Characterizing the role of endocarp a and b cells layers during pod (silique) development in Brassicaceae

ABSTRACT The process of silique dehiscence is essential for the proper dispersal of seeds at the end of a dehiscent fruit plants lifecycle. Current research focuses on genetic manipulation to mitigate this process and enhance shatter tolerance in crop plants, which has significant economic implications. In this study, we have conducted a time-course analysis of cell patterning and development in valve tissues of Arabidopsis thaliana and closely related Triangle of U species (Brassica juncea, Brassica carinata, Brassica napus, Brassica rapa, and Brassica nigra) from Brassicaceae. The goal was to decipher the detailed temporal developmental patterns of the endocarp a and b cell layers of the valve, specifically their degradation and lignification respectively. Additionally, we propose a new classification system for the lignification of the endocarp a cell layer: L1 indicates the cell closest to the replum, with L2 and L3 representing the second and third cells, respectively, each numerical increment indicating lignified cells farther from the replum. Our findings provide a foundational framework absent in current literature, serving as an effective blueprint for future genomic work aimed at modifying valve structures to enhance agronomic traits, such as reducing fiber (lignin) or increasing shatter tolerance.


Introduction
Classically, Arabidopsis silique development has been described through a flower/carpel developmental staging process that describes morphological markers which define the beginning of each stage. 1,2Using this classical method of flower/carpel development, many nuances are lost in silique development since multiple days are combined into a singular stage. 1,2Taking this into consideration, we developed a silique developmental staging method for Arabidopsis that is based on days after pollination (DAP), where each cellular developmental change can be associated with a singular time point, helping to capture previously overlooked stages using the classical method.Additionally, we wanted to expand this understanding by applying the principles learned from Arabidopsis to Brassica napus (canola) and Triangle of U species (B.4][5] Given that canola is an important oil-seed crop in Canada, understanding and characterizing these pods is important for genetic manipulation for advancing trait improvement. To further understand the silique developmental processes, a thorough understanding of the cellular makeup of the silique is essential.During the reproductive process, following successful fertilization, the gynoecium elongates and undergoes regulated developmental processes to form a mature silique which can be split into two distinct components, the valve and replum (Figures 1(a), 3(a)). 4,6The valves are two crescentshaped cell tissues that fuse jointly at a central seam, the replum, with the replum running in parallel down the length of the silique (Figures 1(a), 3(a-b)). 4,68][9] The exocarp is the outermost layer which comprises the epidermis, a protective barrier for the silique (Figures 1(a-b), 3(b-c)). 8,9The middle layer, mesocarp, is made primarily of several rows of parenchymal cells making up the majority of the valves (Figures 1(a-b), 3(b-c)). 8,9he innermost layer comprises the endocarp cells, divided into two distinct cell types, endocarp a and b, with the endocarp a layer being highly vacuolated and the endocarp b layer being lignified (Figures 1(a-b), 3(b-c)). 8,9The replum is the connection point between the two valves on either side (Figures 1(ab), 3(b-c)). 8,9The replum forms the main vascular structure of the silique along with the septum, that is connected to it from both ends (Figures 1(a-b), 3(b-c)). 8,9The fused interaction between the valves and the replum gives rise to the valve margin where a lignified layer of cells connects to the separation layer forming the dehiscence zone (DZ) (Figures 1(a-b), 3 (b-c)). 8,9During the maturation process of the pod, the valve and replum begins to separate across the separation layer due to applied tensile forces and water loss. 5,8In conjunction with the hydrolytic enzymes that aid in the process of cell separation, the tensile forces imparted on the lignified cells within the lignified valve layer and the endocarp b cell layer, from the drying of the mesocarp, lead to separation of the two valves resulting in silique opening (pod shattering). 5,8,10][12] The goal of this study was to develop a time-course characterization of the cellular development within the silique of Arabidopsis and closely related Triangle of U.This work aids in laying down the foundation in silique cellular development for future research.and B. nigra were hand-pollinated and tagged each day for 35 or 36 days consecutively for a total of 9 plants per species.Collection of all pods occurred for all species listed one day after the final day of pollination.

Sectioning, histochemical staining, and whole-mount preparation
Arabidopsis siliques were placed within a general fixative solution of 1.6% paraformaldehyde and 2.5% glutaraldehyde within a 0.05 M phosphate buffer at pH 6.8. 13 Fixation of the siliques lasted 24 h until a 30-min vacuum infiltration at 25 mm Hg.Siliques were subsequently taken through a graded ethanol dehydration series (50%, 75%, 100%) until an additional vacuum infiltration step.After vacuum infiltration, siliques were placed within a graded HEMA-based resin embedding system (Technovit 7100) solution series with ethanol (50%, 75%) until 100% embedding was achieved. 14Double-edged razor blades were used to cut embedded siliques to roughly 1 mm in size within the mid-region to allow for consistent sectioning results (Figure 1(a)).Sections were placed within casts in the desired orientation before the hardening catalyst was added.A Reichert-Jung Autocut 2040 microtome was used to section tissue to 3 µm in size.Sections were placed on microscope slides to dry in place.Sections were stained for 1 min using 0.05% Toluidine Blue O (TBO) in 0.05 M Citrate Buffer pH 4. 15 Coverslips were placed on top of the stained section and microscope slide where they were subsequently imaged using a Nixon Ds-Fi2 camera on a Leitz Aristoplan microscope.
B. juncea, B. carinata, B. napus, B. rapa, and B. nigra siliques were free hand-sectioned using double-edged razor blades around the mid region to allow for consistency of sections (Figure 1(b)). 14A total of 5-10 sections were taken from a total of 3-6 pods at each time point from which a representative sample was imaged.Sections were stained using the aforementioned TBO protocol.Once sections were stained, they were placed on microscope slides with a coverslip on top.Subsequent representative images were taken on a Nikon eclipse E200 microscope using a mounted Samsung S23+ Camera.

Maceration preparation
Maceration fluid was freshly prepared within a clean glass bottle in the fume hood by combining one part of a 30% solution of hydrogen peroxide with four parts distilled water and five parts of glacial acetic acid (1:4:5 ratio). 16B. napus siliques were cut to 5 mm in length and placed within the maceration fluid for 1 to 4 days at 56°C.Once macerated siliques turned whitish in color, they were washed gently with three changes of water with an hour between each change.Cells of the valve were separated by vigorously shaking them within a vial.Cell mixture was placed on to a glass slide with a coverslip and subsequently imaged using a Nixon Ds-Fi2 camera on an Leitz Aristoplan microscope.

Endocarp a and b cellular developmental and lignification patterning in Arabidopsis thaliana
To aid in our understanding of silique developmental patterning in Arabidopsis, a complete time course of development was used to understand the cellular patterning of silique development (Figure 1(a)).Cross-sections were analyzed for each time point of silique development focusing on the different cell layers mentioned in Figure 1(b) with a complete visualization of silique development from −1 to 16 DAP depicted in Figure 1(c).At 3 DAP, the siliques lose the other floral organs (Figure 1(c)).At 6 DAP, the silique reaches its maximum length (Figure 1c).The desiccation process of the silique begins near the tip at 11-12 DAP and continues down until it is fully dried at 14-15 DAP (Figure 1(c)).
Siliques at each individual time point were sectioned from 0 to 16 DAP with the primary focus on the whole cross-section, along with the replum and valve regions (Supplementary Figure S1-5) Silique cross-sections at 3, 6, 9, and 12 DAP were used as representative time points (Figure 2).At 5-6 DAP lignification of the replum and endocarp b cell layer within the wild-type Arabidopsis silique appears, with lignification reaching its maximum at 9-10 DAP (Figure 2).It should be noted that endocarp b lignification begins in the middle of the valve region at 6 DAP, and over the course of the next 3 days, lignification continues until it reaches the replum at 8-9 DAP (Figure 2) 8 DAP marks the first instance of lignification appearing within the walls of the endocarp a 3-Cells until it reaches complete lignification at 10 DAP (Figure 2).
The lignification of the endocarp a cell layer is a unique process of secondary cell wall deposition since it will only deposit within the walls of the first three cells (Figure 2).Owing to this unique lignin deposition compared to the other cells within the endocarp a cell layer, we have decided to classify these cells endocarp a L1, L2, and L3 cells.With L1 indicating the closest lignified cell to the replum with L2 and L3 indicating the second and third lignified cell away from the replum, respectively.This new classification system will allow for a more coherent way to describe these cells moving forward.At 9 DAP, the endocarp a cell layer begins to degrade, with degradation appearing first within the middle of the valve region spreading out toward the replum on both sides (Figure 2).It is not known whether this degradation is due to programmed cell death or mechanistic drying, although previous research has shown potential links to GA application playing a role in early degradation. 4,17nterestingly, endocarp a degradation always appears to follow the lignification of the endocarp a L1/2/3 cells, hinting at a potential mechanism between these two processes.The endocarp a cell layer finally reaches complete degradation at 12 DAP (Figure 2).The desiccation of the silique begins within the mesocarp layer until it reaches complete desiccation at 15-16 DAP (Figure 1(c)).Once complete desiccation of the silique has occurred and along with the lignification of the replum and endocarp b cell layers, the silique progresses to dehiscence at 16 DAP (Figure 1(c)).10]

Endocarp a and b cellular developmental and lignification patterning in Brassica napus
Taking our knowledge gathered from Arabidopsis silique development, we looked to further deepen our understanding of silique (commonly known as pod) developmental patterning in a common oil crop species, canola.Arabidopsis and canola share many similarities between their flower and gynoecium/silique development since they are closely related species (Figures 1(a) and 3(a)). 18 representative whole silique cross-section is shown in Figure 3(b) along with a complete time-course of pod morphology and development from 0 to 35 DAP shown in Figure 3(c).At 4 DAP, the siliques lose the other floral organs and at 9-10 DAP the silique reaches its maximum length (Figure 3(c)).Drying of the siliques is shown to begin at 30-31 DAP until desiccation is complete at 35 DAP (Figure 3(d)).
Similar to Arabidopsis, canola siliques contain two replum regions on opposite ends of the cross-section along with two valves on either side (Figure 3(b-c)).Canola valves expand over top of the replum, covering the separation layer (Figure 3(c)).Lignification can also extend further into the mesocarp layer of the valve as the silique matures (Figure 3(c)).To further expand our knowledge of the cellular makeup of the endocarp a and b cell layers, the valves were macerated from mature canola siliques (Figure 4(a-c)).We found that the endocarp a cells along with the lignified mesocarp layer are predominantly thick-walled parenchyma with identifiable plasmodesmata pits lining the exterior walls (Figure 4(ab)).In contrast, the endocarp b cell layer is comprised of sclerenchyma (Figure 4(c)).From temporal profiling through TBO staining, it can be observed that lignification in the mesocarp layer begins around 20 DAP following initial lignification of the endocarp b cell layer (Figure 4  (d)).The lignification process of the endocarp b cell layer begins at 12 DAP with the walls continuing to thicken over the course of several days (Figure 5).It has previously been shown that the endocarp a cell layer undergoes the same lignification in B. napus and B. juncea that is also observed in Arabidopsis. 19The variation of the amount of these cells differs between section and species. 19n line with this previous research and the new classification system we have put forth in Arabidopsis, we decided to acknowledge these unique lignified cells of the endocarp a layer in a similar manner with the closest lignified cell to the replum as L1, increasing numerically until the final instance of lignification is seen.At 17 DAP, the first instance of lignification occurring in the endocarp a L1 cell is seen (Figure 5).Similar to Arabidopsis, the lignification appears in the first cell closest to the replum before working outward (Figure 5).By 18 DAP, the lignification has progressed to the L2 cell and by 20 DAP the lignification has progressed to L3 (Figure 5).
Between 33 and 35 DAP, the endocarp a lignification has progressed to an L4 and L5 cell, with L5 being the last lignified endocarp a cell from the replum (Figure 5).The endocarp a L1/2/3 cells continue to thicken after 20 DAP leading to an asymmetric deposition of lignin on the inner wall closest to the ovary space, until complete asymmetric deposition of lignin is reached at 35 DAP (Figure 5).Endocarp a cells begin to degrade 25 DAP until they are no longer visible at 35 DAP (Figure 4(d)).Similar to Arabidopsis, the degradation of the endocarp a cell layer follows the lignification of the endocarp a L1/2/3 cells (Figure 5).Between 19 and 22 DAP, a small file of lignified cells was seen to connect between the replum and the junction of the endocarp a and b cell layers (Figure 5).Degradation of the separation layer is shown to begin at 11 DAP, with minimal staining of the pectin polysaccharides (pectin is stained pink/purple) within the separation layer remaining after this time point (Figure 5).1][22][23][24][25] As shown in Figure 5, the first step in silique dehiscence, priming of the middle lamella and cell wall degradation comes much earlier than the complete lignification of the replum and endocarp a/b layers.

Understanding brassicaceae silique cellular developmental patterning
Expanding our understanding of silique development led us to look into the development of other closely related species to Arabidopsis and B. napus within the Brassicaceae family.We decided to focus on species within the Triangle of U, which is an evolutionary theory about the relationship that includes the six most commonly known members of the genus Brassica.Through allopolyploidization events, the three ancestral diploid species combined to create the three common tetraploid crop species. 3We investigated silique cellular development and patterning for the three tetraploid species Brassica juncea (AABB), Brassica carinata (BBCC), and Brassica napus (AACC) as well as two of the three diploid species Brassica rapa (AA), and Brassica nigra (BB).It should be noted that due to difficulties inducing bolting and flowering in Brassica oleracea (CC), we could not obtain a time-course of siliques for this species.
Focusing on the similarities and differences between these species, we observed slight variations in the silique morphology from 0 to 35 DAP (Supplementary Figure 6).B nigra had the flattest pod morphology and B. rapa had the smallest pod over the time-course among the Triangle of U species (Supplementary Figure 6).Additionally, B. carinata has the fattest pods, and B. nigra has longer pods when compared to the other species (Supplementary Figure 6).When   7-11).For all of the Triangle of U species, lignification is seen within the first three cells of the endocarp a cell layer (Supplementary Figures 7-11).The size of the endocarp a L3-cells differs with B. juncea, B. nigra, and B. rapa having a reduction in the overall size and shape of the cell (Supplementary Figure 8, 10-11).The largest endocarp a L3-cell is found within B. napus and B. rapa (Supplementary Figure 7 and 9).These data are in agreement to the work done previously, which also found the size of the endocarp a L3-cells in B. juncea to be much smaller than B. napus. 19Endocarp a degradation in all species begins at 25 DAP and is seen to be completely degraded by 35 DAP (Supplementary Figures 7-11).Complete degradation is shown to occur slightly earlier in B. juncea and B. nigra (Supplementary Figure 8 and 11).This confirms previous results shown in the other Brassicaceae members where L1/2/3 lignification occurs prior to endocarp a degradation.B. nigra has a pronounced bulge near the primary vasculature of the valve beginning at 15 DAP (Supplementary Figure 11).This same phenotype is marginally seen within B. rapa and B. juncea (Supplementary Figure 8  and 9).
The connection between the phenotypic differences can be better understood when comparing it between the two diploid species and the three tetraploid species

Perspectives
Our descriptive work in this study will serve as a blueprint for the scientific community engaged in genomics guided research to manipulate valve structure to enhance agronomic traits such as reducing lignification or enhancing shatter tolerance.

Figure 1 .
Figure 1.Arabidopsis thaliana silique development.A schematic of Arabidopsis thaliana ecotype Col-0 cross-sectional collection.The black dotted line representing the replum region and the red dotted line depicting the mid region of the silique where cross-sections were collected with an accompanying representative cross-sectional image (a).The replum and valve region of a wild-type silique cross-section showcasing different cell layers.The black dotted line indicates the valve.Black arrows indicate the endocarp a lignification.Endocarp a L1-3 cells are indicated on both sides of the replum (b).Whole silique profiling from -1 to 16 days after pollination (DAP)(c).

Figure 3 .
Figure 3. Brassica napus silique development.A schematic of Brassica napus cv.westar cross-sectional collection.The black dotted line represents the replum region and the red dotted line depicts the mid-region of the silique where cross-sections were collected with an accompanying representative cross-sectional image (a-b).The replum and valve region of a wild-type silique cross-section showcasing different cell layers.The black dotted line indicates the valve.Endocarp a L1-4 cells are indicated on both sides of the replum (c).Whole silique profiling from 1 to 35 days after pollination (DAP)(d).
Figures 9 and 11).Interestingly, noticeable gaps appear within the endocarp b cell layer within B. napus, B. juncea, B. rapa,and B. nigra but are not found within B. carinata (Supplementary Figures7-11).For all of the Triangle of U species, lignification is seen within the first three cells of the endocarp a cell layer (Supplementary Figures7-11).The size of the endocarp a L3-cells differs with B. juncea, B. nigra, and B. rapa having a reduction in the overall size and shape of the cell (Supplementary Figure8, 10-11).The largest endocarp a L3-cell is found within B. napus and B. rapa (Supplementary Figure7 and 9).These data are in agreement to the work done previously, which also found the size of the endocarp a L3-cells in B. juncea to be much smaller than B. napus.19Endocarp a degradation in all species begins at 25 DAP and is seen to be completely degraded by 35 DAP (Supplementary Figures7-11).Complete degradation is shown to occur slightly earlier in B. juncea and B. nigra (Supplementary Figure8 and 11).This confirms previous results shown in the other Brassicaceae members where L1/2/3 lignification occurs prior to endocarp a degradation.B. nigra has a pronounced bulge near the primary vasculature of the valve beginning at 15 DAP (Supplementary Figure11).This same phenotype is marginally seen within B. rapa and B. juncea (Supplementary Figure8 and 9).The connection between the phenotypic differences can be better understood when comparing it between the two diploid species and the three tetraploid species.The earlier lignification of B. rapa and B. juncea is potentially due to the A genome since B. nigra only comprises of the B genome.The size of the endocarp a 3-cells differs within the three tetraploid species of B. carinata, B. juncea, and B. napus compared to the two diploid species.Since B. nigra is observed to have the smallest endocarp a 3-cells, this trait is potentially passed on through the B genome to B. carinata and B. juncea which also have relatively small endocarp a 3-cells.B. napus has the largest endocarp a 3-cells potentially due to the C genome in

Figure 5 .
Figure 5. Temporal lignification of Brassica napus silique.Wild-type Brassica napus cv.westar siliques taken 11-22, 25-27 days after pollination (DAP) depicting crosssections of the replum and endocarp a 3-Cell regions.White arrows indicate location of initial lignification within the endocarp b cell layer.Double black arrowheads indicate the initial lignification of the endocarp a cell layer.Green arrowheads indicate the first instance of the lignification linking the replum and endocarp a/b junction.Red arrowheads indicate the asymmetric thickening of the endocarp a L3 cells.Scale bars = 25 µm.
looking at the cellular patterning and development, lignification occurs before 10 DAP in B. rapa and B. juncea compared to later lignification in B. napus, B. carinata, and B. nigra (Supplementary Figures 7-11).Mesocarp lignification within the valve region is pronounced within B. nigra and B. rapa siliques compared to the other homeologs (Supplementary Figures 9 and 11).Interestingly, noticeable gaps appear within the endocarp b cell layer within B. napus, B. juncea, B. rapa, and B. nigra but are not found within B. carinata (Supplementary Figures