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 ovulenucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINESYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomicticPaspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules frompremeiosis to anthesis. Here we transformed sexual Paspalum notatum with a TGS1-like antisense construction under aconstitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominenttrichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealedoccasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisenselines 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 lookedlike a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellarcells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or theMMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced naturefor the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation ofthe legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to controlgenotypes, and formed an equivalent full seed set (~9% of total seeds) after self-pollination. Flow cytometry analyses of caryopsesderived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of25.55 % 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.