Plant Science

Understanding how multicellular organisms are shaped requires high-resolution, quantitative data to unravel how biological structures grow and develop over time. In recent years, confocal live imaging has become an essential tool providing insights into developmental dynamics at cellular resolution in plant organs such as leaves or meristems. In the context of flowers, growth tracking has primarily been limited to sepals, the outermost floral organs, or the post-fertilization gynoecium, which are easily accessible for microscopy. Here, we describe a detailed pipeline for the preparation, dissection, and confocal imaging of the development of internal reproductive floral organs of Arabidopsis thaliana including both the stamen and gynoecium. We also discuss how to acquire high-quality images suitable for efficient 2D and 3D segmentation that allow the quantification of cellular dynamics underlying their development.

Understanding silique and seed morphology is essential to developmental biology. Arabidopsis thaliana is one of the best-studied plant models for understanding the genetic determinants of seed count and size. However, the small size of its seeds, and their encasement in a pod known as silique, makes investigating their numbers and morphology both time consuming and tedious. Researchers often report bulk seed weights as an indicator of average seed size, but this overlooks individual seed details. Removal of the seeds and subsequent image analysis is possible, but automated counts are often impossible due to seed pigmentation and shadowing. Traditional ways of analyzing seed count and size, without their removal from the silique, involve lengthy histological processing (24–48 h) and the use of toxic organic solvents. We developed a method that is non-invasive, requires minimal sample processing, and obtains data in a short period of time (1–2 h). This method uses a custom X-ray imaging system to visualize Arabidopsis siliques at different stages of their growth. We show that this process can be successfully used to analyze the overall topology of Arabidopsis siliques and seed size and count. This new method can be easily adapted for other plant models.

Key features

• No requirement for organic solvents for imaging siliques.

• Easy image capture and rapid turnaround time for obtaining data.

• Protocol may be easily adapted for other plant models.

Graphical overview

Arabidopsis siliques using the Inspex 20i X-ray machine

Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.

In this study, we present a detailed protocol for live imaging and quantitative analysis of floral meristem development in Aquilegia coerulea, a member of the buttercup family (Ranunculaceae). Using confocal microscopy and the image analysis software MorphoGraphX, we were able to examine the cellular growth dynamics during floral organ primordia initiation, and the transition from floral meristem proliferation to termination. This protocol provides a powerful tool to study the development of the meristem and floral organ primordia, and should be easily adaptable to many plant lineages, including other emerging model systems. It will allow researchers to explore questions outside the scope of common model systems.

Soil-surface roots (SORs) in rice are primary roots that elongate over or near the soil surface. SORs help avoid excessive reduction of stress that occurs in paddy, such as in saline conditions. SORs may also be beneficial for rice growth in phosphorus-deficient paddy fields. Thus, SOR is a useful trait for crop adaptation to certain environmental stresses. To identify a promising genetic material showing SOR, we established methods for evaluating SOR under different growth conditions. We introduced procedures to evaluate the genetic diversity of SOR in various growth stages and conditions: the Cup method allowed us to quantify SOR at the seedling stage, and the Basket method, using a basket buried in a pot or field, is useful in quantifying SOR at the adult stage. These protocols are expected to contribute not only to the evaluation of the genetic diversity of SOR, but also the isolation of related genes in rice.

Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.

Cryo-scanning electron microscopy (cryo-SEM) was first introduced for scientific use in the 1980s. Since then, cryo-SEM has become a routine technique for studying the surfaces and internal structures of biological samples with a high water content. In contrast to traditional SEM, cryo-SEM requires no sample pretreatment processes; thus, we can obtain the most authentic images of the sample shape and structure. Cryo-SEM has two main steps: cryoprocessing of samples and scanning electron microscopy (SEM) observation. The cryoprocessing step includes preparation of the cooled slushing station, cooling of the preparation chamber, sample preparation, and sputtering. The sample is then transferred to an SEM cold stage for observation. We used cryo-SEM to study rice root hair tissues, but the methods and protocols can be applied to other root systems. This protocol optimizes the two key operation steps of reducing the humidity in the growth chamber and previewing the samples before sputtering and can more quickly obtain high-quality images.

The ion-selective vibrating probe has been used to detect and quantify the magnitude and direction of transmembrane fluxes of several ions in a wide range of biological systems. Inherently non-invasive, vibrating probes have been essential to access relevant electrophysiological parameters related to apical growth and morphogenesis in pollen tubes, a highly specialized cell where spatiotemporal tuning of ion dynamics is fundamental. Of relevance, crucial processes to the cell physiology of pollen tubes associated with protons and anions have been elucidated using vibrating probes, allowing the identification of diverse molecular players underlying and regulating their extracellular fluxes. The use of Arabidopsis thaliana as a genetic model system posed new challenges given their relatively small dimensions and difficult manipulation in vitro. Here, we describe protocol optimizations that made the use of the ion-selective vibrating probe in Arabidopsis pollen tubes feasible, ensuring consistent and reproducible data. Quantitative methods like this enabled characterizing phenotypes of ion transporter mutants, which are not directly detectable by evident morphological and reproductive defects, providing valuable insights into molecular and cellular mechanisms. The protocol for quantifying extracellular proton and anionic fluxes detailed here can be adjusted to other systems and species, while the sample preparation can be applied to correlated techniques, facilitating the research of pollen tube growth and development.