BackgroundPapain-like and legumain-like proteases are proteolytic enzymes which play key roles in plant development, senescence and defense. The activities of proteases in both families could be inhibited by a group of small proteins called cystatin. Cystatin family genes have been well characterized both in tobacco and rice, suggesting their potential roles in seed development. However, their potential targets, papain-like and legumain-like proteases, have not been well characterized in plants, especially in rice, a model plant for cereal biology.ResultsHere, 33 papain-like and 5 legumain-like proteases have been identified in rice genome, respectively. Gene structure, distribution in rice chromosome, and evolutionary relationship to their counterparts in other plants have been well characterized. Comprehensive expression profile analysis revealed that two family genes display divergent expression pattern, which are regulated temporally and spatially during the process of seed development and germination. Our experiments also revealed that the expression of most genes in these two families is sensitively responsive to plant hormones and different abiotic stresses.ConclusionsGenome-wide identification and comprehensive gene expression pattern analysis of papain-like and legumain-like proteases in rice suggests their multiple and cooperative roles in seed development and response to environmental variations, which provides several useful cues for further in-depth study.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1298-1) contains supplementary material, which is available to authorized users.

The seeds of flowering plants contain three genetically distinct structures: the embryo, endosperm, and seed coat. The embryo and endosperm need to interact and exchange signals to ensure coordinated growth. Accumulating evidence has confirmed that embryo growth is supported by the nourishing endosperm and regulated by signals originating from the endosperm. Available data also support that endosperm development requires communication with the embryo. Here, using single-fertilization mutants, Arabidopsis thaliana dmp8 dmp9 and gex2, we demonstrate that in the absence of a zygote and embryo, endosperm initiation, syncytium formation, free nuclear cellularization, and endosperm degeneration occur as in the wild type in terms of the cytological process and time course. Although rapid embryo expansion accelerates endosperm breakdown, our findings strongly suggest that endosperm development is an autonomously organized process, independent of egg cell fertilization and embryo–endosperm communication. This work confirms both the altruistic and self-directed nature of the endosperm during coordinated embryo-endosperm development. Our findings provide insights into the intricate interaction between the two fertilization products and will help to distinguish the physiological roles of the signaling between endosperm and embryo. These findings also open new avenues in agro-biotechnology for crop improvement.

11 Cystatin genes in rice were identified, and their expression patterns were comprehensively analyzed, which reveals multiple roles in both seed development and plant response to environmental variations. Cystatin is a group of small proteins and known to inhibit the activities of cysteine proteases in the papain C1A and legumain C13 peptidase families in plants. Cystatin family genes have only been well characterized recently in a few plant species such as Hordeum vulgare and Nicotiana tabacum, which show their critical roles in programmed cell death and responses to biotic stresses. Up to now, little is known about cystatin family genes and their roles in Oryza sativa, a model plant for cereal biology study. Here, we identified 11 cystatin genes in rice genome. Comprehensive expression profile analysis reveals that cystatin family genes in rice display diverse expression pattern. They are temporally regulated at different developmental stages during the process of seed production and germination. Our experiments also reveal that the majority of cystatin genes are responsive to plant hormones and different environmental cues including cold, drought and other abiotic stresses, while some others are very stable under different stresses, indicating their fundamental roles in normal plant development. In addition, their distribution in rice chromosomes and their evolutionary relation to the members of Cystatin family in A. thaliana and N. tabacum have also been analyzed. These works suggest multiple roles of cystatin family genes in both seed development and plant response to environmental variations.

Programmed cell death (PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well‐known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase‐like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase‐like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase‐like protease. Recombinant NtTPE8 exhibited legumain and caspase‐like proteolytic activities, both of which could be inhibited by the pan‐caspase inhibitor (Z‐VAD‐FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Down‐regulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase‐like cysteine protease, NtTPE8, exclusively expressed in the integumentary tapetum that is involved in seed development.

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.