A quantitative study of cotyledon positioning in conifer development

Abstract

The number of cotyledons in angiosperm monocots and dicots is tightly constrained. But in the gymnosperm Pinaceae, including conifers, cotyledon number (n c ) can vary widely, commonly between 2 to 12. Conifer cotyledons form in whorled rings, on a domed embryo geometry. We measured embryo diameters and counted cotyledons to determine the radial positioning of the whorl and the circumferential spacing between cotyledons. Results were similar between Douglas fir (Pseudotsuga), Sitka spruce (Picea) and larch (Larix), indicating a common mechanism for cotyledon positioning in conifers. Disrupting transport of the growth regulator auxin (with NPA) led to cup-shaped embryos, indicating that whorl (ring) formation is separable from cotyledon patterning within the ring. NPA inhibits cotyledon outgrowth, but not the spacing (distance) between cotyledons. The NPA effect is direct; it does not operate indirectly on embryo size. These results support a hierarchical model for cotyledon positioning in conifers, in which a first stage (not requiring auxin transport) sets the whorl position, constraining the second stage (which requires auxin transport) to form cotyledons within this whorl. Similarly, recent studies in Arabidopsis have shown that different components of complex developmental patterns can have different transport properties; this aspect of patterning may be shared across plants.

Two-stage patterning dynamics in conifer cotyledon whorl morphogenesis

“…The linear variation of n c with diameter expected for regular spacing in a ring (Fig. 1E, black spots) has been found experimentally in Larix (Harrison and von Aderkas, 2004), Pseudotsuga and Picea (Holloway et al, 2016), indicating that n c variability reflects embryo-to-embryo diameter variability. Without radial constraint, the short wavelength cotyledon spacing (λ) would position cotyledons over the entire embryo, which is not observed: arrangement of primordia in a single whorl involves the combination of a longer spatial scale radial control with a shorter scale circumferential control.…”

“…For the circumferential P2 pattern, the NPA effect appears to be primarily on the outgrowth of cotyledons: while most embryos do not grow cotyledons at moderate NPA concentrations, those that do tend to show normal circumferential spacing (Holloway et al, 2016). This indicates that the outgrowth of distinct cotyledons from the ring is PAT-dependent (perhaps via supply of a critical factor), but that the spacing λ between cotyledons is PAT-independent.…”

“…Within species, n c typically ranges ±1 from these averages for zygotic embryos (Butts and Buchholz, 1940). For somatic cultures (of clonal lines), embryos with n c from 3 to 10 are common in Larix, Pseudotsuga and Picea, which have species averages of 5 to 6 (Harrison and von Aderkas, 2004;Holloway et al, 2016).…”

“…Initiation sites are affected by the positions of older primordia. In conifer embryos, however, a single whorl of cotyledons forms simultaneously, and the single whorl is robust to large changes in radius: experimental measurements show whorl radii increase by about 150 µm over the range of observed n c (Holloway et al, 2016; whorl radius is approx. 125 µm less than embryo radius).…”

The age of black pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) mother trees has no effect on seed germination and on offspring seedling performance

“…In addition, the positive correlations of SDMF with N seeds and GP indicate that pinecones whose weight portion contributed by the internal pine seeds was greater also tended to contain more seeds with a higher germination potential. This could be a consequence of the accumulation and activity of phytohormones, mainly auxins, during embryo and seed development (Holloway et al 2016) that contribute to the pinecone growth when developing seeds are inside (Sauer et al 2013).…”