Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation

Zhao, Lin-lin; Chen, Ru; Bai, Ziyu; Liu, Junyi; Zhang, Yuhao; Zhong, Yicheng; Sun, Meng-xiang; Zhao, Peng

doi:10.1038/s41467-024-46902-8

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Published: 27 March 2024

Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation

Nature Communications volume 15, Article number: 2676 (2024) Cite this article

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Abstract

Autophagy modulates the degradation and recycling of intracellular materials and contributes to male gametophyte development and male fertility in plants. However, whether autophagy participates in seed development remains largely unknown. Here, we demonstrate that autophagy is crucial for timely programmed cell death (PCD) in the integumentary tapetum, the counterpart of anther tapetum, influencing embryo pattern formation and seed viability. Inhibition of autophagy resulted in delayed PCD of the integumentary tapetum and defects in embryo patterning. Cell-type-specific restoration of autophagic activities revealed that the integumentary tapetum plays a non-autonomous role in embryo patterning. Furthermore, high-throughput, comprehensive lipidomic analyzes uncovered an unexpected seed-developmental-stage-dependent role of autophagy in seed lipid metabolism: it contributes to triacylglycerol degradation before fertilization and to triacylglycerol biosynthesis after fertilization. This study highlights the critical role of autophagy in regulating timely integumentary tapetum PCD and reveals its significance in seed lipid metabolism and viability.

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Introduction

Autophagy is an evolutionarily conserved catabolic pathway in eukaryotes that degrades and recycles intracellular contents under normal and stress conditions^1,2. There are three major types of autophagy: macro-autophagy, micro-autophagy, and chaperone-mediated autophagy³. Of these, macro-autophagy (hereafter referred to as autophagy) is the best studied and is characterized by the formation of a double-membrane autophagosome that encloses cytoplasmic components and facilitates their degradation and turnover¹. Autophagy is tightly regulated by a set of conserved core AUTOPHAGY-RELATED (ATG) genes⁴. These core ATG proteins can be categorized into five functional groups during autophagosome formation: the ATG1–ATG13 complex, the ATG9 cycling system, the phosphatidylinositol 3-kinase (PI3K) complex, and ATG8 and ATG12 ubiquitin-like conjugation systems. The ATG8 conjugation system is composed of ATG3, ATG4, ATG7, and ATG8, while the ATG12 conjugation system is composed of ATG5, ATG7, ATG10, ATG12, and ATG16^4,5. In plants, autophagy is critical for adaptation to different abiotic and biotic environmental stresses^6,7. In addition, recent evidence indicates that autophagy also plays crucial roles in normal development, including reproduction, in which it helps to shape pollen germination and anther development^8,9,10,11,12 as well as vegetative growth^13,14. Despite its importance, little is known about the function of autophagy in early seed development¹⁵.

In angiosperms, seeds develop from ovules following double fertilization and consist of three major parts: the embryo, the endosperm, and the seed coat. The embryo and endosperm are derived from two distinct fertilization events in which two sperm cells fertilize one egg cell and one central cell, which are surrounded by the seed coat^16,17,18. The seed coat is a specialized tissue derived from maternal ovule integuments, which are usually composed of outer integument and inner integument layers. In some plant species, such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the innermost layer of the inner integument differentiates into a specific tissue known as the integumentary tapetum or endothelium, which is the counterpart of the anther tapetum^19,20. The anther tapetum provides protection, nutrients, enzymes, and precursors to developing pollen grains; importantly, this tissue degenerates through programmed cell death (PCD)^21,22,23 in a process that is necessary for pollen wall development and male fertility²⁴. Early studies suggest that the integumentary tapetum serves multiple functions, including facilitating nutrient transport to the embryo sac and safeguarding the embryo sac to ensure the development of endosperm and embryo^19,25,26. Similar to the anther tapetum, the integumentary tapetum also undergoes degeneration through PCD after fertilization, which initiates at the pre-embryo stage^25,27. However, in comparison to the extensively studied anther tapetum in relation to pollen development and male fertility, the role of the integumentary tapetum in seed development and molecular mechanisms regulating timely integumentary tapetum degradation are not well understood.

Our previous comprehensive expression profile analysis of ATG genes revealed their relatively high expression level and temporal dynamics during tobacco seed development²⁸, suggesting an important role for autophagy in this process. Here, we provide evidence that autophagy is critical to the timely degradation of the integumentary tapetum and proper embryo pattern formation during seed development. Suppression of autophagy by mutation of the autophagy-specific ATG5 or ATG7, two essential components in the ubiquitin-like conjugation systems necessary for autophagosome formation and maturation²⁹, resulted in delayed PCD of integumentary tapetum and culminated in embryo death. Comparative lipidomic analyzes revealed that autophagy plays a dual role in lipid metabolism during seed development: contributing to lipid degradation before fertilization and supporting lipid biosynthesis after fertilization. Our findings demonstrate that autophagy is an essential mechanism for controlling timely PCD of the integumentary tapetum, which is critical for correct embryonic pattern formation and early seed development.

Results

Autophagy is active in the integumentary tapetum

Autophagy is an evolutionarily conserved intracellular degradation mechanism that is controlled by a set of ATG genes⁴. Our previous genome-wide analysis implicated most ATGs during seed formation based on their temporal expression patterns²⁸, suggesting the potential importance of autophagy in seed development. To explore this possibility, we undertook three different approaches to investigate autophagic activities during seed development. First, we monitored the expression of two autophagy-specific ATGs, ATG5, and ATG7, which are responsible for autophagosome formation, by visualizing the fluorescence of green fluorescent protein (GFP) fused to histone H2B (H2B, to concentrate GFP in nuclei) in proATG5:H2B-GFP and proATG7:H2B-GFP transgenic plants in tobacco. This showed that ATG5 is widely expressed in all layers of the integuments, including the innermost integumentary tapetum (Fig. 1a). We also detected GFP fluorescence in zygotes or early embryos at different developmental stages (Supplementary Fig. 1). ATG7 displayed a similar expression pattern to ATG5, as we observed GFP fluorescence in all layers of integuments including the integumentary tapetum, and early embryos during seed development (Fig. 1b and Supplementary Fig. 1). In addition, both ATG5 and ATG7 proteins are located in the cytoplasm of integumentary tapetum cells (Supplementary Fig. 2a, b). Second, we visualized autophagosomes through ATG8 immunofluorescence^9,30, as well as by inspecting GFP fluorescence from the proTPE8:GFP-ATG8 marker line, in which expression of the GFP-ATG8 cassette is driven from the integumentary tapetum-specific promoter NtTPE8²⁵ (Fig. 1c, d and Supplementary Fig. 3). We detected clear, distinct puncta in the integumentary tapetum by both methods. Transmission electron microscopy (TEM) analysis further confirmed the presence of active autophagy in the integumentary tapetum during seed development. Indeed, we observed double-membrane autophagosomes in the cytoplasm and autophagic bodies in the integumentary tapetum (Fig. 1e). These findings indicate that autophagy is active during seed development, notably in the integumentary tapetum.

Autophagy is essential for seed development

To investigate the role of autophagy in seed development, we generated atg5 and atg7 mutants using the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated nuclease 9 (Cas9)-mediated genome editing system as previously reported⁹. We obtained three independent mutant alleles each of ATG5 and ATG7 for phenotypic analysis (Fig. 2a, b). Western blot analysis revealed that the levels of ATG5 and ATG7 proteins were markedly reduced in their respective mutant lines (Fig. 2c). The autophagic activities of atg5-2 and atg7-1 mutant plants were first assessed by ATG8 immunofluorescence assay (Fig. 2d, e and Supplementary Fig. 4) and with the proTPE8:GFP-ATG8 marker line (Fig. 2f, g). We detected punctate autophagosomes in the integumentary tapetum of wild-type (WT) plants, but hardly the atg5-2 and atg7-1 mutant plants (Fig. 2d–g and Supplementary Fig. 4). We subsequently assessed the extent of ATG8 conjugated with phosphatidylethanolamine (PE), known to be crucial for autophagosome formation, to further determine autophagic activities in atg5-2 and atg7-1 mutants. Consistent with the results from the ATG8 immunofluorescence assay, we observed a notable decrease in the levels of lipidated ATG8 in both atg5-2 and atg7-1 mutants compared to the WT plants (Fig. 2h). These results indicate that mutations in ATG5 or ATG7 impair autophagosome formation and lead to reduced autophagic activities. Moreover, atg5 and atg7 mutants displayed defects in seed development, as we noticed about 30% aborted seeds in all atg5 and atg7 mutant lines, which was significantly higher than in WT plants (Fig. 2i, j). We fully rescued the seed-setting defect of atg5 plants by introducing a proATG5:ATG5-GFP transgene (Fig. 2i, j), demonstrating the essential role of autophagy activity in seed development and the functionality of ATG5-GFP in regulating autophagy in the seed.

**Fig. 2: Mutation in *ATGs* blocks autophagosome formation and leads to seed abortion.**

Autophagy contributes to endothelium and embryo development

Seeds from angiosperms are composed of three major compartments—the seed coat, endosperm, and embryo—whose coordinated development is crucial for seed formation³¹. To investigate the involvement of autophagy in seed development, we carefully analyzed the development of these three compartments. Before fertilization, we observed no obvious differences between WT and atg5 or atg7 ovules. After fertilization, however, seeds from the atg5 and atg7 mutants displayed morphological abnormalities as early as 4 days after pollination (DAP), which prompted us to examine transverse sections of the ovule and seed at different developmental stages (Fig. 3a, b). We observed clear developmental defects in the integumentary tapetum and the embryo of atg5 and atg7 mutants (Fig. 3 and Supplementary Figs. 5 and 6). In WT seeds, the integumentary tapetum expanded from 0 to 4 DAP, and then gradually degraded. In contrast to the WT, the integumentary tapetum of atg5 and atg7 mutant seeds became extremely expanded. The integumentary tapetum of atg5 and atg7 mutant seeds continued to expand after 4 DAP and became extremely thick compared to that in WT seeds at 6 DAP (Fig. 3a, b and Supplementary Fig. 6).

**Fig. 3: Deficiency of autophagy leads to defects in integumentary tapetum degradation and embryo pattern formation.**

To further explore the integumentary tapetum phenotype of atg mutant seeds, we used a modified pseudo-Schiff-propidium iodide (mPS-PI) staining technique (Fig. 3c, Supplementary Fig. 6a, and Supplementary Movies 1 and 2) to visualize integumentary tapetum cells in whole-mount WT and atg ovules and seeds³². The architecture of the integumentary tapetum was indistinguishable between the WT and atg5-2 mutants before fertilization. However, we observed defects in integumentary tapetum degeneration in atg5-2 mutant seeds (Fig. 3c, d). This degradation defect of integumentary tapetum in the atg5-2 mutant was fully rescued by introducing the proATG5:ATG5-GFP transgene into the mutant background (Supplementary Fig. 5). Importantly, a similar phenotype of integumentary tapetum development was also observed in the atg7-1 mutant (Supplementary Fig. 6a–c). We conclude that a defect in autophagy suppresses the degradation of the integumentary tapetum during seed development.

We also detected defects in embryo pattern formation in the atg5 and atg7 mutants. Abnormal seeds were recognizable among atg mutant seeds compared to WT seeds at 6 DAP; we, therefore, subjected 6-DAP seeds to mPS-PI staining. In contrast to the WT, a significantly higher portion of embryos from the atg5-2 (39%) and atg7-1 (40%) mutant seeds exhibited abnormalities in the development of both apical and basal cell lineages (Fig. 3e and Supplementary Fig. 6d). We grouped these embryos from atg5 mutant into four major classes according to their embryonic cell division pattern (Fig. 3e). Compared to WT embryos, 18% of the embryos showed an abnormal cell division pattern in the apical cell lineage, while 13% of the embryos lacked a typical suspensor in atg5-2 mutant. Similar embryo developmental defects were also observed in the atg7-1 mutant, with 19% of the embryos showing abnormal development of apical cell lineage and 12% of the embryos lacking a typical suspensor (Supplementary Fig. 6d). Taken together, these results indicate that autophagy is also critical for the establishment of apical–basal embryo polarity.

Autophagy is crucial for timely integument tapetal PCD onset

Seed development involves a tightly controlled and stage-dependent PCD of the seed coat, endosperm, and suspensor, resulting in their degradation^33,34,35. To investigate the role of autophagy in the PCD of integumentary tapetal cells, we utilized terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL)³⁶ and TEM analyzes. We detected no TUNEL signals in the WT integumentary tapetum at 0 or 2 DAP (Fig. 4a, b). However, TUNEL signals in the integumentary tapetum (52.2% seeds; 47 out of 90 seeds) began to appear at 4 DAP, and nearly all integumentary tapetal cells (94.4% seeds; 85 out of 90 seeds) showed signals at 5 DAP in the WT (Fig. 4a, b), indicating that PCD in the integumentary tapetum is precisely regulated. Compared to the WT, PCD in the integumentary tapetum of the atg5-2 mutant was delayed: only 21.1% seeds (19 out of 90 seeds) of atg5-2 mutant showed initiation of DNA fragmentation in the integumentary tapetum as early as 4 DAP, and we detected significantly fewer TUNEL signals (63.3%; 57 out of 90) in the integumentary tapetum at 5 DAP (Fig. 4a, b). Similar delayed PCD was also detected in the integumentary tapetum of atg7-1 mutants (Fig. 4a, b). Furthermore, TEM analysis revealed that the nuclei of WT integumentary tapetal cells became lobed and their nuclear envelope appeared ruptured at 4 DAP before being progressively disassembled (Fig. 4c); this was accompanied by the typical cytoplasmic vacuolization seen during vacuolar PCD in other plant cells³⁷. By contrast, the nuclear membrane of expanded integumentary tapetal cells remained intact in the atg5-2 mutant even at 6 DAP (Fig. 4c). Vacuolar processing enzyme (VPE), which possesses caspase 1-like activity, is a critical protease in the execution of plant PCD^38,39,40. To determine the relationship between caspase 1-like protease VPE and autophagy in the PCD of the integumentary tapetum, we compared the proteolytic activities of VPE during WT and atg5-2 seed development. Consistent with the delayed PCD in autophagy-deficient mutants, we observed a significant decrease in VPE proteolytic activities in the 4-DAP seeds and subsequent stages in the atg5-2 mutant, but not in the 2-DAP seed prior to PCD, as compared with the WT (Fig. 4d). This suggests that autophagy seems to lie upstream of caspase 1-like protease VPE in the signaling pathway of integumentary tapetum PCD. Overall, our results indicate that autophagy plays a crucial role in the timely initiation of PCD in the integumentary tapetum.

**Fig. 4: Deficiency of autophagy results in the delayed programmed cell death of integumentary tapetum.**

Integumentary tapetum PCD promotes embryo development

Timely PCD in the anther tapetum, the innermost layer of the anther wall that surrounds and supplies nutrients to microspores, is critical for pollen development^23,24, whereas the role of integumentary tapetum in early embryo development has not been well understood. As described above, we observed developmental defects in both integumentary tapetum and embryo in the atg5 mutants, providing us with a unique opportunity to investigate cell communication between integumentary tapetum and embryo. To explore the role of the integumentary tapetum, particularly its timely degradation, in embryo development, we drove ATG5 expression with the integumentary tapetum-specific promoter NtTPE8²⁵ in the atg5-2 mutant background. We detected the specific expression of ATG5-GFP in the integumentary tapetum, as evidenced by GFP fluorescence (Fig. 5a). We examined whether the specific expression of ATG5 in the integumentary tapetum of the atg5-2 mutant could functionally rescue the defects in timely integumentary tapetum PCD and early embryo development. Indeed, expression of ATG5-GFP in atg5-2 integumentary tapetum partially rescued the defects in integumentary tapetum degradation seen in atg5 mutant seeds (Fig. 5b, c), further confirming that autophagy is essential for the PCD of integumentary tapetum. Notably, the frequency of abnormal embryos with defects in cell division pattern also decreased significantly, from approximately 40% to 21%, in the atg5-2 proTPE8:ATG5-GFP complementation lines (Fig. 5d, e). Moreover, specific expression of ATG5-GFP in the atg5-2 integumentary tapetum promoted embryo development and seed formation, as the seed-setting rate rose from 70.1% in the atg5-2 mutant to 82.5% in atg5-2 proTPE8:ATG5-GFP complementation lines (Fig. 5f, g). These results indicate that autophagy-mediated timely PCD of the integumentary tapetum contributes to the correct formation of the embryo pattern in a non-cell-autonomous manner, and suggest a possible cell-to-cell communication between the integumentary tapetum and the embryo.

**Fig. 5: Specific expression of *ATG5-GFP* in the integumentary tapetum of *atg5-2* mutant promotes embryo pattern formation.**

To investigate the molecular connections between autophagy in the integumentary tapetum and embryo pattern formation, we isolated embryos from WT plants, atg5-2 mutant, and atg5-2 mutant expressing proTPE8:ATG5-GFP for RNA sequencing. Overall, the transcripts from the same cell type, but across different biological replicates, exhibited high correlation (r > 0.98). Principal component analysis (PCA) and complete-linkage clustering showed that these nine transcriptomes were classified into three groups (Fig. 6a, b): WT, atg5-2, and atg5-2 proTPE8:ATG5-GFP. The intermediate positioning of the atg5-2 proTPE8:ATG5-GFP group between WT and atg5-2 suggests that specific expression of ATG5 in the integumentary tapetum of atg5-2 could partially rescue the gene expression profile of atg5-2 embryos. Consistent with the PCA results, a comparison of differentially expressed genes (DEGs) between WT/atg5-2 and atg5-2 proTPE8:ATG5-GFP/atg5-2 revealed that the specific expression of ATG5 in the integumentary tapetum of atg5-2 mutant leads to a significant decrease in the number of DEGs in the atg5-2 embryos (Fig. 6c–e and Supplementary Data 1). This reduction suggests that specific expression of ATG5 in the integumentary tapetum can partially restore the aberrant embryonic gene expression profile observed in the atg5-2 mutant. We then focused our analysis on the set of genes in embryos that are regulated by autophagy in the integumentary tapetum (Fig. 6f). Gene ontology (GO) analysis identified several well-known pathways associated with embryo pattern formation, including pattern specification and auxin-related pathways, which are regulated by autophagy in the integumentary tapetum (Fig. 6f). This finding further confirms the non-cell-autonomous role of autophagy in the integumentary tapetum during embryo development.

**Fig. 6: Comparative transcriptomic analysis of 8-cell embryos from WT, *atg5-2* mutant, and *atg5-2* mutant complemented with integumentary tapetum-specific *ATG5* expression.**

Autophagy plays dual roles in seed lipid homeostasis

Previous reports have revealed a close relationship between autophagy and lipid metabolism in animals and plants^41,42. In particular, lipids can serve as regulatory molecules and degradation substrates for autophagy^43,44, as well as play significant roles in seed development^45,46. To investigate the potential role of autophagy in lipid metabolism during early seed development, we performed a comparative lipidomic analysis of ovules and seeds at 4 DAP from the WT and atg5-2 mutant using ultra-high-performance liquid chromatography followed by tandem mass spectrometry (UPLC–MS/MS). Five biological replicates were performed for both the WT and atg5-2 ovules, and 4-DAP seeds. Thus, a total of 20 independent samples were included for comparative lipidomic analysis. As a result, we detected 703–727 individual lipids in the lipidomic data of different samples, which could be grouped into 30 classes including triacylglycerol (TG), phosphatidylcholine (PC), phosphatidylinositol (PI), and PE (Supplementary Data 2). TG was the most abundant lipid in the ovules and seeds at 4 DAP, followed by PE, PC, and PI. PCA (Fig. 7a) uncovered a significant separation between ovules and seeds along the first principle component, explaining 50.1% of the standing variation, with the atg5-2 and WT samples being separated along PC2 (explaining 17.9% of the variation), revealing the dynamics of lipid metabolism as a function of seed age and the effect of autophagy on lipid homeostasis during early seed development.

**Fig. 7: Impact of autophagy deficiency on lipid metabolism during seed development.**

We focused on the differences in lipid composition between WT ovules and 4-DAP seeds to investigate lipid metabolism during early seed development (Fig. 7b and Supplementary Figs. 7 and 8). We detected a significant accumulation of numerous TG species (77 out of 225; Supplementary Data 2), the main components of stored lipids in plants, in seeds after fertilization (Fig. 7b and Supplementary Fig. 7a, b). We also observed the accumulation of 29 of 76 diacylglycerol (DG) species, the intermediates in TG biosynthesis, in the seed compared to unfertilized ovules (Fig. 7b and Supplementary Fig. 7a, b), consistent with the essential role of TG biosynthesis in seed development⁴⁵. In addition, the two phospholipids PE and PI, which are involved in the formation of autophagosomes⁴³, accumulated in the seeds compared to the ovules (Fig. 7b and Supplementary Fig. 7a, b). However, when we compared the lipid compositions of atg5-2 ovules and 4-DAP seeds (Supplementary Fig. 7c, d), we noted that a significant fraction of lipid species (163 out of 320; Supplementary Data 2) are differentially regulated compared to those in WT ovules and seeds (Supplementary Fig. 7e). Specifically, 29 out of 77 TG species were specifically more abundant following fertilization in WT plants, but not in the atg5-2 mutant, confirming that autophagy has considerable influence on lipid metabolism during early seed development.

To explore this effect in more detail, we compared the lipid compositions of ovules and seeds from the WT and the atg5-2 mutant (Fig. 7b and Supplementary Fig. 8). We detected hundreds of metabolites whose abundances differed between WT and atg5-2 ovules (Fig. 7c) or between WT and atg5-2 4-DAP seeds (Fig. 7d), representing the consequences of autophagy on lipid metabolism during early seed development. We identified only 11 lipid species commonly upregulated by autophagy at different developmental stages (Fig. 7e), suggesting that autophagy plays a distinct developmental-stage-dependent role in lipid metabolism. The levels of TGs were significantly higher in atg5-2 ovules than in the WT, whereas we observed decreased TG levels with a concomitant increase in free fatty acids in atg5-2 seeds at 4 DAP relative to WT seeds (Fig. 7b, f, and g), suggesting a dual role for autophagy in lipid metabolism during early seed development.

As mentioned above, autophagy-mediated degradation of the integumentary tapetum is critical for seed development. We thus focused on the role of autophagy in integumentary tapetum lipid metabolism. Consistent with the lipidomic analysis, BODIPY 493/503 staining for neutral lipids in ovules and 4-DAP seeds demonstrated that autophagy contributes to TG metabolism in the integumentary tapetum. Indeed, we observed higher TG contents in both atg5-2 and atg7-1 ovules before fertilization, but lower TG levels in the seeds after fertilization (Fig. 8a–e), suggesting that autophagy has a distinct role in the lipid metabolism of the integumentary tapetum during early seed development.

**Fig. 8: Altered triacylglycerol (TG) levels in the integumentary tapetum of autophagy-deficient mutant.**

Discussion

Autophagy is an important pathway for the degradation of intracellular components and has been implicated in PCD^47,48,49. PCD in plants can be further divided into environment-induced PCD (ePCD) and developmental PCD (dPCD)⁵⁰. Whereas the relationship between autophagy and ePCD is relatively well understood, the role of autophagy in dPCD is still largely unknown. One recent study reported that this role is cell type-specific: autophagy is necessary for timely dPCD in proximal root cap cells, but dispensable for dPCD execution of distal lateral root cap cells, suggesting a cell death promotion role for autophagy in dPCD in proximal root cap cells¹⁴. It has also been suggested that autophagy is involved in dPCD of suspensor cells in somatic embryos⁵¹, as well as in the tracheary elements of the xylem⁵² and the anther tapetum⁸, implying a common role for autophagy in promoting dPCD in different cell types.

Timely PCD of the anther tapetum, the counterpart of the integumentary tapetum, is essential for microsporogenesis and male gamete fertility. Tapetum PCD defects result in the formation of abnormal pollen grains, hence, male sterility^24,53. In rice (Oryza sativa), for example, a mutation in TAPETUM DEGENERATION RETARDATION (TDR), encoding a basic helix–loop–helix transcription factor, delays tapetum PCD, leading to aborted pollen development and male sterility²⁴. Autophagy also contributes to PCD in the rice anther tapetum. Indeed, mutation in ATG7 leads to tapetum degradation defects, causing complete sporophytic male sterility, indicating that autophagy-mediated anther tapetum PCD is crucial in male gametophyte development⁸. Here, we demonstrated that the integumentary tapetum also undergoes degeneration during seed development in tobacco, displaying typical characteristics of plant dPCD. Our present results are consistent with the involvement of autophagy in dPCD, demonstrating significant autophagic activities in the integumentary tapetum that promote dPCD during seed development. Blocking autophagy by mutation of autophagy-specific genes, such as ATG5 or ATG7, resulted in delayed PCD in the integumentary tapetum, indicating that autophagy plays a critical role in promoting the initiation of dPCD in the integumentary tapetum.

Seed formation is a complex program that entails the growth of three genetically distinct entities: the seed coat, the embryo, and the endosperm³¹. The integumentary tapetum is a layer of cells specified from the innermost integuments that surround each developing embryo sac in flowering plants. Integumentary tapetal cells exhibit cytological characteristics similar to those of secretory cells and share similar features with the anther tapetum¹⁹. The anther tapetum acts as a safeguard for the developing pollen grains by providing them with essential nutrients, enzymes, and precursors for pollen development^23,54. However, the role of the integumentary tapetum in seed development has long been overlooked. It has been speculated that this tissue serves multiple roles in seed development, such as providing the necessary nutrients and supporting embryo and endosperm development, based on its strategic location and cytological characteristics. Consistent with this notion, a recent study revealed the significance of symplastic signals through plasmodesmata of integuments for the pre-fertilization development of the embryo sac⁵⁵. In addition, another recent research demonstrated that micropylar integumentary-tapetum-generated gibberellin serves as a maternal signal regulating PCD initiation in the embryonic suspensor through the NtGID1-NtCRF1-NtCYS-NtCP14 signaling cascade⁵⁶. Gibberellin transiently increases in abundance in the micropylar integumentary tapetum after fertilization and is transported to the basal suspensor cell, where it regulates the module responsible for suspensor PCD. Studies on the cysteine protease gene NtTPE8 specifically expressed in integumentary tapetum cells and the serine proteinase inhibitor II gene SaPIN2 from American black nightshade (Solanum americanum) also support a critical role for integumentary tapetum in early embryonic development^25,26. Silencing of NtTPE8 resulted in embryo abortion despite the exclusive expression of this cysteine protease gene in the integumentary tapetum, suggesting a non-cell-autonomous role for the integumentary tapetum in regulating embryo development²⁵. In the present study, we discovered that timely PCD in the integumentary tapetum is crucial for correct embryo pattern formation, similar to the role of anther tapetum PCD in microsporogenesis. Specific expression of ATG5 in the integumentary tapetum partially rescued the defects in embryo pattern formation, suggesting that cell-to-cell communication between the seed coat and the embryo is crucial for proper embryo development. Elucidating the connections between integumentary tapetum and embryo in further studies will facilitate our understanding of the mechanisms of early seed development.

Research in animal systems has revealed an intricate interplay between autophagy and lipids^41,44. TGs stored in lipid droplets can be degraded through a form of selective autophagy called lipophagy. Suppressing autophagy significantly increases TG storage in lipid droplets⁴¹. In addition to lipid degradation, autophagy appears to also be implicated in TG biogenesis, although the exact mechanisms are still largely unknown⁵⁷. Compared to the amount of research conducted on animals, the amount of research on the connection between autophagy and lipids in plants lags behind^58,59,60. Similar to animals, recent evidence also suggests a dual role of autophagy in lipid metabolism, involving participation in triglyceride degradation and biogenesis, which is exhibited not only under different growth conditions but also at different developmental stages^61,62. In Arabidopsis, blocking basal autophagy activities by mutating ATG2 or ATG5 led to a significant decrease in TG content in mature seeds and 4-day-old seedlings, whereas lipid droplets were degraded through starvation-induced lipophagy, suggesting different roles for autophagy in lipid metabolism between basal autophagy under normal development and stress-induced autophagy under stress conditions⁶¹. Similarly, in Chlamydomonas, a recent study suggests distinct roles of autophagy in lipid metabolism at different starvation stages, with its potential contribution to lipid droplet formation during the early stages and likely involvement in lipid droplet degradation during the later stages⁶². Our present study revealed that the role of autophagy in lipid metabolism during integumentary tapetum development is dependent on the developmental stage in tobacco. Autophagy-deficient mutants experienced a significant increase in the TG contents of their ovules before fertilization, whereas we observed a marked decrease in TG contents in atg5 mutant seeds after fertilization. This finding uncovers a previously underappreciated developmental-stage-dependent role for autophagy in lipid metabolism. Additionally, the role of autophagy in lipid metabolism is also likely cell type and development dependent. This idea is supported by the lower TG contents observed in the mature anthers of the Osatg7 mutant⁸, whereas the abundance of TGs increases significantly in autophagy-deficient pollen grains⁹. Taken together, the role of autophagy in plant lipid metabolism is complex, as it plays a cell-type- and developmental-stage-dependent role under normal growth conditions. However, the regulatory mechanisms regarding the distinct roles of autophagy in lipid biosynthesis or degradation in different cell types are worthy to be elucidated in further studies.

Methods

Plant materials

N. tabacum L. cv. Petite Havana SR1 plants were grown under 12 h light/12 h dark at 25 °C in a glasshouse.

Vector construction and plant transformation

To generate proATG5:H2B-GFP and proATG7:H2B-GFP constructs, the promoter sequences of ATG5 and ATG7 were amplified from tobacco genomic DNA and inserted into the pART27 vector upstream of the H2B-GFP cassette. CRISPR/Cas9-based genome editing system was introduced to generate atg5 and atg7 mutants. The gRNAs for ATG5 and ATG7 were designed and cloned into the pYLCRISPR/Cas9P35S-N according to the previous protocol⁶³. Briefly, suitable target sites were selected, and then PCR primers were designed according to the target sites. Next, the target sequences were introduced into corresponding sgRNA expression cassettes through PCR. Finally, the sgRNA expression cassettes were inserted into the pYLCRISPR/Cas9P35S-N vector. To generate proATG5::ATG5-GFP and proTPE8:ATG5-GFP constructs, the coding sequences of ATG5 amplified from the cDNA prepared from the seeds and cloned into the pART27 vector upstream of GFP to generate ATG5-GFP fusion constructs. The promoters of ATG5 and TPE8 were inserted upstream of ATG5-GFP to generate proATG5:ATG5-GFP and proTPE8:ATG5-GFP, respectively. To generate proTPE8:GFP-ATG8, the ATG8 coding sequence was amplified and cloned into the pART27 vector downstream of GFP, and then the promoter of TPE8 was inserted upstream of GFP-ATG8. Primers for vector construction are listed in the Supplementary Table 1. Agrobacterium tumefaciens-mediated transformation of tobacco was performed to generate transgenic plants.

Immunofluorescence

Ovules and seeds from WT and atg mutants were collected to prepare sections for subsequent immunofluorescence assay according to the previous protocol with minor modifications⁶⁴. Briefly, the ovules and seeds were fixed in 4% paraformaldehyde for 12 h and then washed three times with PBS. After the fixation, the samples were dehydrated using an alcohol gradient from 10% to 100% and replaced with a gradient of xylene from 25% to 100%. The samples were then embedded under a series of paraffin xylene mixtures. Sections at 10 µm were prepared using a rotary microtome (Leica RM2245, Germany). After de-paraffinization, rehydration, and antigen retrieval, the sections were then used for immunofluorescence. The slides were incubated in 5% BSA solution at 4 °C for 12 h. The samples were then washed and incubated with the anti-ATG8A antibody (Abcam, ab77003; 1:100 dilution) dilution or anti-ATG7 antibody (produced by Abclonal, E3633; 1:100 dilution) for 1 h at room temperature. After washing with PBS three times, the samples were stained with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:200 dilution; Abcam, ab150077) for 2 h at room temperature. For double immunofluorescence, ovules from proTPE8:GFP-ATG8 plants were collected, and paraffin sections were prepared as described above. The slides were then incubated with both the anti-ATG8A antibody (Abcam, ab77003; 1:100 dilution) and the anti-GFP antibody (DIA-AN, 2057; 1:100 dilution) for 1 h at room temperature. After washing with PBS three times, the samples were stained with an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific, A-11012; 1:200 dilution) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, A-11001; 1:200 dilution) for 2 h at room temperature. The samples were observed under a confocal laser scanning microscope (CLSM) (Leica TCS SP8, Germany) with the excitation at 488 nm/emission at 500 ± 20 nm for Alexa Fluor 488 and excitation at 561 nm/emission at 600 ± 20 nm for Alexa Fluor 594.

TUNEL assay

Sections of ovules and seeds were prepared following the protocol described above for immunofluorescence assay. DNA fragmentation was detected using the DeadEnd™ Fluorometric TUNEL System (Promega, G3250) according to the manufacturer’s instructions. In brief, the paraffin sections were dewaxed with xylene, rehydrated with ascending grades of alcohols, fixed in 4% paraformaldehyde for 15 min, and washed with PBS. Subsequently, the samples were digested with 20 μg/mL Proteinase K for 10 min, fixed again in 4% paraformaldehyde for 5 min, washed with PBS, treated with Equilibration buffer for 10 min, and incubated in 100 μL rTdT incubation buffer at 37 °C for 60 min. Finally, the reaction was terminated in 2× SSC solution for 15 min, and the samples were washed by fresh PBS. The samples were then observed under a confocal microscope (Leica TCS SP8, Germany) under excitation with a 488 nm laser, and emission measured at 520 ± 20 nm.

BODIPY staining

The ovules and seeds were collected and embedded in the low-melting-point agarose (Promega, V2111). The fresh sections of ovules and seeds were prepared with the fully automated Leica VT1200 S vibrating blade microtome (Leica, Germany) for BODIPY staining. Fresh sections were then stained with the BODIPY 493/503 (Thermo Fisher Scientific, D3922) and SCRI Renaissance 2200 (SR2200) for 10 min. After washing with PBS three times, the sections of ovules and seeds were observed under a CLSM (Leica TCS SP8, Germany) with the excitation at 405 nm/emission at 435 ± 20 nm for SR2200 and excitation at 488 nm/emission at 515 ± 15 nm for BODIPY.

CLSM observation and image analysis

Samples observation and image acquisition were performed by a CLSM (Leica TCS SP8, Germany). To observe GFP fluorescence in the ovules and seeds of proATG5:H2B-GFP, proATG7:H2B-GFP, proTPE8:GFP-ATG8, and proTPE8:ATG5-GFP transgenic plants, the fresh sections were prepared using the fully automated Leica VT1200 S vibrating blade microtome (Leica, Germany) for CLSM observation. Samples were excited at 552 nm laser with emission measured at 620 ± 25 nm for PI observation, and excited at 488 nm laser with emission measured at 520 ± 20 nm for GFP observation. The images used for quantitative comparisons were acquired under identical parameter settings. Image processing was performed using the Leica Application Suite X (LAS X 3.5.5) and ImageJ (1.53k).

TEM analysis

The procedure for TEM assay was performed according to our previous protocol with minor modifications⁹. Samples were embedded through high-pressure freezing and freeze substitution. For high-pressure freezing, isolated ovules and seeds were collected into the cryoprotectant (Promega, V2111), transferred to the carrier, and then fixed by a high-pressure freezing system (Leica EM HPM100). Freeze substitution was then carried out using a freeze substitution processor (Leica EM AFS2) with acetone containing 2% osmic acid for a 5-day workflow as follows: −90 °C for 72 h, 3.75 °C per hour raise to −60 °C and lasts 12 h, 3.75 °C per hour raise to −30 °C and lasts 12 h, 3.75 °C per hour raise to 0 °C and lasts 2 h. After extensive washing with acetone, the samples were then embedded under a series of Spurr’s resin acetone gradient mixtures (30–100%). The embedding block was trimmed by a milling system (Leica EM TRIM2), and samples were sectioned into 70 nm ultra-thin slices with an ultramicrotome (Leica EM UC7). After staining with 2% uranyl acetate, the samples were observed with a TEM (JEM-1400, JEOL) equipped with the supporting camera system DS-L1 (Gatan).

Lipidome analysis

Ovules and 4-DAP seeds from both WT and atg5 plants were collected for lipid extraction and subsequent lipidome profiling. The extraction and lipidome profiling was carried out using a previously published protocol⁶⁴. All samples were ground in liquid nitrogen and freeze-dried using SCIENTZ-100F. Then, the lipid was extracted by a solvent system of methanol and methyl tert-butyl ether (MTBE). In detail, 1 mL of lipid extract (MTBE:MeOH = 3:1) was added to 20 mg of freeze-dried sample and vortexed for 30 min. Subsequently, 300 µL of ultra-pure water was added and vortexed for 1 min. The supernatant was obtained by centrifugation and concentrated to dryness at 20 °C. Finally, 200 µL of a lipid solution (CAN:IPA = 1:1) was added to redissolve the dried extract. During the experiment, we used a mixture of test samples as the quality control sample. We analyzed a total of four quality control samples during the experiment.

The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC, ExionLC^TM AD, SCIEX) and Tandem Mass Spectrometry (MS/MS, QTRAP® 6500+, SCIEX). Liquid phase conditions are listed as follows: column, Thermo Accucore™C30; mobile phase A, acetonitrile/water, 60/40 (V/V), 0.1% formic acid, 10 mmol/L ammonium formate; mobile phase B, acetonitrile/isopropanol, 10/90 (V/V), 0.1% formic acid, 10 mmol/L ammonium formate; gradient elution program, A/B (V/V), 80/20 at 0 min, 70/30 at 2 min, 40/60 at 4 min, 15/85 at 9 min, 10/90 at 14 min, 5/95 at 15.5 min, 5/95 at 17.3 min, 80/20 at 17.5 min, and 80/20 at 20 min; flow velocity, 0.35 mL/min; column temperature, 45 °C; sample volume, 2 µL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. LIT and triple quadrupole scans were obtained on a triple quadrupole-linear ion trap mass spectrometer (QTRAP® 6500+ LC–MS/MS System), equipped with an ESI Turbo Ion–Spray interface, operating in 500 °C source temperature, 5500 V positive ion spray voltage, −4500 V negative ion spray voltage, 45 psi ion source gas 1, 55 psi gas 2, 35 psi curtain gas and controlled by Analyst 1.6.3 software. Quantification was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with the MS1 mass resolution 3000–5000. Differential metabolites are determined by both VIP ≥ 1 and absolute Log₂FC (fold change) ≥ 1. VIP values were extracted from OPLS-DA results, which were generated using the R package MetaboAnalystR.

Embryo isolation

Living embryo isolation was performed according to our previously established protocol^34,65. Briefly, seeds at 6 DAP were treated with an enzyme solution comprising 1% Cellulase R-10 and 0.8% Macerozyme R-10 (Yakult Honsha Co. Ltd, Tokyo, Japan) dissolved in 11% mannitol for 30 min. Embryo sacs containing 8-cell embryos were first released by gentle grinding under an inverted microscope. Subsequently, 8-cell embryos were mechanically isolated from the embryo sacs after a second enzymatic treatment for 10 min with 0.25% Cellulase R-10 and 0.2% Macerozyme R-10 dissolved in 11% mannitol. Following extensive washing with 11% mannitol three times, the isolated 8-cell embryos were collected for subsequent mRNA extraction and library construction.

mRNA extraction and RNA-seq library construction

mRNA extraction and cDNA preparation were performed according to our previous protocol⁶⁶. mRNA extraction was carried out using the Dynabeads mRNA DIRECT Micro Kit (Life Technologies, USA), and cDNA preparation was performed using SMART-seq v4 Ultra Low Input RNA Kit (Takara Bio USA). cDNA was purified using AMPure XP beads (Beckman Coulter, A63880). Purified cDNA was then quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA-seq libraries were prepared using the VAHTS Universal Plus DNA Library Prep Kit for MGI (Vazyme Biotech) and then sequenced on an MGI DNBSEQ-T7 with a 2 × 150 bp paired-end model.

RNA-seq data analysis

The raw reads were filtered using fastp with the option “--detect_adapter_for_pe” to eliminate low-quality reads and adapters⁶⁷. Subsequently, the clean reads were aligned to the N. tabacum reference genome using STAR (v.2.7.11a) with the parameter “--outSAMtype BAM SortedByCoordinate”⁶⁸. The reads count and fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using RSEM (v.1.3.3) with “--paired-end --no-bam-output”⁶⁹. Differential gene expression analysis was conducted using the DESeq2 package⁷⁰ according to the following criteria: fold change ≥ 2; Benjamini–Hochberg adjusted P value < 0.05. K-means cluster analysis of DEGs was performed using the ClusterGVis package (https://github.com/junjunlab/ClusterGVis). GO enrichment analysis were performed on the PANTHER website (https://pantherdb.org/) with “Fisher’s exact test” and “Calculate False Discovery Rata”. The plots were generated using the R package ggplot2 (v.3.4.3).

Immunoblot assay

To characterize ATG protein levels in WT and atg mutants, total proteins were extracted using the buffer composed of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetamide, and proteinase inhibitor cocktail (Roche, 4693132001). The extracts were then subjected to SDS-PAGE and subsequently transferred to nitrocellulose membrane (GVS North America, 1212590) for immunoblotting with rabbit anti-ATG5 antibody (produced by Abclonal, E3631; 1:2000 dilution) or rabbit anti-ATG7 antibody (produced by Abclonal, E3633; 1:2000 dilution)⁹. ATG8–PE assay was performed according to the previous protocol⁹. Briefly, the extracts were subjected to SDS-PAGE on 12% polyacrylamide gels in the presence of 6 M urea (Sigma-Aldrich, U6504). The separated proteins were then transferred to nitrocellulose membranes (GVS North America, 1212590), and probed with anti-ATG8A antibody (Abcam, ab77003; 1:1000 dilution). Antibody against β-actin (Abbkine, A01050; 1:2000 dilution) was used as the loading control in western blot analysis. HRP Goat Anti-Mouse IgG (Abclonal, AS003; 1:4000 dilution) or HRP Goat Anti-Rabbit IgG (Abclonal, AS014; 1:4000 dilution) were used as secondary antibodies in the immunoblot assay.

VPE activity assay

VPE proteolytic activity assay was performed according to the previous protocols with minor modifications³⁸. Briefly, total proteins were extracted from seeds at various stages using an extraction buffer composed of 50 mM MES (pH 6.0), 2 mM EDTA, 10% glycerol, 0.1% CHAPS, 0.01% Brij-35, 2% polyvinylpolypyrrolidone, 10 mM L-cysteine, and 10 mM sodium metabisulphite. Proteolytic activities were subsequently measured in a reaction buffer consisting of 50 mM sodium acetate (pH 5.5), 10 μg of total protein, 50 μΜ substrate Z-AAN-AMC (Bachem, 4033201), 10 mM L-cysteine, 1 mM EDTA, and 0.01% Brij-35. The fluorescence levels of the released AMC were monitored using a SPECTRAmax iD5 microplate reader (Molecular Devices, USA) with an excitation wavelength of 360 nm and an emission wavelength of 455 nm.

Plot preparation and statistical analysis

Bar–dot and box–whisker plots were prepared using GraphPad Prism 9. Two-sided Student’s t test and Tukey–Kramer multiple comparison test were performed for statistical analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Lipidomics data are provided with this paper. RNA-seq data have been uploaded to the NCBI Gene Expression Omnibus (GEO) under accession GSE248624. All seeds and other materials related to the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (31991201, 31970340), the Major Project of Hubei Hongshan Laboratory (2022hszd017), and the Science and Technology Department of Hubei Province (2022CFA071). We thank Prof. Y.G. Liu (South China Agricultural University) for the kind offer of the CRISPR/Cas9 vectors.

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Authors and Affiliations

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
Lin-lin Zhao, Ru Chen, Ziyu Bai, Junyi Liu, Yuhao Zhang, Yicheng Zhong, Meng-xiang Sun & Peng Zhao
Hubei Hongshan Laboratory, Wuhan, China
Peng Zhao

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Lin-lin Zhao
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Ru Chen
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Ziyu Bai
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Junyi Liu
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Yuhao Zhang
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Yicheng Zhong
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Contributions

P.Z. and M.-x.S. designed the experiments. L.-l.Z., R.C., Z.B., J.L., and Y.Z. performed the experiments. L.-l.Z., Y.Z., P.Z., and M.-x.S. contributed to the data analysis and wrote the manuscript.

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Correspondence to Peng Zhao.

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Zhao, Ll., Chen, R., Bai, Z. et al. Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation. Nat Commun 15, 2676 (2024). https://doi.org/10.1038/s41467-024-46902-8

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Received: 28 May 2023
Accepted: 13 March 2024
Published: 27 March 2024
DOI: https://doi.org/10.1038/s41467-024-46902-8

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