0 Q&A 218 Views Jun 5, 2024

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques.

0 Q&A 338 Views Apr 5, 2024

Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, Agrobacterium tumefaciens–mediated genetic transformation of Hongkong kumquat (Fortunella hindsii Swingle) has been widely employed for gene function analysis. However, the perennial nature of woody plants results in the generation of transgenic fruits taking several years. Here, we show the procedures of Agrobacterium-mediated transient transformation and live-cell imaging in kumquat (F. crassifolia Swingle) fruit, using the actin filament marker GFP-Lifeact as an example. Fluorescence detection, western blot analysis, and live-cell imaging with confocal microscopy demonstrate the high transformation efficiency and an extended expression window of this system. Overall, Agrobacterium-mediated transient transformation of kumquat fruits provides a rapid and effective method for studying gene function in fruit, enabling the effective observation of diverse cellular processes in fruit biology.

0 Q&A 598 Views Sep 5, 2023

Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant physiology/performance. Despite its significance, the classical cytogenetic study is now frequently avoided because of its tedious job. In general, root meristems are used to study the mitotic chromosome number, even though the use of root tips was restricted because of sample availability, processing, and lack of standard protocols. Moreover, to date, a protocol using shoot tips to estimate chromosome number has not yet been achieved for tree species’ germplasm with a large number of accessions, like mulberry (Morus spp.). Here, we provide a step-by-step, economically feasible protocol for the pretreatment, fixation, enzymatic treatment, staining, and squashing of meristematic shoot tips. The protocol is validated with worldwide collections of 200 core set accessions with a higher level of ploidy variation, namely diploid (2n = 2x = 28), triploid (2n = 3x = 42), tetraploid (2n = 4x = 56), hexaploid (2n = 6x = 84), and decosaploid (2n = 22x = 308) belonging to nine species of Morus spp. Furthermore, accession from each ploidy group was subjected to flow cytometry (FCM) analysis for confirmation. The present protocol will help to optimize metaphase plate preparation and estimation of chromosome number using meristematic shoot tips of tree species regardless of their sex, location, and/or resources.

0 Q&A 1636 Views Sep 5, 2023

Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membranes.

Graphical overview

0 Q&A 377 Views Sep 5, 2023

Since the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell–specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.

0 Q&A 526 Views Mar 5, 2023

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins.

Graphical abstract

0 Q&A 484 Views Feb 20, 2023

Chloroplast movement has been observed and analyzed since the 19th century. Subsequently, the phenomenon is widely observed in various plant species such as fern, moss, Marchantia polymorpha, and Arabidopsis. However, chloroplast movement in rice is less investigated, presumably due to the thick wax layer on its leaf surface, which reduces light sensitivity to the point that it was previously believed that there was no light-induced movement in rice. In this study, we present a convenient protocol suitable for observing chloroplast movement in rice only by optical microscopy without using special equipment. It will allow researchers to explore other signaling components involved in chloroplast movement in rice.

0 Q&A 645 Views Dec 20, 2022

MicroRNAs (miRNA) are small (21–24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves.

Graphical abstract

0 Q&A 940 Views Dec 5, 2022

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.

0 Q&A 1853 Views Aug 20, 2022

Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating mature autophagosomes and therefore is regarded as a bona fide autophagic marker; colocalization assays with ATG8 are wildly used as a reliable method to identify the components of autophagy machinery or autophagic substrates. Here, we describe a colocalization assay with fluorescent-tagged ATG8 using a tobacco (Nicotiana benthamiana)-based transient expression system.