An Arabidopsis single-nucleus atlas decodes leaf senescence and nutrient allocation

1. Summary

With rapid advancements in single-cell RNA-seq technologies, exploration of the systemic coordination of critical physiological processes has entered a new era. Here we generated a comprehensive Arabidopsis single-nucleus transcriptomic atlas using over one million nuclei from tissues encompassing multiple developmental stages. Our analyses identified cell types that have not been characterized in previous single-protoplast studies, and revealed cell-type conservation and specificity across different organs. Through time-resolved sampling, we revealed highly coordinated onset and progression of senescence among the major leaf cell types. We originally formulated two molecular indexes to quantify the ageing state of leaf cells at single-cell resolution. Additionally, facilitated by weighted gene co-expression network analysis (WGCNA), we identified hundreds of novel hub genes that may integratively regulate leaf senescence. Inspired by the functional validation of novel hub genes, we built a systemic scenario of carbon and nitrogen allocation among different cell types from source leaves and sink organs.

Figure 1 A comprehensive cell atlas of Arabidopsis. (A) Schematic illustration of the sampling strategy in this study. A total of 20 tissues (T1-T20) were collected from vegetative growth to reproductive growth, including two seedling stages (6 and 11 DAG) and six different rosette stages (S1-S6) from 14 to 49 DAG. T1-T2: whole root from two stages of seedling (6d and 11d after germination); T3: shoot at 6d (including cotyledon, SAM and leaf primordia); T4: stem (whole stem including apical, middle and basal regions) at 42d; T5: cauline leaf at 42d; T6-T11: six stages of the second pair of true leaves from expansion to senescence (as shown by arrows); T12-T14: three stages of flowers (flower bud to fully opened flowers) at 49d; T15-T20: six successive timepoints of siliques (0 to 5d after anthesis with 1 day interval). Tissues T6-T11 are indicated by arrows in the individuals from S1-S6. (B) Numbers of profiled nuclei and captured genes of each sample. DAG, days post germination. DPA, days post anthesis. (C) UMAP of global clustering of all cells colored by organs. (D) UMAP of single-nucleus atlas of Arabidopsis colored by major cell types.

2. Data description

2.1 Raw data

  In total, 20 samples were collected at indicated day post-germination for all tissues including seedling, cotyledon, hypocotyl, root, rosette leaf, stem, lateral leaf, flower, and silique. Then we used an in-house nuclei isolation protocol (see method) and DNBelab C Series Single-Cell Library Prep Set (MGI, 1000021082) for snRNA data generation as previously described (Han et al., 2022). The concentration of DNA library was measured by Qubit (Invitrogen). Libraries were sequenced by DNBSEQ-T7RS.

2.2 Expression matrix

  The raw sequencing reads were filtered and demultiplexed by PISA (Version 1.1.0) (https://github.com/shiquan/PISA), and aligned to the TAIR10 reference genome using STAR (version 2.7.4a) (Dobin et al., 2013) with default parameters. Over one million nuclei from 20 samples passed the accepted droplet-based single nuclei filtering criteria with > 200 genes/nucleus. Following the majority of previous studies, we used a more rigorous criteria with > 500 genes/nucleus to obtain high-quality nuclei for downstream analyses. A total of 913,769 nuclei passed our quality control, with 1610 mean genes and 2451 mean UMIs captured in each nucleus. Cells from each of the 20 samples were independently clustered to obtain single-sample maps , then integrated to generate multi-stage organ level maps and a comprehensive all-cell transcriptome atlas.

3. Results

3.1 Root (6 DAG)

SnRNA-seq profiling of root (6 DAG).

UMAP clustering of root (6 DAG) and annotation of cell types. DAG, days after germination.

3.2 Root (11 DAG)

SnRNA-seq profiling of root (11 DAG).

UMAP clustering of root (11 DAG) and annotation of cell types. DAG, days after germination.

3.3 Shoot (6 DAG)

SnRNA-seq profiling of shoot (6 DAG).

UMAP clustering of shoot (6 DAG) and annotation of cell types.

3.4 Stem

snRNA-seq profiling of stem (42 DAG).

UMAP clustering of stem (42 DAG) and annotation of cell types. DAG, days after germination.

3.5 Cauline

snRNA-seq profiling of cauline (42 DAG).

UMAP clustering of cauline (42 DAG) and annotation of cell types. DAG, days after germination.

3.6 Rosette at stage 1 (14 DAG)

snRNA-seq profiling of rosette at stage 1 (14 DAG).

UMAP clustering of rosette at stage 1 (14 DAG) and annotation of cell types. DAG, days after germination.

3.7 Rosette at stage 2 (21DAG)

snRNA-seq profiling of rosette at stage 2 (21 DAG).

UMAP clustering of rosette at stage 2 (21 DAG) and annotation of cell types. DAG, days after germination.

3.8 Rosette at stage 3 (28DAG)

snRNA-seq profiling of rosette at stage 3 (28 DAG).

UMAP clustering of rosette at stage 3 (28 DAG) and annotation of cell types. DAG, days after germination.

3.9 Rosette at stage 4 (35 DAG)

snRNA-seq profiling of rosette at stage 4 (35 DAG).

UMAP clustering of rosette at stage 4 (35 DAG) and annotation of cell types. DAG, days after germination.

3.10 Rosette at stage 5 (42 DAG)

snRNA-seq profiling of rosette at stage 5 (42 DAG).

UMAP clustering of rosette at stage 5 (42 DAG) and annotation of cell types. DAG, days after germination.

3.11 Rosette at stage 6 (49 DAG)

snRNA-seq profiling of rosette at stage 6 (49 DAG).

UMAP clustering of rosette at stage 6 (49 DAG) and annotation of cell types. DAG, days after germination.

3.12 Early flower

snRNA-seq profiling of early flower.

UMAP clustering of early flower and annotation of cell types.

3.13 Middle flower

snRNA-seq profiling of middle flower.

UMAP clustering of middle flower and annotation of cell types.

3.14 Late flower

snRNA-seq profiling of late flower.

UMAP clustering of late flower and annotation of cell types.

3.15 Silique (0 DPA)

snRNA-seq profiling of silique (0 DPA).

UMAP clustering of silique (0 DPA) and annotation of cell types. DPA, days post anthesis.

3.16 Silique (1 DPA)

snRNA-seq profiling of silique (1 DPA).

UMAP clustering of silique (1 DPA) and annotation of cell types. DPA, days post anthesis.

3.17 Silique (2 DPA)

snRNA-seq profiling of silique (2 DPA).

UMAP clustering of silique (2 DPA) and annotation of cell types. DPA, days post anthesis.

3.18 Silique (3 DPA)

snRNA-seq profiling of silique (3 DPA).

UMAP clustering of silique (3 DPA) and annotation of cell types. DPA, days post anthesis.

3.19 Silique (4 DPA)

snRNA-seq profiling of silique (4 DPA).

UMAP clustering of silique (4 DPA) and annotation of cell types. DPA, days post anthesis.

3.20 Silique (5 DPA)

snRNA-seq profiling of silique (5 DPA).

UMAP clustering of silique (5 DPA) and annotation of cell types. DPA, days post anthesis.

4. Sampling strategy and cell type annotation

Detailed sampling strategy of each tissue

As stated by the title of the manuscript, this work aims to present a single-nucleus atlas resource of the model plant Arabidopsis with particular biological focus on leaf senescence and nutrient allocation from the rosette source to the flower and silique sinks. So, the major motivation of tissue selection is to cover as many different organs as durable, while also including enough dynamic samples for the focused rosette leaves, flowers and siliques. To the end, we have covered root, stem, leaf, flower and silique with developing seeds in it. Overall, our work already covered all the organ-types of Arabidopsis, which to our knowledge is the most comprehensive in formally published works. A comprehensive sampling of all organs at the whole life-cycle will provide even more information for the community. Nevertheless, some tissues are incompatible for current single-cell or single-nucleus sequencing approaches, such as dried seeds and completely yellow leaves, and some are impractical to collect, such as roots of adult plants in soil. Following are more detailed explanations for each tissue and timing selection:

The 6-day-old and 11-day-old roots from petri dish

The 6-day-old roots from sterile seedlings were widely used in previous single-cell sequencing works1, 2. We included this tissue and time point so that we can make a parallel comparison with previous work to check the feasibility of our method and the quality of our datasets (Figure S3E–S3J). The 11-day-old roots already developed lateral root primordia and emerged lateral roots, which will provide single-cell gene expression information on root architecture formation for the community, although this topic is beyond the scope of this work3. Seedlings older than 14 days are usually too large for petri dish culture, and may induce major space stress to the seedlings. So, we usually transplanted seedlings to soil after 7 days post seed germination, and from that point on, sampling intact roots became impractical.

The 6-day-old shoot from petri dish

The 5~7-day-old cotyledons from sterile seedlings were also widely used in previous single-cell sequencing works4,5. Besides, this tissue also includes small but recognizable SAM and leaf primordia5. To keep a consistency within the study, we used 6-d-old shoot part of sterile seedlings in addition to the 6-d-root. We believe this stage together with our early expansion leaf stage (S1) would also provide the community with an important resource to study early-stage leaf development, although this neither was our major interest.

The 6-stage rosette leaves

In order to fully explore the transition of leaves from anabolic to catabolic, we collected 6 stages of the second pair of rosette leaves, which covered growth phases from leaf expansion to mature, and to onset and progression of leaf senescence6, 7. Namely, S1 is 4 days after the leaf emergence, which represents the vigorous expansion phase of leaf; S2 is near full expansion, and S3 is full expansion, which represent the near-mature and mature phases of leaf; S4 is with marginal yellowing, representing the typical phenotype of leaf senescence onset; S5 and S6 carries gradually enlarged yellow area, representing the progression of leaf senescence. After S6, leaves become completely yellow and dried, which are no longer compatible for any approach of single-cell sequencing. More importantly, at any stage of leaf development, leaves are composed of highly heterogenous cells. As indicated by our pseudo-time analysis (Figure S4S), the 6-stage rosette leaves encompassed large overlap during successive stages, which nicely demonstrated our sampling density was sufficient to cover all phases of leaf cell development.

The cauline leaf at 42 days after germination (DAG)

The cauline leaf basically take similar roles as the rosette leaf to be a source organ (Figure S4U–S4V), and the cell types are also similarly distributed between cauline leaf and rosette leaf (Figure S1E and S2E)8. So, we just collected cauline leaf at 42 DAG, when it was fully expanded but does not start senescence.

The stem at 42 DAG

The inflorescence stem links the rosette leaves to flowers and siliques, and is the major routine for water and nutrient transport. We collected stems at 42 DAG when the rosette leaves start to senesce and flowers and siliques start to develop. In another words, this stage encompasses active transport activity in the stem (Figure 5A)9. On the other hand, we collected the whole stem along its axis, including the apical, middle and basal regions, but excluded the cauline leaf and buds, so that sufficient heterogeneity of cell development in the stem could be captured.

The 3-stage flowers and 6-stage siliques

The flower and silique are major sink organs when the rosette leaves transit from anabolic to catabolic. Focusing on the nutrient allocation from rosette to flowers and siliques, we collected 3-stage flowers and 6-stage siliques, which represented the successive development of reproductive tissues, with the third stage flowers about one or half day prior to the first stage siliques. According to literature reports10, pollination occurs at the third stage of flower, when it is fully opening. Male and female gametes and flower ancillary organs gradually developed in the other two stages. Post anthesis, the siliques elongated within 6 days, and the fertilized eggs in the silique gradually developed into torpedo embryos, after which the siliques gradually dried out and became incompatible for nucleus extraction and sequencing. Therefore, our sampling strategy also provided the community with valuable resource for the investigation of reproductive tissue development on single-cell level, although this again was beyond the scope of this work.

Detailed description for cell type annotation

To construct a comprehensive cell atlas of the model plant Arabidopsis, we generated snRNA data from 20 tissues that represent the key developmental stages and transitions throughout the entire life cycle (Figure 1A). Cells from each of the 20 tissues were both independently clustered to generate 20 single-tissue atlases (Figures S1 and S2A-S2T; Table S1) and integrated to generate a comprehensive integrative atlas (Figures 1C–1D and S2U). For root, rosette, flower and silique tissues that were sampled with multiple time points, cells were also clustered to generate integrative atlas at tissue level (Figures 2E, 2F, S3A, S3B, and S3L–S3P). Independent clustering of each dataset using Seurat R Package resulted in a total of 367 clusters. Cluster-enriched gene lists were detected using “FindAllMarkers”. To annotate the cell-type identity of each cluster, a curated list of cell-type-specific marker genes, supported by genetic studies, tissue-specific expression patterns, or newly identified in previous single-cell studies, was compiled (Table S1). The marker gene list was also cross compared with the cluster enriched gene list (Table S1).

Cell type annotation for root

The 6-day, 11-day and integrative root nucleus populations were grouped into 24, 23 and 32 clusters, respectively (Figures S1A, S2A, S1B, S2B, S3A and S3B). In the integrative root nucleus population, cluster 14 and 19 were assigned as atrichoblast due to the enriched expression of the non-hair root epidermis marker AT1G79840 (GL2)11, while cluster 8 was assigned as trichoblast based on the enriched expression of root hair elongation factors AT4G33880 (RSL2)12 and AT5G58010 (LRL3)13. Multiple clusters, including cluster 0, 1, 4, 7, 9, 10, 11, 16, 17, 21, and 28 exhibited enriched expression of cortex marker genes such as AT1G05570 (ATGSL06) and AT5G27350 (SFP1), AT5G03570 (ATIREG2) and AT3G12700 (NANA)2, 14, indicating their cortex identity. Similarly, multiple clusters, including cluster 2, 18, 20, 23, 24 and 27 showed enriched expression of endodermis marker genes. The partial overlapping expression patterns of these marker genes indicated developmental sequence of these clusters. For instance, MYB36 is the upstream regulation of casparian strip formation15, indicating cluster 20 is more distally located in the root tip, while cluster 24 expresses several CASPs16, suggesting casparian strip is actively established in these cells and they are located in late elongation zone or mature zone. Suberin synthesis-related gene GPAT5 was enriched in cluster 23 and 27, suggesting these cells are fully differentiated and start secondary cell wall biogenesis17. Enriched expression of AT4G30450 (XPP)18 and AT2G36120 (DOT1)19 indicated cluster 5, 12,13 and 25 are pericycle cells. Cluster 22 was assigned as procambium/xylem based on mixed expression of both xylem marker AT4G35350 (XCP1)20 and procambium marker AT5G61480 (TDR/PXY)9. Cluster 6 and 15 were assigned as phloem parenchyma and companion cell, respectively, based on their specific expression of AT5G24800 (bZIP9)21 and AT1G79430 (APL)22. The enriched expression of multiple cell-cycle-related genes, such as AT1G76310 (CYCB2;4)23, AT1G76540 (CDKB2;1)22, AT3G11520 (CYCB1;3)23 and AT4G37490 (CYCB1;1)1, indicated cluster 26 and 29 are dividing cell. Cluster 30 and 31 were both assigned as root cap based on the enriched expression of cap cell markers, with AT2G47000 (ABCB4)24 in cluster 30, AT1G79580 (SMB)14 and AT1G33280 (BRN1)14 in cluster 31. Cluster 3 specifically expressed two initial cell markers AT5G63660 (PDF2.5) and AT3G60390 (HAT3), that were identified by previous single-cell study1, so we assigned these clusters as initial cell. In the 6-day and 11-day root nucleus population, all the above cell types were annotated with similar marker genes.

Cell type annotation for shoot

The 6-day shoot nucleus population was grouped into 21 clusters (Figures S1C and S2C). Cluster 3, 6, 7, 11 and 17 were assigned as epidermis based on the enriched expression of the epidermis-specific marker genes AT4G21750 (ATML1)5 and AT1G68530 (CER6)9, 25, which is involved in wax synthesis and cuticle formation in plants. Enriched expression of AT3G24140 (FAMA)26, which promotes differentiation of stomatal guard cells and halts proliferative divisions in their immediate precursors, indicated that cluster 16 corresponds to guard cell. The trichome identity of cluster 15 was supported by the enriched expression of the well-known trichome-specific marker gene AT1G79840 (GL2)11. We also identified multiple genes specifically expressed in trichome as novel trichome-specific markers (Figure 2D). Multiple clusters, including cluster 0, 1, 2, 4, 10 and 12 show enriched expression of mesophyll specific genes that had been established in previous single-cell studies27, such as AT1G37130 (NIA2), AT2G38230 (PDX11) and AT2G39470 (PNSL1), indicating these clusters are mesophyll. Cluster 8 was assigned as bundle sheath based on the enriched expression of an established marker AT2G42530 (COR15B)28. Enriched expression of AT3G48740 (SWEET11)28, 29 and AT3G1193028 indicated cluster 5 and 14 are phloem parenchyma. Enriched expression of AT1G22710 (SUC2)30 indicated cluster 9 is companion cell. Cluster 19 was assigned as xylem based on the enriched expression of three established markers AT2G37090 (IRX9)31, AT5G12870 (MYB46)32 and AT1G71930 (VND7)9. The enriched expression of multiple cell-cycle related genes, such as AT1G08560 (KN)33, AT1G76310 (CYCB2;4)23 and AT5G16250 (MERCY1)23 indicated cluster 13 and 18 are dividing cell. Cluster 20 specifically expressed AT3G21710 (VUP1)34, which was established to express in vascular tissues and was required for normal xylem vessel development, indicating its vascular identity.

Cell type annotation for stem

The 42-day stem nucleus population was grouped into 15 clusters (Figures S1D and S2D). Cluster 2 was assigned as epidermis due to the enriched expression of a well-documented epidermis-specific marker gene AT1G68530 (CER6)9, 25. Enriched expression of AT3G24140 (FAMA)26 indicated cluster 13 is guard cell. Transgenic studies suggested that photosynthesis-related genes such as AT2G05070 (LHCB2.2) and AT5G54270 (LHCB3) are highly abundant in cortex of stem12. In our stem dataset, multiple clusters, including cluster 1, 3, 5 and 12 show enriched expression of photosynthesis-related genes, suggesting these clusters are cortex. Enriched expression of AT3G48740 (SWEET11)28, 29 and AT1G22710 (SUC2)30 indicated that cluster 6 and 8 are phloem parenchyma and companion cell, respectively. Cluster 7 and 14 were assigned as xylem/interfascicular fiber due to the enriched expression of three established markers AT2G37090 (IRX9)31, AT5G12870 (MYB46)32 and AT1G32770 (NST3)9. Cluster 4 and 10 were assigned as procambium and starch sheath, due to the enriched expression of AT2G01950 (VH1)35 and AT1G68570 (NPF3.1)5 respectively. Cluster 11 specifically expressed AT3G49780 (PSK3)36, which was established to abundantly express in vascular bundles of cotyledons and leaves, indicating vascular identity of this cluster. No well-known markers were detected in cluster 0. However, we detected pith enriched genes AT1G70830 (MLP28) and AT1G80170 identified by Stereo-seq in this study, supporting the annotation of cluster 0 as pith. For cluster 9, no marker genes were detected and no cluster-enriched genes identified by Stereo-seq data, we therefore remained this cluster as “unknown”.

Cell type annotation for leaf samples

The integrative rosette nucleus population was grouped into 28 clusters (Figures S3L and S3M), and the stage 1 to stage 6 (S1-S6) rosette nucleus populations were grouped into 18 (S1), 11 (S2), 15 (S3), 13 (S4), 10 (S5) and 14 (S6) clusters (Figures S1F–S1K and S2F–S2K), respectively. The cauline leaf nucleus population was grouped into 12 clusters (Figures S1E and S2E). For the integrative rosette nucleus population, cluster 6, 7, 9, 10, 11 and 13 were assigned as epidermis based on the enriched expression of the epidermis-specific marker genes AT4G21750 (ATML1)5 and AT1G68530 (CER6)9, 25. Enriched expression of AT3G24140 (FAMA)26 indicated cluster 24 is guard cell. Multiple clusters, including cluster 0, 1, 2, 3, 4, 8, 12, 16, 18, 19, 21 and 25 show enriched expression of mesophyll specific genes that had been established in previous single-cell studies27, such as AT5G38420 (RBCS2B), AT3G50480 (HR4) and AT2G45180 (DRN1), indicating these clusters are mesophyll. Cluster 5 and 20 were assigned as bundle sheath based on the enriched expression of an established marker AT2G42530 (COR15B)28. Enriched expression of AT3G48740 (SWEET11)29 and AT5G24800 (bZIP9)29 indicated cluster 22 is phloem parenchyma. Enriched expression of AT1G22710 (SUC2)30 and AT1G79430 (APL)9, 37 indicated cluster 15 and 26 are companion cell. Cluster 17 was identified as xylem, supported by enriched expression of two xylem marker genes AT5G46730 and AT5G61660 (validated by FISH5). The enriched expression of cell-cycle related genes, such as AT1G08560 (KN)27, AT3G27060 (TSO2)38 and AT4G32830 (AUR1)23 indicated cluster 23 and 27 are dividing cell. Cluster 14 specifically expressed AT3G49780 (PSK3)39, which was established to abundantly express in vascular bundles of cotyledons and leaves, indicating vascular identity of this cluster. Most of the cell types, including epidermis, guard cell, mesophyll, phloem parenchyma, companion cell, were identified by marker-based annotation in all the leaf nucleus populations. However, no clusters in S2, S5 and S6 stage leaves exhibited clear xylem marker gene signatures. By tracing the sample-of-origin for the xylem nuclei in the integrative rosette dataset, we were able to identify subsets of nucleus populations in S2, S5 and S6 rosette leaves, and manually assigned these nuclei as xylem.
Nevertheless, trichome was only identified in the cluster 16 of S1 leaf, supported by the enriched expression of the well-known trichome-specific marker gene AT1G79840 (GL2)11. Moreover, no dividing cells were identified in S4-S6 rosette datasets or in the cauline leaf, suggesting cell proliferation is mainly absent in these tissues.

Cell type annotation for flower

In flower, there are 23, 18, and 19 clusters identified in early, middle and late flower, respectively, resulted in 27 clusters in integrated flowers (Figures 2F, S1L–S1N, S2L–S2N and S3P). In the integrated landscape, cluster 7 was annotated as epidermis based on preferentially expressed gene AT2G26250 (FDH)40, which contributes to adhesion response and cell differentiation in the epidermis. Enriched expression of markers AT3G24140 (FAMA)26 and AT5G26000 (TGG1)41 in cluster 9 indicated its identify of guard Cell. Photosynthesis-related genes such as AT3G54890 (LHCA1) and AT1G67090 (RBCS1A)42, 43 are highly abundant in clusters 0, 23, and 24, suggest their identity of cortex. Cluster 10 was assigned as companion cell due to the dominant expression of AT1G22710 (SUC2)44. Cluster 8 and 15 were annotated as phloem parenchyma. Among them, cluster 8 showed the enriched expression of AT1G77690 (LAX3)45, which is involved in proton-driven auxin influx. Cluster 15 was characterized by preferentially expressed gene AT5G24800 (bZIP9)29, 46 and AT3G48740 (SWEET11)28, 29 which are shown to be cell-specific markers of phloem parenchyma in previous single cell studies29, 46. Cluster 14 and 16 were assigned as procambium due to the enriched expression of marker genes AT3G45610 (DOF3.2)47 and AT5G61480 (TDR)9. Integument marker genes AT1G23420 (INO)48, AT2G42830 (SHP2)49, and AT3G58780 (SHP1)49 showed abundant expression in cluster 6, suggesting its identity of integument. AT2G39060 (SWEET9)50 is a nectary-specific sugar transporter mediating the secretion of sucrose from the nectary parenchyma to the extracellular space. It was preferentially expressed in clusters 17 and 20, suggested the identify of nectary. However, stigma marker gene AT1G65450 (GLAUCE)51 showed dominant expression as well, these two clusters were therefore annotated as nectary/stigma cell. Clusters 12 and 21 were annotated as sperm due to the enriched expression of AT1G19890 (MGH3)52. Cluster 5 was identified as vegetative nucleus based on the enriched genes AT2G33240 (XID) and AT5G20690 (LURE1)52. Cluster 19 was assigned as microsporocyte due to the specific expression of AT5G40260 (SWEET8)53. Clusters 2, 3, 11 showed abundant expression of pollen-specific markers AT3G06830 (PME23) and AT5G07410 (PME48)54, suggested the identity of pollen. Cluster 1, 4, 13, 22, and 25 were assigned as tapetum due to the preferentially expressed genes of several GRP genes (AT5G07510 (GRP14), AT5G07540 (GRP16), AT5G07530 (GRP17), AT5G07520 (GRP18), AT5G07550 (GRP19), AT5G07560 (GRP20))55, and TKPR genes (AT4G35420 (TKPR1) and AT1G68540 (TKPR2))56. Specific expression of well-known markers was not detected in three clusters 18, 23 and 26, which were therefore remained as “unknown”. In early, middle and late flower, dividing cell was detected, with the enriched expression of cell-cycle related genes AT1G47210 (CYCA3;2)57 and AT1G04020 (ATBARD1)58. Abscission zone was detected in late flower cluster 15, in which AT4G28490 (RLK5)59 and AT5G65710 (HSL2)60 genes were preferentially expressed.

Cell type annotation for silique

At each time point, the siliques nucleus populations were clustered into distinct groups, specifically 24 clusters at 0 DPA, 22 clusters at 1 DPA, 26 clusters at 2 DPA, 20 clusters at 3 DPA, 20 clusters at 4 DPA, and 20 clusters at 5 DPA (Figures S1O–1T and S2O–S2T). When considering the integrative siliques nucleus population, it was clustered into a total of 29 groups (Figures 2E and S3N). For the integrative siliques nucleus population, cluster 5 was identified as epidermis based on the enriched expression of three well-studied epidermis-specific genes, including AT5G15310 (MYB16)61 AT1G27950 (LTPG1)62, and AT2G26250 (FDH)63. MYB16 is a MIXTA-like MYB transcription factor and regulate epidermal cell morphology as well as cuticle development in Arabidopsis. Cluster 11 was identified as guard cells based on the enriched expression of guard cell marker genes AT3G24140 (FAMA)26 and AT5G26000 (TGG1)41. The designation of cluster 24 as nectary cells was based on the enriched expression of AT2G36190 (CWINV4)64 and nectary-specific sugar transporter AT2G39060 (SWEET9)50. Cluster 23 was classified as stigma/transmitting tract cells because of the enriched expression of AT2G02850 (ARPN)65 and AT3G01530 (MYB57)66. ARPN is a single plantacyanin gene and strongly expressed in the stigma/style in Arabidopsis. Clusters 8 and 20 were annotated as cortex cells owing to the enriched expression of AT3G18830 (PLT5)67. Cluster 10 was designated as companion cells based on the enriched expression of AT1G22710 (SUC2)44 and AT4G19840 (ATPP2-A1)68. The assignment of cluster 9 as phloem parenchyma cells was based on the enriched expression of AT5G24800 (bZIP9)69. Cluster 13 was identified as xylem cells due to the enriched expression of AT5G44030 (CESA4)70 and AT5G17420 (CESA7)71. Cluster 17 was classified as procambium cells based on the enriched expression of AT1G52150 (ATHB15)72 and AT4G32880 (ATHB8)73. ATHB15 is homeobox-leucine zipper protein and probable transcription factor involved in the regulation of meristem development to promote lateral organ formation. Cluster 1 was designated as seed coat (L1)/integument cells because of the enriched expression of AT1G23420 (INO)48, AT2G42830 (SHP2)49, and AT3G58780 (SHP1)49. Clusters 0, 14, and 25 were identified as seed coat (L2) cells due to the enriched expression of AT4G12960 (GILT)74, AT1G16390 (OCT3)75, and AT5G49180 (PME58)54. GILT is gamma-interferon-responsive lysosomal thiol protein and expressed in the outer integument of seed coat. Cluster 15 was classified as seed coat (L3/4) cells based on the enriched expression of AT1G11590 (PME19)54 and AT3G20210 (dVPE)76. Clusters 2 and 12 were designated as seed coat (L5) cells owing to the enriched expression of AT1G61720 (BAN)77 and AT3G59030 (TT12)78. BAN specifically expressed in the endothelium of seed coat. TT12 showed specific expression in the endothelium layer of the ovule and the seed coat. Clusters 7 and 26 were identified as embryo cells because of the enriched expression of AT2G30470 (VAL1)79 and AT3G20740 (FIE)80. Clusters 4, 18, and 21 were classified as endosperm cells based on the enriched expression of AT1G02580 (MEA)80, AT4G25530 (HDG6)81, and AT1G71890 (SUC5)82. MEA expressed in unpollinated siliques that contain maturing gametophytes. Cluster 3 was designated as dividing cell due to the enriched expression of AT1G04020 (ATBARD1)58, AT1G50490 (UBC20)83, and AT3G25980 (MAD2)84. MAD2 expresses in actively dividing tissues, early in organ development, in young leaves, lateral root primordia and root meristems. Clusters 6, 16, 19, 22, and 27 were identified as pollen cells based on the enriched expression of AT2G19770 (PRO4)85 and AT5G07410 (PME48)54. PRO4 specifically expressed in mature pollen grains. PME48 expressed in mature pollen grains in the anthers and on the stigma. Cluster 28 was assigned as unknown due to no enrichment of specific marker genes.

References for supplemental note:

1. Wendrich, J.R., Yang, B., Vandamme, N., et al. (2020). Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions. Science 370, 4970. https://doi.org/10.1126/science.aay4970
2. Denyer, T., Ma, X., Klesen, S., et al. (2019). Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing. Dev Cell. 48, 840–852 https://doi.org/10.1016/j.devcel.2019.02.022
3. Lewis, D.R., Negi, S., Sukumar, P., et al. (2011). Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers. Development 138, 3485–3495. https://doi.org/10.1242/dev.065102
4. Liu, Z., Zhou, Y., Guo, J., et al. (2020) Global dynamic molecular profiling of stomatal lineage cell development by single-cell RNA sequencing. Mol. Plant. 13, 1178–1193. https://doi.org/10.1016/j.molp.2020.06.010
5. Zhang, T.Q., Chen, Y., and Wang, J.W. (2021). A single-cell analysis of the Arabidopsis vegetative shoot apex. Dev. Cell 56, 1056–1074. https://doi.org/10.1016/j.devcel.2021.02.021
6. Breeze, E., Harrison, E., McHattie, S., et al. (2011). High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. Plant Cell. 23, 873–894. https://doi.org/10.1105/tpc.111.083345
7. Woo, H.R., Koo, H.J., Kim, J., et al. (2016). Programming of plant leaf senescence with temporal and inter-organellar coordination of transcriptome in Arabidopsis. Plant Physiol. 171, 452–67. https://doi.org/10.1104/pp.15.01929
8. Xia, K., Sun, H.X., Li, J., et al. (2022). The single-cell stereo-seq reveals region-specific cell subtypes and transcriptome profiling in Arabidopsis leaves. Dev. Cell 57, 1299–1310. https://doi.org/10.1016/j.devcel.2022.04.011
9. Shi, D., Jouannet, V., Agustí, J., et al. (2021). Tissue-specific transcriptome profiling of the Arabidopsis inflorescence stem reveals local cellular signatures. Plant Cell 33, 200–223. https://doi.org/10.1093/plcell/koaa019
10. Cai, S., and Lashbrook, C.C. (2008). Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis ZINC FINGER PROTEIN2. Plant Physiol. 146, 1305–1321. https://doi.org/10.1104/pp.107.110908
11. Lin, Y., and Schiefelbein, J. (2001). Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis. Dev. (Camb.) 128, 3697–3705. https://doi.org/10.1242/dev.128.19.3697
12. Yi, K., Menand, B., Bell, E., et al. (2010). A basic helix-loop-helix transcription factor controls cell growth and size in root hairs. Nat. Genet. 42, 264–267. https://doi.org/10.1038/ng.529
13. Jia, Z., Giehl, H., Hartmann, A., et al. (2023). A spatially concerted epidermal auxin signaling framework steers the root hair foraging response under low nitrogen. Curr. Biol. 33, 3926–3941. https://doi.org/10.1016/j.cub.2023.08.040
14. Shahan, R., Hsu, W., Nolan, M., et al. (2022). A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants. Dev. Cell 57, 543–560. https://doi.org/10.1016/j.devcel.2022.01.008
15. Kamiya, T., Borghi, M., Wang, P., et al. (2015). The MYB36 transcription factor orchestrates Casparian strip formation. Proc. Natl. Acad. Sci. U.S.A. 112, 10533–10538. https://doi.org/10.1073/pnas.1507691112
16. Nakamura, M., and Grebe, M. (2018). Outer, inner and planar polarity in the Arabidopsis root. Curr. Opin. Plant Biol. 41, 46–53. https://doi.org/10.1016/j.pbi.2017.08.002
17. Beisson, F., Li, Y., Bonaventure, G., et al. (2007). The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19, 351–368. [https://doi.org/10.1105/tpc.106
18. Andersen, G., Naseer, S., Ursache, R., Wybouw, B., Smet, W., De Rybel, B., Vermeer, M., and Geldner, N. (2018). Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells. Nature 555, 529–533. https://doi.org/10.1038/nature25976
19. Petricka, J., Clay, K., and Nelson, M. (2008). Vein patterning screens and the defectively organized tributaries mutants in Arabidopsis thaliana. Plant J. 56, 251–263. https://doi.org/10.1111/j.1365-313X.2008.03595.x
20. Funk, V., Kositsup, B., Zhao, C., and Beers, E. P. (2002). The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol. 128, 84–94. https://doi.org/10.1104/pp.010514
21. Lara, P., Oñate-Sánchez, L., Abraham, Z., Ferrándiz, C., Díaz, I., Carbonero, P., and Vicente-Carbajosa, J. (2003). Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. J. Biol. Chem. 278, 21003–21011. https://doi.org/10.1074/jbc.M210538200
22. Bonke, M., Thitamadee, S., Mähönen, P., Hauser, T., and Helariutta, Y. (2003). APL regulates vascular tissue identity in Arabidopsis. Nature 426, 181–186. https://doi.org/10.1038/nature02100
23. Apelt, F., Mavrothalassiti, E., Gupta, S., Machin, F., Olas, J., Annunziata, G., Schindelasch, D., and Kragler, F. (2022). Shoot and root single cell sequencing reveals tissue- and daytime-specific transcriptome profiles. Plant Physiol. 188, 861–878. https://doi.org/10.1093/plphys/kiab537
24. Terasaka, K., Blakeslee, J., Titapiwatanakun, B., Peer, A., Bandyopadhyay, A., Makam, N., Lee, R., Richards, L., Murphy, S., Sato, F., and Yazaki, K. (2005). PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots. Plant Cell 17, 2922–2939. https://doi.org/10.1105/tpc.105.035816
25. Millar, A. A., Clemens, S., Zachgo, S., Giblin, E. M., Taylor, D. C., and Kunst, L. (1999). CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11, 825–838. https://doi.org/10.1105/tpc.11.5.825
26. Ohashi-Ito, K., and Bergmann, D. C. (2006). Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18, 2493–2505. https://doi.org/10.1105/tpc.106.046136
27. Tang, B., Feng, L., Hulin, M. T., Ding, P., and Ma, W. (2023). Cell-type-specific responses to fungal infection in plants revealed by single-cell transcriptomics. Cell Host Microbe 31, 1732–1747. https://doi.org/10.1016/j.chom.2023.08.019
28. Procko, C., Lee, T., Borsuk, A., Bargmann, B. O. R., Dabi, T., Nery, J. R., Estelle, M., Baird, L., O'Connor, C., Brodersen, C., et al. (2022). Leaf cell-specific and single-cell transcriptional profiling reveals a role for the palisade layer in UV light protection. Plant Cell 34, 3261–3279. https://doi.org/10.1093/plcell/koac167
29. Kim, Y., Symeonidi, E., Pang, Y., Denyer, T., Weidauer, D., Bezrutczyk, M., Miras, M., Zöllner, N., Hartwig, T., Wudick, M., et al. (2021). Distinct identities of leaf phloem cells revealed by single cell transcriptomics. Plant Cell 33, 511–530. https://doi.org/10.1093/plcell/koaa060
30. Machin, Q., Beckers, M., Tian, X., Fairnie, A., Cheng, T., Scheible, R., and Doerner, P. (2019). Inducible reporter/driver lines for the Arabidopsis root with intrinsic reporting of activity state. Plant J. 98, 153–164. https://doi.org/10.1111/tpj.14192
31. Peña, J., Zhong, R., Zhou, K., Richardson, A., O'Neill, A., Darvill, G., York, S., and Ye, H. (2007). Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 19, 549–563. https://doi.org/10.1105/tpc.106.049320
32. Zhong, R., Richardson, E. A., and Ye, Z. H. (2007). The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosynthesis in Arabidopsis. Plant Cell 19, 2776–2792. https://doi.org/10.1105/tpc.107.053678
33. Lukowitz, W., Mayer, U., and Jürgens, G. (1996). Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61–71. https://doi.org/10.1016/s0092-8674(00)80993-9
34. Grienenberger, E., and Douglas, J. (2014). Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 regulates xylem development and growth by a conserved mechanism that modulates hormone signaling. Plant Physiol. 164, 1991–2010. https://doi.org/10.1104/pp.114.236406
35. Han, E., Geng, Z., Qin, Y., Wang, Y. and Ma, S. (2024). Single-cell network analysis reveals gene expression programs for Arabidopsis root development and metabolism. Plant Commun. 5, 100978. https://doi.org/10.1016/j.xplc.2024.100978
36. Matsubayashi, Y., Ogawa, M., Kihara, H., Niwa, M., and Sakagami, Y. (2006). Disruption and overexpression of Arabidopsis phytosulfokine receptor gene affects cellular longevity and potential for growth. Plant Physiol. 142, 45–53. https://doi.org/10.1104/pp.106.081109
37. Han, X., Zhang, Y., Lou, Z., Li, J., Wang, Z., Gao, C., Liu, Y., Ren, Z., Liu, W., Li B., et al. (2023). Time series single-cell transcriptional atlases reveal cell fate differentiation driven by light in Arabidopsis seedlings. Nat. Plants 9, 2095-2109. https://doi.org/10.1038/s41477-023-01544-4
38. Wang, C., and Liu, Z. (2006). Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development. Plant Cell 18, 350–365. https://doi.org/10.1105/tpc.105.037044
39. Matsubayashi, Y., Ogawa, M., Kihara, H., Niwa, M., and Sakagami, Y. (2006). Disruption and overexpression of Arabidopsis phytosulfokine receptor gene affects cellular longevity and potential for growth. Plant Physiol. 142, 45–53. https://doi.org/10.1104/pp.106.081109
40. FISH, Yephremov, A., Wisman, E., Huijser, P., Huijser, C., Wellesen, K., and Saedler, H. (1999). Characterization of the FIDDLEHEAD gene of Arabidopsis reveals a link between adhesion response and cell differentiation in the epidermis. Plant Cell 11, 2187-2201.https://doi.org/10.1105/tpc.11.11.2187
41. Thangstad, O. P., Gilde, B., Chadchawan, S., Seem, M., Husebye, H., Bradley, D., and Bones, A. M. (2004). Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum. Plant Mol. Biol. 54, 597-611.https://doi.org/10.1023/B:PLAN.0000038272.99590.10
42. Wientjes, E., van Stokkum, H., van Amerongen, H., and Croce, R. (2011). The role of the individual Lhcas in photosystem I excitation energy trapping. Biophys. J. 101, 745-754. https://doi.org/10.1016/j.bpj.2011.06.045
43. Izumi, M., Tsunoda, H., Suzuki, Y., Makino, A., and Ishida, H. (2012). RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity. J. Exp. Bot. 63, 2159-2170.https://doi.org/10.1093/jxb/err434
44. Schulze, X., Reinders, A., Ward, J., Lalonde, S., and Frommer, B. (2003). Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system. BMC Biochem. 4, 1-10.https://doi.org/10.1186/1471-2091-4-3
45. Swarup, K., Benková, E., Swarup, R., et al. (2008) The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 10, 946-954.https://doi.org/10.1038/ncb1754
46. Liu, Z., Wang, J., Zhou, Y., Zhang, Y., Qin, A., Yu, X., Zhao, Z., Wu, R., Guo, C., Bawa, G., Rochaix, D., et al. (2022). Identification of novel regulators required for early development of vein pattern in the cotyledons by single-cell RNA-sequencing. Plant J. 110, 7-12. https://doi.org/10.1111/tpj.15719
47. Miyashima, S., Roszak, P., Sevilem, I., Toyokura, K., Blob, B., Heo, J., Mellor, N., Rahko, H., Otero, S., Smet, W., et al. (2019). Mobile PEAR transcription factors integrate positional cues to prime cambial growth. Nature 565, 490-494. https://doi.org/10.1038/s41586-018-0839-y
48. Villanueva, M., Broadhvest, J., Hauser, A., Meister, J., Schneitz, K., and Gasser, S. (1999). INNER NO OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules. Genes Dev. 13, 3160-3169. https://doi.org/10.1101/gad.13.23.3160
49. Ehlers, K., Bhide, S., Tekleyohans, G., Wittkop, B., Snowdon, J., and Becker, A. (2016). The MADS Box genes ABS, SHP1, and SHP2 are essential for the coordination of cell divisions in ovule and seed coat development and for endosperm formation in Arabidopsis thaliana. PLoS One 11, e0165075. https://doi.org/10.1371/journal.pone.0165075
50. Lin, W., Sosso, D., Chen, Q., Gase, K., Kim, G., Kessler, D., Klinkenberg, M., Gorder, K., Hou, H., Qu, Q., et al. (2014). Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 508, 546-549. https://doi.org/10.1038/nature13082
51. Swanson, R., Clark, T., and Preuss, D. (2005). Expression profiling of Arabidopsis stigma tissue identifies stigma-specific genes. Sex. Plant Reprod. 18, 16-171. https://doi.org/10.1007/s00497-005-0009-x
52. Misra, S., Sousa, G., Barros, M., Kermanov, A., and Becker, D. (2023). Cell-type-specific alternative splicing in the Arabidopsis germline. Plant Physiol. 192, 85-101. https://doi.org/10.1093/plphys/kiac574
53. Yu, J., Hogan, P., and Sundaresan, V. (2005). Analysis of the female gametophyte transcriptome of Arabidopsis by comparative expression profiling. Plant Physiol. 139,1853-1869. https://doi.org/10.1104/pp.105.067314
54. Louvet, R., Cavel, E, Gutierrez, L., Guénin, S., Roger, D., Gillet, F., Guerineau, F., and Pelloux, J. (2006). Comprehensive expression profiling of the pectin methylesterase gene family during silique development in Arabidopsis thaliana. Planta 224, 782-791. https://doi.org/10.1007/s00425-006-0261-9
55. Frank, W., Joseé, R., Maércio, A., and Elliot, M. (2004). Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 16, 1314-1326. https://doi.org/10.1105/tpc.021741
56. Grienenberger, E., Kim, S., Lallemand, B., Geoffroy, P., Heintz, D., Souza, A., Heitz, T., Douglas, J., and Legrand, M. (2010). Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollen in monomer biosynthesis. Plant Cell 22, 4067-4083. https://doi.org/10.1105/tpc.110.080036
57. Vandepoele, K., Raes, J., De, L., Rouzé, P., Rombauts, S., Inzé, D. (2002) Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell 14, 903-916. https://doi.org/10.1105/tpc.010445
58. Han, P., L,i Q. and Zhu, X. (2008). Mutation of Arabidopsis BARD1 causes meristem defects by failing to confine WUSCHEL expression to the organizing center. Plant Cell 20, 1482-1493. https://doi.org/10.1105/tpc.108.058867
59. Jinn, TL., Stone, JM., Walker, JC. (2000). HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission. Genes Dev. 14, 108-117.
60. Cho, K., Larue, T., Chevalier, D., Wang, H., Jinn, L., Zhang, S. and Walker, C. (2008). Regulation of floral organ abscission in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 105, 15629-15634. https://doi.org/10.1073/pnas.0805539105
61. Oshima, Y., Shikata, M., Koyama, T., Ohtsubo, N., Mitsuda, N., and Ohme-Takagi, M. (2013). MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. Plant Cell 25, 1609-1624. https://doi.org/10.1105/tpc.113.110783
62. Lee, B., Go, S., Bae, J., Park, H., Cho, H., Cho, J., Lee, S., Park, K., Hwang, I., and Suh, C. (2009). Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Physiol. 150, 42-54. https://doi.org/10.1104/pp.109.137745
63. Pruitt, E., Vielle-Calzada, P., Ploense, E., Grossniklaus, U., and Lolle, J. (2000). FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proc. Natl. Acad. Sci. U.S.A. 97, 1311-1316. https://doi.org/10.1073/pnas.97.3.1311
64. Ruhlmann, M., Kram, W., and Carter, J. (2010). CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis. J. Exp. Bot. 61, 395-404. https://doi.org/10.1093/jxb/erp309
65. Dong, J., Kim, S. T., and Lord, E. M. (2005). Plantacyanin plays a role in reproduction in Arabidopsis. Plant Physiol. 138, 778-789. https://doi.org/10.1104/pp.105.063388
66. Cheng, H., Song, S., Xiao, L., Soo, H. M., Cheng, Z., Xie, D., and Peng, J. (2009). Gibberellin acts through jasmonate to control the expression of MYB21, MYB24, and MYB57 to promote stamen filament growth in Arabidopsis. PLoS Genet. 5, e1000440. https://doi.org/10.1371/journal.pgen.1000440
67. Klepek, Y. S., Geiger, D., Stadler, R., Klebl, F., Landouar-Arsivaud, L., Lemoine, R., Hedrich, and R., Sauer, N. (2005). Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose. Plant Cell 17, 204-218. https://doi.org/10.1105/tpc.104.026641
68. Dinant, S., Clark, A. M., Zhu, Y., Vilaine, F., Palauqui, J. C., Kusiak, C., Thompson, A. (2003). Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms. Plant Physiol. 131, 114-128. https://doi.org/10.1104/pp.013086
69. Weltmeier, F., Rahmani, F., Ehlert, A., Dietrich, K., Schütze, K., Wang, X., Chaban, C., Hanson, J., Teige, M., Harter, K., et at al. (2009). Expression patterns within the Arabidopsis C/S1 bZIP transcription factor network: availability of heterodimerization partners controls gene expression during stress response and development. Plant Mol. Biol. 69, 107-119. https://doi.org/10.1007/s11103-008-9410-9
70. Holland, N., Holland, D., Helentjaris, T., Dhugga, K. S., Xoconostle-Cazares, B., and Delmer, P. (2000). A comparative analysis of the plant cellulose synthase (CesA) gene family. Plant Physiol. 123, 1313-1324. https://doi.org/10.1104/pp.123.4.1313
71. Taylor, G., Laurie, S., and Turner, R. (2000). Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell 12, 2529-2540. https://doi.org/10.1105/tpc.12.12.2529
72. Kyoko Ohashi-Ito, and Hiroo Fukuda (2003). HD-zip III homeobox genes that include a novel member, ZeHB-13 (Zinnia)/ATHB-15 (Arabidopsis), are involved in procambium and xylem cell differentiation, Plant Cell Physiol. 44, 1350–1358. https://doi.org/10.1093/pcp/pcg164
73. Prigge, J., Otsuga, D., Alonso, M., Ecker, R., Drews, N., and Clark, E. (2005). Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development. Plant Cell, 17, 61-76. https://doi.org/10.1105/tpc.104.026161
74. Wu, L., El-Mezawy, A. and Shah, S. (2011). A seed coat outer integument-specific promoter for Brassica napus. Plant Cell Rep. 30, 75-80. https://doi.org/10.1007/s00299-010-0945-2
75. Küfner, I., and Koch, W. (2008). Stress regulated members of the plant organic cation transporter family are localized to the vacuolar membrane. BMC Res. Notes 11,1-43. https://doi.org/10.1186/1756-0500-1-43
76. Nakaune, S., Yamada, K., Kondo, M., Kato, T., Tabata, S., Nishimura, M. and Hara-Nishimura, I. (2005). A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development. Plant Cell 17, 876-87. https://doi.org/10.1105/tpc.104.026872
77. Devic, M., Guilleminot, J., Debeaujon, I., Bechtold, N., Bensaude, E., Koornneef, M., Pelletier G. and Delseny M. (1999). The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J. 19, 387-398. https://doi.org/10.1046/j.1365-313X.1999.00529.x
78. Marinova, K., Pourcel, L., Weder, B., Schwarz, M., Barron, D., Routaboul, J. M., Debeaujon, I. and Klein, M. (2007). The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat. Plant Cell 19, 2023-2038. https://doi.org/10.1105/tpc.106.046029
79. Tsukagoshi, H., Saijo, T., Shibata, D., Morikami, A. and Nakamura, K. (2005). Analysis of a sugar response mutant of Arabidopsis identified a novel B3 domain protein that functions as an active transcriptional repressor. Plant Physiol. 138, 675-685. https://doi.org/10.1104/pp.104.057752
80. Luo, M., Bilodeau, P., Dennis, E., Peacock, W. and Chaudhury, A. (2000). Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. U.S.A. 97, 10637-10642. https://doi.org/10.1073/pnas.170292997
81. Yoko, I., Yasushi, K., Ayako, Y., Mitsutomo, A. and Takashi, A. (2007). Molecular basis of late-flowering phenotype caused by dominant epi-alleles of the FWA locus in Arabidopsis. Plant Cell Physiol. 48, 205–220. https://doi.org/10.1093/pcp/pcl061
82. Pommerrenig, B., Popko, J., Heilmann, M., Schulmeister, S., Dietel, K., Schmitt, B., Stadler, R., Feussner, I. and Sauer, N. (2013). SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation. Plant J. 73, 392-404. https://doi.org/10.1111/tpj.12037
83. Criqui, M., de Almeida Engler, J., Camasses, A., Capron, A., Parmentier, Y., Inzé, D. and Genschik, P. (2002). Molecular characterization of plant ubiquitin-conjugating enzymes belonging to the UbcP4/E2-C/UBCx/UbcH10 gene family. Plant Physiol. 130, 1230-1240. https://doi.org/10.1104/pp.011353
84. Caillaud, M., Paganelli, L., Lecomte, P., Deslandes, L., Quentin, M., Pecrix, Y., Le Bris, M., Marfaing, N., Abad, P. and Favery, B. (2009). Spindle assembly checkpoint protein dynamics reveal conserved and unsuspected roles in plant cell division. PLoS One 27, e6757. https://doi.org/10.1371/journal.pone.0006757
85. Kandasamy, M., McKinney, E., Meagher, R. (2002). Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues. Cell Motil. Cytoskelet. 52, 22-32. https://doi.org/10.1002/cm.10029