Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco (Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos. Similar to animals, plants eliminate tissues and organs that have transient function via programmed cell death (PCD). This is especially apparent during plant embryogenesis, when a plant zygote divides into an apical cell, which develops into a mature embryo, and a basal cell, which generates a single organ called the suspensor. During early seed development, the suspensor connects the embryo to the surrounding seed tissues and transports the nutrients and hormones required for its early development. Once its functions are fulfilled, the suspensor is subsequently eliminated by PCD. In this study, we answer a long-standing question in the field by elucidating the mechanism that is responsible for initiating suspensor PCD at a specific time during embryogenesis. Our findings show that in tobacco plant embryos, suspensor PCD is controlled by two antagonistically acting proteins—the pro-death cathepsin protease NtCP14 and its inhibitor cystatin NtCYS, which co-localize to the basal-most cell of the suspensor. High expression levels of NtCYS during early embryogenesis confer suspensor growth and viability by suppressing NtCP14 activity. When the suspensor ceases to grow, NtCYS expression is downregulated, leading to increased NtCP14 activity and to the initiation of PCD. The genetic modulation of this NtCYS/NtCP14 expression ratio either delays or hastens suspensor PCD, demonstrating its indispensable role in plant embryo development.

A comparison of eudicot and monocot model plants explores recent advances and open questions on gene regulatory networks during zygote development, parental inf

In plants, macroautophagy/autophagy has mainly been associated with stress-related processes but how it impacts normal physiological and developmental processes remains largely unexplored. Pollen germination is the critical first step towards fertilization in flowering plants. It is metabolically demanding and relies on high levels of cytoplasmic reorganization activities to support a dramatic morphological transformation that underlies the development of a pollen tube as the conduit to deliver sperm for fertilization. The role of autophagy in this process remains unclear. Here we provide evidence that pollen germination is accompanied by elevated autophagic activity and successful pollen tube emergence depends on autophagy-mediated cytoplasmic deletion. Genetic and cytological experiments demonstrate that inhibition of autophagy prevents pollen germination while induces the persistence of a layer of undegraded cytoplasm at the germination aperture. Together, these results unveil a novel compartmentalized autophagy. Furthermore, high-throughput comparative lipidomic analyses show that suppressed autophagy-induced inhibition of pollen germination is accompanied by altered profiles of stored and signaling lipids. Proteomic analyses reveal that autophagy likely exert its role in pollen germination via downstream mitochondria-related pathways. These findings reveal a critical role for autophagy in initiating pollen germination and provide evidences for compartmental cytoplasmic deletion being crucial for male fertility.

Sexual reproduction involves the fusion of two gametes of opposite sex. Although the sperm-expressed fusogen HAPLESS 2 (HAP2) or GENERATIVE CELL SPECIFIC 1 (GCS1) plays a vital role in this process in many eukaryotic organisms and an understanding of its regulation is emerging in unicellular systems [J. Zhang et al. , Nat. Commun. 12, 4380 (2021); J. F. Pinello et al. Dev. Cell 56, 3380–3392.e9 (2021)], neither HAP2/GCS1 interactors nor mechanisms for delivery and activation at the fusion site are known in multicellular plants. Here, we show that Arabidopsis thaliana HAP2/GCS1 interacts with two sperm DUF679 membrane proteins (DMP8 and DMP9), which are required for the EGG CELL 1 (EC1)-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization. Our studies in Arabidopsis and tobacco provide evidence for a conserved function of DMP8/9-like proteins as HAP2/GCS1 partner in seed plants. Our data suggest that seed plants evolved a DMP8/9-dependent fusogen translocation process to achieve timely acquisition of sperm fusion competence in response to egg cell–derived signals, revealing a previously unknown critical step for successful fertilization.