The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development 1 Sunil Kumar Singh, a Vajinder Kumar, b Ramamurthy Srinivasan, b Paramvir Singh Ahuja , a,2 Shripad Ramchandra Bhat , b,3 and Yelam Sreenivasulu a,3,4 a Biotechnology Division, Council of Scientific and Industrial Research CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India b Indian Council of Agricultural Research ICAR-National Research Centre on Plant Biotechnology, New Delhi 110012, India ORCID IDs: 0000-0002-6234-0276 (S.K.S.); 0000-0002-8825-3517 (V.K.); 0000-0002-3875-6650 (R.S.); 0000-0002-8349-6691 (S.R.B.); 0000-0003-2610-3916 (Y.S.). In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants. The life cycle of higher plants comprises a short, haploid, gametophyte phase and a long, diploid, mul- ticellular, sporophyte phase. The transition to the gametophyte phase is initiated when a diploid spore mother cell differentiates and undergoes meiosis to give rise to four haploid megaspores. In the ovule, of the four spores, the three that are closest to the micropylar end of the ovule degenerate while the fourth, closest to the chalazal end, becomes a functional megaspore and differentiates into a megagametophyte after typically three cycles of free nuclear mitotic divisions followed by highly polarized cellularization (Yang et al., 2010; Sprunck and Gross-Hardt, 2011). The resulting embryo sac is the mature female gametophyte (FG). A typical FG comprises a seven-celled embryo sac containing six haploid cells (an egg cell, two synergids, and three an- tipodals) with one diploid central cell. Likewise, fol- lowing meiosis, the male microspore undergoes an asymmetric cell division resulting in a male gameto- phyte comprising one vegetative cell and two sperm cells (Twell, 2011). The haploid gametes develop in the midst of diploid maternal tissue, which requires constant communication between the two types of cells. After fertilization, inter- communications among the embryo, the endosperm, and the seed coat, each with a unique genetic identity, is essential for proper seed development (Figueiredo and Köhler, 2016). During the alternation of generations, cells 1 This work was funded by the Council of Scientific and Industrial Research (project nos. MLP-072 and BSC-0107), New Delhi, India, and the Indian Council of Agricultural Research, New Delhi, India, through the National Agricultural Innovation Project (NAIP-4157). 2 Deceased 3 Address correspondence to srbhat@nrcpb.org and sreenivasulu@ ccmb.res.in. 4 Current address: Council of Scientific and Industrial Research- Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Yelam Sreenivasulu (sreenivasulu@ccmb.res.in). Y.S. conceived the original screening and research plans and su- pervised the experiments; S.K.S. performed most of the experiments; V.K. performed microarray experiments and qRT-PCR analysis.; R.S., P.S.A., S.R.B., and Y.S. designed the experiments and analyzed the data; S.R.B. and Y.S. conceived the project and wrote the article with contributions of all the authors; Y.S. supervised and complemented the writing. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00275 1220 Plant Physiology Ò , November 2017, Vol. 175, pp. 1220–1237, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.org on June 5, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.