Basis of single-seed formation in chestnut: cytomorphological observations reveal ovule developmental patterns of Castanea henryi

Plant materials

The experimental site was located on the western campus of the Central South University of Forestry and Technology, Changsha, Hunan Province, China. The Chinese Chinquapin (Castanea henryi) cultivar Huali 4 (Variety No.: XiangS-SC-SH-010-2015) was used for observation, and the cultivar Huali 2 (Variety No.: XiangS-SC-SH-008-2015) was used as the pollen donor. The female inflorescences were collected between June and September 2021 for processing and preservation. The trees were approximately 3–4 m tall and grown for 8 years, and they exhibited normal blossoming and fruit-bearing capacities. Pollination and non-pollination treatments were applied to the flowers of the same tree. Three trees were treated, and about 120–150 flowers were sampled from each tree.

Pollination treatment

Male inflorescences of experimental materials were removed, and the female flowers which stigmas were just present were bagged to avoid the entry of exogenous pollen. A total of 400 female flowers were treated to ensure sufficient material for subsequent sampling. The anthers were peeled off from the staminate catkin with toothbrush, and were spread over paper sheets in a ‘pollen room’ (28 °C, 40% RH, 4,000 lx) for 4 h in order to loosen the pollen. Pollen was collected in dry covered 1.5 mL centrifuge tubes, and temporarily stored at 4 °C. On days 5 to 7 of flowering, when the angle between the stigma and the flower mid-axis was about 30° to 45°, pollination treatment was carried out on 200 female flowers in sunny and windless weather, and bags were put on immediately after pollination. The other 200 female flowers were not pollinated. In both treatments, the bags were removed 2 weeks after pollination when the stigmata of the female flowers turned yellow-brown. Samples were taken weekly after treatment. The two main developmental stages we investigated were the mature flower and the young fruit after fertilization.

Paraffin sectioning

The ovary was peeled off from the involucrum and fixed in an ethanol-acetic acid mixture (3:1, v/v), pumped, fixed in an acetic acid mixture for 6 h, and stored in 70% ethanola t 4 °C until needed. Using the conventional paraffin section method described by Fan et al. (2015), samples were sectioned 8 µm thickness with a Leica rotary microtome (RM2235; Leica, Heidelberg, Germany). The dewaxed sections were stained with safranin and Fast Green, and the slides were sealed with neutral balsam. Sections were examined, photographed, and imaged using an optical microscope (BX-53; Olympus, Tokyo, Japan).

Semi-thin sectioning

The samples were fixed in 2.5% glutaraldehyde in 1× phosphate-buffered saline. The samples were then dehydrated with an ethanol gradient series and embedded in EMbed 812 resin. The embedded samples were cut to 2 µm with a glass knife in an ultramicrotome (UC7, Leica) and stained with a mixture of 0.5% safranin and 1% methyl violet (1:1, v/v) (Tolivia, Navarro & Tolivia, 1994). Stained sections were viewed and photographed using the BX-51 optical microscope.

Ovaries and nuts

C. henryi is a monoecious species, bearing two types of catkins: the staminate catkins and bisexual catkins with female flowers that usually occur singly (Fig. 1A). In this species, a ring of green acuminate bracts covers the female flowers, and placental hairs or trichomes in the placenta protect the ovary. The involucrum progressively hardens into a burr when the female flower differentiates into the fruit (Fig. 1B). The pistils of C. henryi comprise the stigma, style, and a large ovary (Fig. 1C). When mature, the burr naturally cracks open, revealing the nut inside. Mature C. henryi nuts are conical in shape and their shells are smooth, hard, reddish brown, or bright brown (Fig. 1D).

Development of the ovule

In the early stages, there was no evident difference in ovule size within the same ovary (Fig. 2A). At 5–6 weeks after pollination, ovules continued to develop with enlargement of the ovary (Figs. 2B , 2C). At week 7 after pollination, the volume of one ovule was visibly larger than that of other ovules, and it was full and rounded (Fig. 2D); this ovule has already been fertilized. With the continuous expansion of the ovary, the fertilized ovule continued to develop, showing a milky white color, and the volume becoming much larger than that of the other ovules. Conversely, the other ovules gradually browned (Figs. 2E, 2F). In flowers that were bagged and not pollinated did not have a single prominent ovules, indicating fertilization. All ovules in these ovaries gradually browned, atrophied, and finally formed empty structures (Figs. 2G–2I).

Double fertilization

We observed that the ovules of C. henryi were enveloped by inner and outer integuments to form a micropyle. The development type of embryo sac was typically polygonum-type with double integuments and thick nuclear tissue. C. henryi had delayed fertilization and immature ovules during blooming. Approximately 6 weeks after pollination, they developed into mature embryo sacs with typical seven-cell and eight-nuclear structures (Fig. 3A). The egg apparatus comprised an egg cell and two synergids near the micropyle, and the nucleus of the egg cell was inclined toward the chalazal end. The synergid cell was pear-shaped, and its nucleus was inclined toward the micropyle end. The central cell was in the middle of the embryonic sac (Fig. 3A). The antipodal cells were close to the chalazal end and degenerated shortly after the embryo sac matured.

Two sperm cells appeared in the embryo sac (Figs. 3B, 3C). The sperm nucleus was seen in some images in close proximity to the egg nucleus (Figs. 3D, 3E), and in other images the sperm nuclei appeared attached to the egg nuclear membrane and undergoing the process of fusion (Fig. 3F). In images where karyogamy appeared to be taking place, the chromatin of the male nucleus was diffuse, and a small nucleolus appeared (Fig. 3G). In some images, the egg sac appeared to contain a zygote (Figs. 3H, 3I). In some sections, a sperm nucleus was seen near the polar nucleus in the central cell (Figs. 3J–3L ), and in some images, the sperm nucleus and the nuclear membrane of the polar nucleus were apposed and appeared to be fusing (Fig. 3M). The chromatin in the sperm nucleus was either displaced from one side to the polar nucleus or spread throughout (Figs. 3N, 3O). A nucleolus was also observed (Fig. 3P). The two nuclei fused to form the primary endosperm nucleus, which was located near the middle of the embryo sac (Fig. 3Q). The contact between the sperm and secondary nuclei preceded the contact between the sperm and egg nuclei.

Embryonic development

The egg cells formed zygotes after successful double fertilization. After the dormancy period and multiple divisions, the globular embryo gradually formed at week 8 after pollination (Figs. 4A–4E). The globular embryos were relatively regular (Figs. 4D–4F). The cotyledon primordia then developed on both sides of its top, forming a heart-shaped embryo (Figs. 4G, 4H). The final mature embryo and integument grew synchronously and filled the entire ovary. The nuts finally matured at week 20 after pollination.

The embryonic development of C. henryi did not include the formation of an obvious radicle. The development type of the endosperm was nuclear. The endosperm first formed in the middle of the ovule and approached the end of the micropyle at the globular embryo stage. At the heart-shaped embryo stage, the endosperm cells began to degrade their cell walls, and became distributed in the embryo sac in a free state (Figs. 4G, 4H). The endosperm presumably provided nutrients to the embryo during development, there was no endosperm when the kernel was mature (Fig. 4I).

Ovule abortion before double fertilization

Abortions in plant seed formation are complex and diverse. Abortion may occur at any stage, ranging from the archesporial cell to embryonic development and maturation. In C. henryi, some ovules prior to fertilization had embryo sacs that appeared to be abnormal even tough the integument appeared to be developing normally. In a single ovary at the mature embryo sac stage, approximately half of the ovules exhibited abnormal embryo sac development (Table 1). The abnormalities can be categorized into three types: the embryo sac cavity was elongated and narrow (Figs. 5A, 5B); there was a normal embryo sac cavity, but the structure of the egg apparatus structure was missing or incomplete (Fig. 5C); and embryo sac degeneration occurred. Embryo sac degeneration was manifested as follows: the embryo sac sometimes developed to the four-nucleate stage (Fig. 5D); sometimes, at the late stage of embryo sac development, the central cell (Fig. 5E), antipodal cell (Fig. 5F), and egg apparatus could appear to be degenerated (Figs. 5G, 5H), and finally, sometimes abortive cells with abnormal embryo sac development showed degeneration of embryo sac, shrinkage, and deformation of nucellus tissue cells, as well as degeneration and disintegration of nucellus tissue (Fig. 5I).

To investigate the impact of pollination on ovule development, we subjected female chestnut flowers to two treatments: pollination and non-pollination. Compared to pollinated ovules, non-pollinated ovules exhibited relatively delayed development, with a reduction in the number of ovules appearing with normal development at each stage. Approximately 53.53% of ovules in pollinated ovaries developed into mature embryo sacs with a seven-cell eight-nucleus structure (Table 1). Without pollination, only about 16.55% of ovules in a single ovary had a mature embryo sac structure (Table 1). Pollination partially influences the developmental outcome of the embryo sac.

Abortion of ovules after double fertilization

Not all ovules formed seeds after successful fertilization. In the semi-thin sections, approximately 16% of the ovules appeared to show post-fertilization abortion, which can be classified into three types: first, a normal proembryo structure could not be formed after fertilization, no normal zygote appeared, and only a clump of flocculent tissue was present (Figs. 6A–6C); second, the zygote formed, but it did not split (Figs. 6D–6F); and thirds, the zygote was formed, and the development of the zygote stagnated after the first normal division to form the second proembryo (Figs. 6G, 6H). If the ovule formed a zygote, then its endosperm developed normally from the primary endosperm nucleus to the free endosperm nucleus (Figs. 6I, 6J). Moreover, after the formation of the primary endosperm nucleus followed by a very short dormancy period multiple free endosperm nuclei were formed.