A Plant-Specific TGS1 Homolog Influences Gametophyte Development in Sexual Tetraploid Paspalum notatum Ovules

Abstract

Aposporous apomictic plants form clonal maternal seeds by inducing the emergence of non-reduced (2n) embryo sacs in the ovule nucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomictic Paspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis. Here we transformed sexual P. notatum with a TGS1-like antisense construction under a constitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominent trichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealed occasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisense lines showed 12.93–15.79% of ovules bearing extra nuclei, which can be assigned to aposporous-like embryo sacs (AES-like) or, alternatively, to gametophytes with a misguided cell fate development. Moreover, around 8.42–9.52% of ovules showed what looked like a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellar cells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or the MMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced nature for the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation of the legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to control genotypes, and formed an equivalent full seed set (∼9% of total seeds) after self-pollination. Flow cytometry analyses of caryopses derived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of 25.55% in the germination percentage was detected when comparing antisense lines with controls. Our results indicate that PN_ TGS1-like influences ovule, gametophyte and possibly embryo development.

Apomixis Technology: Separating the Wheat from the Chaff

“…Among these findings, particularly diverse are those in Paspalum, such as the PnTgs1-like protein (a trimethylguanosine synthase-like protein), whose function has been associated gametophyte and possibly embryo development [115,116], QUI-GON JINN (a gene showing homology with mitogen-activated protein kinase kinase kinases (MAP3K/MAPKKK/MEKK)) having probable roles in the acquisition of a gametophytic cell fate by AIs, and the development of aposporous embryo sacs [110], PsORC3a (a pseudogene with homology to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3)) with a probable role regulating expression of its functional homolog and with the development of apomictic endosperm [64], PN_LNC_N13 (a member of a family of long non-coding RNAs (lncRNAs)) involved in splicing regulation [117], or a number of small RNAs differentially represented in sexual and apomictic flowers likely involved in diverse regulatory pathways-including auxin signaling-important in promoting apomixis [118].…”

How to Become an Apomixis Model: The Multifaceted Case of Paspalum

“…For instance, apomictic seeds of tetraploid pseudogamous Paspalum notatum show a 2:5 embryo:endosperm ratio (4x embryo and 4x + 4x + 2x = 10x endosperm). This method was successful during the last two decades for determining reproductive behaviors in individual or bulked seeds of several Paspalum species [ 23 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ]. Another distinctive feature of seed development in Paspalum apomicts is the lack of deleterious response to deviations from the 2:1 maternal-to-paternal genomic ratio in the endosperm strictly required for sexual reproduction [ 39 ].…”

“…In connection with this, in situ RNA hybridization protocols were developed for Paspalum , and gradually optimized to analyze the spatio-temporal plus sense/antisense expression distribution within the ovule [ 38 , 58 , 90 , 96 , 97 , 98 , 99 , 100 , 101 ]. An example of expression characterization in reproductive tissues at early megasporogenesis involving the apomixis candidate gene ORC3 is shown in Figure 5 .…”

“…Since the nucellus is the site of aposporous initials (AIs) differentiation, we proposed that PN_TGS1- like might be preventing the differentiation of apospory initials in sexual P. notatum plants [ 97 ]. Thereafter, a full-sexual P. notatum genotype was transformed with a TGS1 -like antisense construction under a constitutive promoter, to obtain lines with a reduced transcript representation [ 38 ]. Antisense plants developed prominent trichomes on the adaxial leaf surface, occasionally formed twin ovules, and showed around 15% of ovules bearing what looked like supernumerary aposporous-like gametophytes (i.e., numerous female gametophytes with a typical Paspalum unreduced megagametophyte morphology, including an egg cell, one-two synergids, two polar nuclei, and no antipodal cells).…”

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation

“…Since then, clearing protocols have been used to analyze ovule, ES, and embryo development. Various basic studies aimed at understanding the reproductive biology in several species (Herr, 1982;Herr, 1992;Musiał et al, 2015;Wilkinson and Tucker, 2017;Barke et al, 2018;Hoshino et al, 2018;Colono et al, 2019;Tofanelli et al, 2019); as well as for applied studies for plant breeding (Ogburia and Adachi, 1994;Ogburia and Adachi, 1995;Ogburia and Adachi, 1996;Ponitka and S´lusarkiewicz-Jarzina, 2004;Janowicz and Wojciechowski, 2010;Hoshino et al, 2018;Kwiatkowska et al, 2019). Clearing techniques have also been used to study morphology and gene expression for understanding developmental and other biological processes (Kurihara et al, 2015;Musielak et al, 2016).…”