growth of arabidopsis thaliana plants and preparation of flowers for pollen hydration assay

שיטה משופרת למדידת פרופילי הידרציה של אבקה ב- Arabidopsis thaliana

Successful sexual reproduction in angiosperms typically relies on the transfer of intraspecific pollen grains from the anther to the stigma, either within or between individuals (i.e. pollination). This transfer of pollen grains to a receptive flower is usually mediated by pollinators or abiotic factors; as such, this also frequently results in the deposition of heterospecific pollen under natural conditions. With a few exceptions, the progression of pollination by heterospecific pollen is evolutionarily disadvantageous, reducing reproductive fitness through lost mating opportunities, with most resulting hybrid progeny either failing to develop appropriately or being sterile1. Thus, mechanisms have evolved to block pollination by &#39;incompatible&#39; heterospecific pollen2. Rapid recognition of compatible pollen is therefore arguably the most important process in the early stages of sexual reproduction in many flowering plants.
In the Brassicaceae family, where stigmas are of the &#39;dry&#39; type, a series of molecular checkpoints act at multiple stages in the reproductive process regulating pollination, such that only compatible pollen is successful. Pollen hydration is one of the most important checkpoints (Figure 1), as pollen that fails to hydrate cannot progress to produce a pollen tube and subsequently deliver sperm to the female gametophyte. Frequently, incompatible grains fail to pass this first pollination checkpoint, and thus do not gain access to stigmatic water3. Amongst members of the Brassicaceae family, the recognition of pollen occurs rapidly, with compatibility being established within minutes of pollen grain attachment to the pistil4,5. In recent years, much progress has been made, and we are now beginning to understand the molecular mechanisms that regulate key pollination checkpoints.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65280/65280fig01.jpg" /> 
Figure 1: Overview of key events during compatible pollination. These stages, such as pollen hydration and pollen tube germination, are also pollination &#39;checkpoints&#39; that must be successfully navigated to effect compatible pollination. The diagram represents a &#39;dry&#39; type stigma, which is typical to species from the Brassicaceae family2,20. <a href="https://www.jove.com/files/ftp_upload/65280/65280fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Pioneering research on the Brassica self-incompatibility (SI) system, where &#39;self&#39; pollen is recognized and rejected, established the paradigm for pollen-stigma recognition in Brassicaceae6,7,8,9,10. SI in Brassica and its relatives is mediated by &#39;recognition&#39; proteins that reside on the surface of pollen and at the stigmatic plasma membrane that, upon interaction, lead to pollen rejection. SI pollen rejection operates by disruption of the basal pollen-stigma compatibility system which, when fully activated by the perception of compatible pollen, leads to targeted secretion by the stigma, thus driving pollen hydration (for reviews of the pollen compatibility mechanism, see11,12). In the example of SI, the pollen-borne ligand is a small cysteine-rich protein, S-locus cysteine rich (SCR/SP11), and the stigmatic receptor is the S-locus receptor kinase (SRK).
Recently, in Arabidopsis thaliana, another group of small cysteine-rich pollen-borne proteins, pollen coat protein class Bs (AtPCP-Bs), have been found to be important regulators of pollen acceptance through the activation of pollen hydration13. Stigmatic receptors of the AtPCP-Bs and aspects of the downstream regulatory pathway have also recently been described14,15. Interestingly, mutational studies of genes encoding potential pollen-borne and stigmatic signaling mediators of pollen hydration (including AtPCP-Bs) have failed to generate plants that have a complete block to the pollen hydration checkpoint. This strongly suggests that multiple other, yet undiscovered, factors play a role in the regulation of pollen hydration. Building upon the method first described by Wang et al.13, here we describe an improved high-resolution in vivo bioassay suitable for the identification of subtle pollen hydration defects in candidate mutant A. thaliana lines.

מחבר זרקור: ברזולוציה גבוהה, גרגר יחיד, In Vivo אבקה הידרציה bioassay עבור Arabidopsis thaliana  

בדיקה ביולוגית ברזולוציה גבוהה, מדגן יחיד, In Vivo אבקת הידרציה עבור Arabidopsis thaliana

Watch this Scientific Journal Video about Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay at JoVE.com

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay   

צמיחה של צמחי Arabidopsis thaliana והכנת פרחים לבדיקת הידרציה אבקה

התחילו בריבוד זרעי A.thaliana במי מילי-Q סטריליים למשך שלושה ימים בארבע מעלות צלזיוס. מעבירים את הזרעים המרובדים לעציצי הקומפוסט על ידי זיוף הזרעים המרובדים. מניחים את העציצים בתא גידול מבוקר סביבה למשך כשישה שבועות עד שהתפרחות מבוססות היטב.כדי להבטיח פריחה סינכרונית, זרעו יחד את הצמחים התורמים ומקבלי האבקה וכל צמח בקרה מתאים אחר. יום לפני הביואסיה, בחרו את ניצני הפרחים בשלב 12 מהצמח המקבל, לאחר ניצני פרחים שלא נפתחו שיראו את פתיחת הפרח ואת הגחלת ביום המחרת. לאחר מכן, הניחו שקופית זכוכית מתחת למיקרוסקופ מנתח סטריאו.הדביקו את אזור הגבעול הקרוב לפרח הנמען שנבחר על שקופית זכוכית זו. בעזרת זוג מלקחיים עדינים, פתחו בזהירות את ניצן הפרח והוציאו את כל עלי הכותרת והאנתרים. לאחר שסיימתם, ודאו שהפיסטיל לא ניזוק והסטיגמה נקייה מאבקנים מזהמים.

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Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay

579 Views.  University of Bath. התחילו בריבוד זרעי A.thaliana במי מילי-Q סטריליים למשך שלושה ימים בארבע מעלות צלזיוס. מעבירים את הזרעים המרובדים לעציצי הקומפוסט על ידי זיוף הזרעים המרובדים. מניחים את העציצים בתא גידול מבוקר סביבה למשך כשישה שבועות עד שהתפרחות מבוססות היטב.כדי להבטיח פריחה סינכרונית, זרעו ...

Video: Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay   

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Begin by stratifying A.thaliana seeds in sterile Milli-Q water for three days at four degrees Celsius. Transfer the stratified seeds to the compost pots by pipetting the stratified seeds. Place the pots in an environmentally controlled growth chamber for approximately six weeks until the inflorescences are well established.To ensure synchronous flowering, sow the pollen donor and recipient plants and any other appropriate control plant lines together. One day prior to the bioassay, select these stage 12 floral buds from the recipient plant, Having unopened flower buds that will show the opening of the flower and the anther dehiscence on the following day. Next, place a glass slide under a stereo dissecting microscope.Tape the region of the stem close to the selected recipient flower on that glass slide. Using a pair of fine-tipped forceps, carefully tease open the flower bud and remove all the flower petals and anthers. Once done, ensure that the pistil is undamaged and the stigma is free of contaminating pollen.

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements  

Begin the assay by laying the labeled A.thaliana pollen recipient plant on its side, positioning the emasculated flower on the stage of an inverted microscope to image the stigma. To fix the position of the emasculated recipient flower, immobilize the stem using strips of masking tape. From the pollen donor plant, remove a healthy and freshly-opened flower and place the pollen donor plant under a dissecting microscope.Gather pollen grains using fine-tipped forceps. Remove excess pollen grains by lightly touching the forceps against the donor petals until the pollen grain's monolayer is formed. Return to the pollen recipient plant and use a low power objective lens to focus the inverted microscope on the stigma.Holding the forceps along the opening between the arms of the forceps, carefully approach and unpollinated stigmatic papilla cell. Continue approaching the unpollinated stigmatic papilla cell until the suitably positioned pollen grain on the forceps makes light contact with its surface. Once pollen attachment is confirmed, slowly withdraw the forceps.Orient the pollen grain with its equatorial axis visible and in sharp focus, and immediately switch to a higher power objective lens, like 20x. Capture the first pollen grain image as T-0, and continue capturing images at one-minute intervals for 10 minutes. Adjust the focus to accommodate small movements in the pollen grain or stigma.Record the room's ambient temperature and relative humidity every two minutes, allowing future comparisons between experimental replicates. Save the images in a lossless format, such as ND2, before repeating the pollination and imaging steps for additional pollen grains to acquire data for control and experimental pollinations. Begin the data measurements for pollen hydration assay using image analysis software.Use similar parameters of digital zoom and the approach defining the pollen boundary for all measurements in the datasets. Record the semiminor axis values for all time points in the dataset. Once the measurements are complete for a dataset, export the raw semiminor axis values of each individual time point to a spreadsheet.Present the data in columns per image stack and calculate the percentage semiminor axis change. Repeat the steps and obtain data from at least 15 hydrated pollen grains for each plant line. A few pollen grains may fail to hydrate or may hydrate significantly slower than expected.They're known as dud grains. Exclude these from the dataset. Calculate the mean values for each time point per plant using the specialized software package GraphPad Prism.Analyze the hydration data from wild-type and mutant lines at each time point using unpaired T-test and one-way ANOVA to compare the means of the specific time point of interest. To compare the means across all the time points, perform multiple T-tests between wild-type and mutant lines across multiple time points. The pollen hydration time series data for wild-type plants collected on different days showed that the minimum and maximum values for the means between replicates for all time points were within 3%This representative data for wild-type pollinations demonstrated a high degree of consistency for relatively low sample numbers and across different days.