ORIGIN OF RECOGNITION COMPLEX 3 controls the development of maternal excess endosperm in the Paspalum simplex agamic complex (Poaceae)

Abstract

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

Keywords: ORC3; Paspalum simplex; Apomixis; cell cycle; interploidy crosses; maternal excess endosperm.

Fig. 1.

Phenotypic analysis of the P. simplex transformed plants. (A) Representative photographs of the aerial part of wild type [39(G) 4n WT c^– and 53(E) 4n WT c⁺] and RNAi transgenic (1E 4n RNAi c^– and B7 4n RNAi c⁺) plants in the first and second year of growth. In the second year, B7 4n RNAi c⁺ developed a transient phenotype with stoloniferous structures (white arrows), which disappeared later when this plant developed reproductive branches (second year, right picture). (B) Root apparatus of WT and RNAi genotypes; black bar length is 10 cm. (C) GUS colorimetric assay on sections of plant leaves and roots. Black arrows indicate the blue color of the transformed plant vascular tissue.

Fig. 2.

Flow cytometric analysis of mature seeds and chromosome counts in P. simplex hybrids. (A) Histogram of leaf tissue from a control diploid (2n=2x) individual. (B) Histogram of mature seed of a diploid cytotype. The 2C (2x) peak corresponds to the DNA content of nuclei in the embryo and the smaller 3C peak to nuclei in the endosperm. The 4C and 6C peaks correspond to nuclei from the embryo and endosperm that have undergone endoreduplication. (C) Histogram of a sexual tetraploid (2n=4x) seed showing a 2C peak corresponding to the embryo (4x) and a smaller peak related to the endosperm 3C (6x). The 4C peak accounts for the endoreduplication of some nuclei of the embryo. (D) Histogram of a WT pseudogamous apomictic tetraploid seed showing a 2C peak corresponding to a parthenogenetic embryo (4x) and a 5C peak corresponding to a maternal excess endosperm; the 4C peak is related to embryo endopolyploidization. (E) Histogram of a seed derived from the 1E 4n RNAi c^–×Bulk 2n WT c^± interploidy cross (Table 2) showing a 3C:5C peak pattern. This embryo:endosperm genome ratio is derived from the double fertilization of a reduced (n=2x) egg cell and both polar nuclei (n=2x+n=2x) by reduced (n) male gametes to form a triploid embryo (2n+n) and a pentaploid (2n+2n+n) maternal excess endosperm. (F) Histogram of a seed derived from the B7 4n RNAi c⁺×Bulk 2n WT c^± interploidy cross (Table 2) showing again a 3C:5C peak pattern. (G) Histogram of seed derived from the 27(C.1) 4n WT c^–×D9 2n WT c⁺ control interploidy cross (Table 2) showing a 3C:5C pattern similar to that described in (E). A 6C peak in (E–G) can be attributed to endoreduplication of the embryo cells. (H) Root tip metaphase chromosome spread of a seed from the control cross described in (G). Arrows indicate three metacentric homologous chromosomes. PI, propidium iodide sensor.

Fig. 3.

qRT–PCR transcriptional profiles of PsORC3b and PsORC3c isogenes in developing endosperms of P. simplex. (A) Relative expression profile of the PsORC3b isogene in interploidy crosses of WT and RNAi-inactivated tetraploid mother plants pollinated with diploid genotypes. The numbers followed by HAP indicate the hours after pollination of sample collection and mark the endosperm developmental stages schematized below the graph corresponding to: 6 HAP (first mitotic divisions), 24 HAP (syncytium with nuclei localized at periphery of the embryo sac), 48 HAP (late cellularization), 120 HAP (late proliferative stage), and 240 HAP (onset of endoreduplication). (B) Relative expression profile of PsORC3c in the same material as in the last two stages of (A). (C) Relative expression profile of PsORC3b and PsORC3c isogenes in inter- and homoploid crosses in which the mother plant differs for the presence of the PsORC3c isogene. The same letters on the top of the histograms indicate non-significant differences for the expression for P≤0.005, c⁺ or c^– indicate presence or absence of the PsORC3c isogene in the parent lines, and c^± indicates bulked pollen collected from mixed 2n genotypes. Bars on the top of histograms indicate the SEs. The relative expression level refers to the expression value of the PsORC3b isogene of the sample 53(E) 4n WT c⁺ in (A) (6 HAP) and (C), and of the PsORC3c isogene of the same sample in (B) arbitrarily set to 1.

Fig. 4.

Histological analysis of developing seeds from WT and RNAi transgenic plants after homo- and interploidy crosses at 12, 24, 48, 120, and 240 hours after pollination (HAP). (A, E, I, M, Q) Longitudinal sections of developing seeds obtained by open pollination of the WT sexual tetraploid plant 27(C.1) 4n WT c^– with tetraploid pollen donors (balanced endosperm). (B, F, J, N, R) Developing seeds form natural apomictic tetraploid plant 31A 4n WT c⁺ derived from open pollination with tetraploid sexual plants (natural maternal excess endosperm). (C, G, K, O, S) Developing seed from the tetraploid sexual RNAi-inactivated PsORC3 RNAi-inactivated plant B7 4n RNAi c⁺ crossed with diploid pollen donors (induced maternal excess endosperm). (D, H, L, P, T) Developing seeds from the natural tetraploid sexual plant 53(E) 4n WT c⁺, crossed with diploid genotypes (induced maternal excess endosperm). Scale bar=30 µm in A–D, 50 µm in E–L, and 150 µm in (M–T).

Fig. 5.

Model of maternal excess endosperm development controlled by PsORC3 in P. simplex. (A, B) In apomictic plants, the presence of PsORC3a lncRNA in antisense orientation inhibits both PsORC3c (when present) and PsORC3b expression. This fact always allows the formation of viable seeds with maternal excess endosperm. (C, D) In interploidy control crosses, if the sexual tetraploid mother plants harbor the PsORC3c isogene (C), PsORC3b is up-regulated and no maternal excess endosperm is produced, whereas in mother plants null for PsORC3c, PsORC3b is maintained at its basal expression and the formation of triploid seeds with maternal excess endosperm is allowed (D). (E, F) In interploidy crosses in which ORC3 RNAi plants are used as seed parents, the expression of RNAi transcript mimics the action of PsORC3a in natural apomicts (by silencing both PsORCb and c); in both cases of PsORC3 genotype configuration, generation of triploid seeds with maternal excess endosperm is allowed.