Annis E. Richardson

In plants, final organ shape and position influence function and whole plant productivity. In Zea mays (maize), leaf, ear, and tassel morphology are all key agronomic features influencing yield. These aerial structures originate from stem cell populations within meristems. Understanding how meristems are regulated and how stem cells differentiate over developmental time to form mature leaves, ears, and tassels is important for identifying novel routes for maize improvement. High-resolution imaging of meristem morphology is a key component of developmental studies. Scanning electron microscopy (SEM) is widely used to visualize meristem topology; however, conventional SEM preparation requires fixation, dehydration, drying, and conductive coating. These steps are time-consuming, technically demanding, and can introduce damage. Here, we present a rapid protocol for imaging freshly dissected meristems from maize using a benchtop scanning electron microscope. Fresh tissues are mounted directly onto conductive adhesive and imaged under high vacuum at low accelerating voltage, enabling high-quality micrographs to be captured within minutes of dissection. Although dehydration and tissue collapse can occur under vacuum, rapid sample handling and image acquisition minimize these effects and produce images comparable to those obtained from conventionally prepared specimens. This streamlined workflow substantially reduces preparation time, lowers material requirements, and facilitates medium-throughput morphological analysis. The method provides a practical and reproducible approach for rapid developmental phenotyping and is readily adaptable to other plant species and other tissues.

The plant kingdom displays a wide array of shapes, and this morphology is tightly linked to function and productivity. For example, leaf size, shape, and arrangement around the stem define canopy architecture, influencing light interception and photosynthetic capacity. Understanding how final morphology is determined is, therefore, not only important for our basic understanding of various plant species, but also an essential tool in the development of new crop varieties. All aerial organs in flowering plants arise on the flanks of meristems throughout development. In Zea mays (maize) , these meristems are deeply enclosed within successive leaf layers, making their identification and isolation technically challenging. Successful dissection requires accurate staging, recognition of anatomical landmarks, and careful tissue handling to avoid damage or desiccation. These challenges can present a significant barrier to researchers attempting maize developmental biology studies. Here, we provide a practical, step-by-step guide for identifying and dissecting vegetative shoot apical, tassel, and ear meristems. The protocol integrates external morphological cues, nodal position, stem texture, and diagnostic cross-sectional features to distinguish cuts made above, through, or below the meristem. Detailed strategies are described to sequentially remove enclosing leaf primordia, while preserving meristem integrity. By formalizing techniques often transmitted informally within laboratories, this protocol lowers the barrier to entry for maize meristem research, supporting downstream applications including imaging, gene expression analysis, and developmental phenotyping.

The final morphology of a plant is determined by much earlier events that occur during development. Meristems are critical for the development of the aerial parts of plants, such as leaves and stems. The morphology of these meristems, which can vary significantly across genetic backgrounds, correlates with phenotypic traits in Zea mays (maize) that influence yield, such as leaf initiation rate and kernel row number. Traditional methods for morphological analysis of meristems, including histological sectioning and fine dissection, often require considerable technical expertise. To address these challenges, we present a detailed protocol employing methyl-salicylate for tissue clearing, combined with Differential Interference Contrast (DIC) microscopy to visualize maize shoot apical meristems. This method enables the intact visualization of vegetative meristems without the need for extensive dissection, thereby preserving the natural morphology of the meristem. Using this approach, the meristem is coarsely dissected, fixed, optionally stained, and cleared to enable visualization through leaf layers. By improving the accessibility and quality of meristem imaging, this protocol facilitates accurate quantification of meristem morphology, critical for genetic and developmental studies. Optimized primarily for maize, the outlined techniques can also be adapted for other plant species, providing a versatile tool in the field of plant developmental biology.

Maize ( Zea mays ) is a cornerstone of agriculture and global genetics research. Every year more than 200 million hectares of maize are grown worldwide for human and animal consumption, and biofuel and energy production. Maize has also been a key research model organism since the 1800s, and over 150 years of research has led to many fundamental discoveries in genetics and developmental biology. These findings include “jumping genes” (transposons) and the first homeobox gene in plants, KNOTTED1 , whose discovery overturned the hypothesis that homeobox genes were unique to animals. Research in maize has not only deepened our fundamental understanding of the world around us but has also driven crop improvements. Maize yield and resilience are intimately linked with plant morphology. Developmental biology aims to understand how morphology arises through the coordinated action of gene expression, hormones, cell behaviors, and biophysics across space and time. This review delves into the foundations of developmental analyses, spanning three components: morphology, time, and space. We also discuss the challenges inherent to maize developmental research, such as the inaccessibility of meristems and developing organs, and introduce techniques that address these challenges.

Zea mays (maize) is a critical global crop, generating more than 1 billion metric tons of grain annually. Plant morphology is tightly associated with final yield and is determined by the process of development. Accurate assessment and recording of mature plant morphology is essential for linking genotype to phenotype and for assessing environmental influences on plant development and yield. In maize, imaging of fully emerged aerial organs provides an accessible and informative developmental end point for comparative phenotypic analyses. Here, we provide a protocol for capturing high-quality images of mature maize plants and detached leaf blades, suitable for publication and downstream quantitative trait extraction. We emphasize standardized imaging conditions, including high-contrast backgrounds, diffuse lighting, fixed camera positioning, and consistent scale calibration, to minimize technical variation and to facilitate automated image processing. Guidance is provided for both laboratory- and field-based imaging setups and uses widely available equipment. Images generated using this protocol can be analyzed using platforms such as FIJI to extract morphometric traits, including length, width, and outline-based shape descriptors. By prioritizing standardization at the point of acquisition, this protocol supports reproducible, scalable phenotyping of mature maize morphology for developmental studies in maize.

Summary: The grass leaf plays a critical role in global food security, generating the carbon stores in cereal grains, which provide > 50% of global calories. As the global population grows, there is an urgent need to increase food production using fewer resources and to develop more resilient agricultural systems to withstand variable climate conditions and rising socio‐economic and environmental costs. Precision engineering of cereal crops, tailored to diverse environmental conditions and agronomic practices, is a vital strategy for achieving food security. Given the fundamental importance of the leaf in driving cereal productivity, it is an ideal engineering target. Leaf development occurs over large temporal and spatial scales and is environmentally regulated, posing significant challenges for predictive engineering approaches and limiting the feasibility of a one‐size‐fits‐all approach. In this review, we synthesise current understanding of cereal leaf development and identify critical developmental biology questions that must be resolved to facilitate the truly programmable plants of the future.

The plant hormone auxin regulates a wide variety of transcriptional responses depending on the cell type, environment and species. How this diversity is achieved may be related to the specific complement of auxin-signalling components in each cell. The levels of activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) are particularly important. Tight regulation of ARF protein levels is probably key in determining this balance. Through comparative analysis of novel, dominant mutants in maize and the moss Physcomitrium patens, we have discovered a ~500-million-year-old mechanism of class-B ARF protein-level regulation mediated by proteasome degradation, important in determining cell fate decisions across land plants. Thus, our results add a key piece to the puzzle of how auxin regulates plant development.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein’s output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.