) Representative confocal images of P3 and/or P4 showing the expression of Cyclin-D2B-YFP, a reporter for the G1/S transition in the cell cycle. (F) Relative amounts of cell proliferation in P3/P4 leaf primordia of normal and vt2, quantified by measuring the integrated density of the Cyclin-D2B-YFP signal over the area of the primordium using ImageJ/FIJI. The bars represent the mean measurements ± standard error of the mean. Scale bar = 500 µm (A,D,E); 200 µm (B); 100 µm (C). Figure 4A-C has been modified and reproduced with permission from Robil and McSteen24, while Figure 4D-F is unpublished data from the authors. Abbreviations: DAG = days after germination; P = plastochron; vt2 = vanishing tassel2; d3 = dwarf plant3; YFP = yellow fluorescent protein; RFP = red fluorescent protein; PIN1a-YFP = pZmPIN1a::ZmPIN1a:YFP; TCS-Tomato = TCSv2::NLS-tdTomato; DR5-RFP = DR5rev::mRFPer; GAR2-YFP = pZmGAR2::ZmGAR2:YFP; Cyclin-D2B-YFP = pZmCyclin-D2B::ZmCyclin-D2B:YFP; ns = no significant difference; au = arbitrary unit. Please click here to view a larger version of this figure.
Supplementary File 1: Examples of leaf development staging in maize seedlings. Please click here to download this File.
Supplementary Figure S1: Plant samples and materials used in the protocols. (A,B) A maize seedling 7-8 DAG with the upper leaf whorl removed using a wire stripper. (B) The inset shows a close-up of the shoot with the exposed P6 primordium. (C-G) Shoots of maize seedlings with the upper whorls removed for transverse section analysis (C,D) and surrounding leaves fully removed for whole-mount analysis (E-G). (H) A roll of polyurethane gel clear double-sided nano tape. (I,J) P6 leaf primordium (I) and proximal region of P8 leaf (J) unrolled and mounted on glass slides with the nano tape. (K) A glass slide with an unrolled leaf primordium mounted on the stage of an epifluorescence microscope. Abbreviation: DAG = days after germination. Please click here to download this File.
We present two methods for preparing maize leaf primordia for cellular imaging. The first method (protocol section 2) allows measurement of the primordium for transverse section analysis, while the second method (protocol section 3) enables unrolling and flattening of the primordium for whole-mount analysis. These methods facilitate the cellular imaging of FPs in maize leaf primordia24 (as shown in Figure 4 and Figure 5) and provide simple solutions to the challenges of imaging developing maize leaves. Protocol section 2 reduces dissection time and improves sampling accuracy by measuring the primordia prior to sectioning rather than relying solely on staging parameters9,16. With commercially available nano tape, protocol section 3 solves the long-standing problem of imaging whole-leaf primordia in maize. This protocol improves on the previous method, which used dialysis tubing31, and is a much cheaper alternative to CT and MRI11,32,33. However, when it comes to visualizing leaf anatomical traits and producing optimal results, both protocols have some limitations, which are outlined in Table 2 and are discussed in more detail below.
In protocol section 2, we encountered difficulty visualizing cell outlines in thick transverse sections of the leaf primordia, and counterstaining with cell wall- or plasma membrane-binding fluorescent dyes did not provide satisfactory results. For instance, FM 4-64 produced suboptimal results compared to the plasma membrane FP marker, pZmPIP2-1::ZmPIP2-1:CFP39 (PIP2-1-CFP; Figure 3A-D). To overcome this limitation, we recommend using a vibratome to produce thinner tissue sections (~0.1 mm)58 which will allow for a vivid bright-field imaging of cell outlines or optimizing the counterstaining protocol47,59.
In protocol section 3, the main limitation is the difficulty of mounting the leaf without tearing, damage, or air bubbles, as detailed in protocol steps 3.2.5-3.2.6 (Figure 3E-K). Because the maize leaf is bilaterally symmetrical, a half-leaf mount rather than a whole-leaf mount may be sufficient for visualization9. To do this, the primordium can be cut with a razor blade along the longitudinal axis after unrolling it up to the midrib, allowing only half of the leaf to be mounted. Another limitation of protocol section 3 is that the thickness of the leaf can limit the optical resolution of the fluorophore signal during deep imaging. To address this issue, it is possible to employ a tissue clearing technique60. However, we found that ClearSee61, a commonly used clearing reagent for imaging plant tissues, is not compatible with the protocol because it causes the sample and coverslip to detach from the nano tape. A potential solution to this problem could be to apply a semipermeable membrane31 over the leaf sample, allowing it to be treated with the clearing solution while being held in place by the nano tape. Such a method that allows liquid solutions to be applied to the unrolled leaf could also be used for whole-mount RNA in situ hybridization and immunolocalization techniques, which have previously been optimized for developing maize inflorescences but not for whole-leaf primordia62,63.
We described protocols for maize, which has large leaf primordia even at the seedling stage. Other grass species with much smaller leaf primordia, such as rice, barley, wheat, Setaria, and Brachypodium16,23,64,65,66, may require the use of additional precision tools to effectively apply these protocols. Furthermore, these protocols were not intended for live cell imaging, which captures real-time dynamic processes of tissue formation and cellular responses. However, as fluorescent probes, imaging technologies, and computing capabilities continue to advance in live cell imaging for plants67, future research could build on these protocols to develop live cell imaging strategies tailored to the unique features of grass leaf primordia.
The authors do not have any conflicts of interest to disclose.
The authors would like to thank the Maize Genetics Cooperation, the Maize Cell Genomics Project, Dave Jackson (Cold Spring Harbor Laboratory, NY), Anne W. Sylvester (Marine Biological Laboratory, University of Chicago, IL), Andrea Gallavotti (Rutgers University, NJ), and Carolyn G. Rasmussen (University of California, Riverside) for providing the mutant and transgenic stocks, as well as Robert F. Baker and Alexander Jurkevich of the Advanced Light Microscopy Core at the University of Missouri-Columbia for their assistance with confocal microscopy. JMR was supported by the J. William Fulbright Fellowship, The Diane P. and Robert E. Sharp Fund, and the National Science Foundation's Plant Genome Research Program (IOS-1546873) to PM. CDTC, CMRV, EDCDP, and RJRR are supported by the Ateneo College Scholarship Program. CDTC, EDCDP, and RJRR are supported by the DOST-SEI S&T Undergraduate Scholarship. DODL is supported by Fr. Thomas Steinbugler SJ Academic Scholarship. RJRR is supported by Aiducation International–Pathways to Higher Education Scholarship. This work was supported by the School of Science and Engineering and the Rizal Library, Ateneo de Manila University.
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