Live Confocal Imaging of Brachypodium Spikelet Meristems

[Abstract] Live confocal imaging of fluorescent reporters and stains in plant meristems provides valuable measurements of gene expression, protein dynamics, cell polarity, cell division, and growth. The spikelet meristem in the grass Brachypodium distachyon (Brachypodium) is well suited to live imaging because of the ease of dissection, small meristem size, simple arrangement of organs, and because each plant provides abundant spikelet meristems. Brachypodium is also far easier to genetically transform than other grass species. Presented here is a protocol for the growth, staging, dissection, mounting, and imaging of Brachypodium spikelet meristems for live confocal imaging. awn. Plants at this early “first-awn” stage are the easiest to dissect. C. The end of the ideal stage occurs when the terminal spikelet completely emerges from the last vegetative leaf. C shows the very end of the permissible stage. detail of the first awn of the terminal spikelet. D. Inflorescences where the terminal spikelet has fully emerged are suitable for Brachypodium two lateral

general, we try to accelerate the transition to flowering while still maintaining a healthy plant with several lateral branches. Using a 20 h light/4 h dark cycle and small pot sizes stimulates flowering, but in these conditions proper watering is important. Larger pot sizes and shorter day lengths will lengthen the time to flowering, but may yield more spikelets for imaging per plant.
Plants can be grown at higher densities if they are only used for harvesting spikelets. If plants are grown for seeds, a lower planting density or culling before flowering is recommended. See the Materials and Reagents section for pot sizes and soil recommendations. Below you will find general growth guidelines.

Seed preparation (optional)
For some batches of seed, germination can be improved by removing the lemma. The lemma is the outer leaf-like organ with a long protruding tendril-like mid-vein called the awn ( Figure 3E).
Peel off the lemma by gripping the awn and opening away from the seed.

Sowing
Ensure soil is well watered before sowing. Place 2 seeds per pot to ensure adequate germination. Plants will need to be culled down to 1 per pot if they are being grown for seed.
Sow by pushing the seed vertically just below the soil surface with the awn end facing up.

Stratification (optional)
After sowing place trays at 4 °C for 2-4 days to synchronize germination.

Germination
Ensure trays are well watered before placing into the light, and cover with clear plastic lids until plants are germinated, up to one week.

Fertilization
Include fertilizer with every second watering according to manufacturer's instructions.

Watering before flowering
Watering is performed by pouring ~1 cm of water into the bottom of each tray.

Watering during flowering and seed set
After flowering and especially during seed-set, plants will need plenty of water. After the first addition of water, allow plants to soak for 15-20 min before adding a small amount of additional water. At this stage, plants can sit in a small amount of water to ensure they do not dry out (e.g., over the weekend). Maintaining adequate watering late in development is critical to seed set.
Watering can be cut off at the first sign of senescence (brown/yellowing starting in the inflorescence) to accelerate seed drying. Alternatively, watering can be extended during most of senescence to increase seed yield.

B. Staging
Proper staging will ensure consistent results and facilitate dissection. In our growth conditions, Brachypodium has three main branching axes at floral transition. The central apical branch will generally flower first, and it is this branch that we focus on to harvest spikelet meristems. Upon the transition to flowering the apical meristem will first initiate several lateral spikelet (LS) meristems before transitioning into the terminal spikelet (TS) meristem (  The key to staging is to identify branches where the floral transition has just occurred, but before the terminal spikelet fully emerges. This stage is easily identified because the awn of the terminal spikelet is visible emerging from the sheath of the last vegetative leaf (Figures 3B and 3C). At this stage the lateral spikelets will be immature and largely exposed. We have termed this stage the "first-awn" stage. The first-awn stage in the main axis occurs in our growth conditions starting approximately 3 weeks after moving plants to light, after the production of approximately 7 leaves.
The earlier the first-awn stage is identified, the easier the lateral spikelets will be to dissect. It may be necessary to unroll the last vegetative leaf in order to see the first awn at the earliest stages ( Figure 3B). Branches where the entire terminal spikelet has emerged from the last vegetative leaf ( Figure 3D) are too old and are not suitable for harvesting spikelet meristems. At this late stage, the lateral spikelets are covered and tend to break off the main axis during dissection.
We have not noticed significant differences between lateral and terminal spikelet meristems, but harvesting meristems from a consistent position is recommended.  Figure   1.

Video 1. Video of dissection and mounting of Brachypodium spikelet meristem. Video
shows how to identify the first awn stage, dissect out the spikelet meristem sample, remove organs covering the meristem, and mount for confocal imaging.
1. Identify branches at the "first-awn" stage ( Figure 4A) 2. Remove the sample from the main plant by cutting just below the last visible node. Node tissue is generally white and is covered in a collar of trichomes, hereafter called the "fuzzy collar" (FC) ( Figure 4A).
3. Release the internal tissues by cutting through the entire stem just above the fuzzy collar of the node ( Figure 4B). 6. Once again, cut just above the fuzzy collar of the node (Figure 4C), carefully grip the leaf base with your fingers and pull out the inflorescence ( Figure 4D). 7. Under the dissecting scope grip the sample by the terminal spikelet and select one of the lateral spikelets for dissection. Generally, the lowest lateral spikelet is the easiest to dissect.
8. Using a probe, syringe needle, or fine forceps start at the base of the spikelet and remove the immature bracts ( Figure 4E) and any immature lemmas covering the spikelet meristem.
Note: The immature bracts and lemmas wrap around the spikelet, so it is often easier to remove them if you insert the probe on the opposite side of the spikelet meristem from the midvein and unwrap. The organ will break at the base. 9. Remove immature organs until the spikelet meristem is visible ( Figure 4F). 10. For the last few lemmas that have not grown enough to cover the spikelet meristem, only the awns should be removed by gripping with forceps and bending away from the spikelet meristem apex until the awn breaks off.
Note: If these awns are not removed a bubble will form around the spikelet meristem making staining and imaging under water impossible.
11. The exposed spikelet meristem is now ready for optional staining or mounting ( Figure 4G). inner nodes are removed from the sheath by pulling out, repeat by carefully cutting just above the fuzzy collar (FC) of the next node. D. Repeat until the entire inflorescence is released by pulling on the end of the terminal spikelet (TS). The basal most lateral spikelets (arrows) are typically the easiest to dissect because they are less likely to break off, and the meristems are more exposed. E. Expose the meristem by removing the glume (GL) and lemma primordia wrapped around the spikelet meristem. F. Remove the remaining lemma awns (arrows) by carefully bending down and breaking off or by using the sharp end of a syringe needle to cut off.
G. Once all lemmas and lemma awns are removed the meristem is fully exposed (arrow) and ready for mounting or optional counterstaining. H. After optional counterstaining, the inflorescence is trimmed and mounted in a Petri dish by adhering the ends with drops of 1% agarose. Immerse the sample in water to prevent drying and image immediately.

D. Counterstains (optional)
After dissection, samples can be counterstained by placing directly into a 1.5 ml microfuge tube containing the stain of choice ( Figure 5A). Make sure a bubble does not surround the spikelet meristem and that it does not contact the side of the tube.
We have successfully used propidium iodide to stain cell walls ( Figures 5B and 5D, Propidium iodide:10 μg/ml in water for 10-20 min). Propidium iodide is normally excluded from the inside of the cell by the plasma membrane. Thus, damaged or dead cells will stain internally, especially in the nucleus, and damaged samples are easily identified.
We also regularly use the vital stain FM4-64 to stain cell membranes and endocytic vesicles ( Figure 5C, FM4-64 ( Figure 5C): 50 µg/ml in water for 5-15 min). Short stain times (5-10 min) will mark only the cell membrane, while longer stain times will mark vesicles as the stain is endocytosed. Note: Be sure to leave enough tissue at both ends to act as mounting points ( Figure 4H).
2. Using molten 1% agarose and a Pasteur pipette, "glue" the two ends of the sample down in the center of a ~5 cm Petri dish ( Figure 4H).
3. Working quickly before the agarose solidifies, adjust the sample so the target meristem sits in the plane of the dish. This may involve twisting the main branch axes quite severely. 4. After the agarose completely solidifies cover with water and image immediately, do not allow the sample to dry out.

F. Imaging
Imaging parameters will depend on the specific reporter, sample, confocal, objective, and other factors, but general recommendations are provided here.
Most confocals will have settings to automatically optimize XY and Z resolutions. These are a good starting point, but these settings often oversample for most experiments and lead to long scan times and possible bleaching. For routine moderately-high resolution imaging, we use around 1024 x 1024 resolution and a field of view of 200 µm or less. Lower resolutions around 512 x 512 may be suitable for some experiments and will greatly increase the scanning speed. We recommend a bit depth of at least 12, especially if quantifying florescence. We generally use a zoom of approximately 3x with a 20x objective and a Z step-size of 0.5-3 µm with the pinhole set to 1 airy unit, although these settings should be adjusted to the particular sample and experimental goal.
Because the spikelet meristem is symmetrical, only one half of the depth needs to be imaged in order to get a good sense of a particular expression domain. The sample can be turned 90°, remounted with agarose and re-imaged to get a different view (Figures 2B and 2C). The relatively small size of the Brachypodium spikelet meristem means it is generally easy to image deep into the tissue. However, the fluorescent signal will decrease deeper into the tissue, especially at the base of the spikelet meristem ( Figure 5D).