Chestnut (Castanea mollissima) is a deciduous tree species with major economic and ecological value that is widely used in the study of floral development in woody plants due its monoecious and out-of-proportion characteristics. Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that plays an important role in floral development. In this study, a total of 18 SPL genes were identified in the chestnut genome, of which 10 SPL genes have complementary regions of CmmiR156. An analysis of the phylogenetic tree of the squamosa promoter-binding protein (SBP) domains of the SPL genes of Arabidopsis thaliana, Populus trichocarpa, and C. mollissima divided these SPL genes into eight groups. The evolutionary relationship between poplar and chestnut in the same group was similar. A structural analysis of the protein-coding regions (CDSs) showed that the domains have the main function of SBP domains and that other domains also play an important role in determining gene function. The expression patterns of CmmiR156 and CmSPLs in different floral organs of chestnut were analyzed by real-time quantitative PCR. Some CmSPLs with similar structural patterns showed similar expression patterns, indicating that the gene structures determine the synergy of the gene functions. The application of gibberellin (GA) and its inhibitor (Paclobutrazol, PP₃₃₃) to chestnut trees revealed that these exert a significant effect on the number and length of the male and female chestnut flowers. GA treatment significantly increased CmmiR156 expression and thus significantly decreased the expression of its target gene, CmSPL6/CmSPL9/CmSPL16, during floral bud development. This finding indicates that GA might indirectly affect the expression of some of the SPL target genes through miR156. In addition, RNA ligase-mediated rapid amplification of the 5′ cDNA ends (RLM-RACE) experiments revealed that CmmiR156 cleaves CmSPL9 and CmSPL16 at the 10th and 12th bases of the complementary region. These results laid an important foundation for further study of the biological function of CmSPLs in the floral development of C. mollissima.

Strawberry (Fragaria × ananassa) is widely used as a model for fleshy fruit biology research; nevertheless, the pathway of phloem unloading in strawberry fruit remains unknown. Using transmission electron microscopy (TEM) and the symplastic tracer, carboxyfluorescein, the phloem unloading pathway was studied in ‘Akihime’ strawberry fruit from post-anthesis to the ripening stage. Ultrastructural analysis showed that many plasmodesmata existed between the sieve element cells (SEC) and the companion cells (CC), but plasmodesmata were absent between the sieve element cell – CC complexes and their surrounding phloem parenchyma cells (PPCs). Interestingly, an abundance of plasmodesmata was observed between parenchyma cells. Imaging of carboxyfluorescein unloading revealed that the dye was confined only to the phloem vascular bundles in developing receptacles. In conclusion, the results obtained from the present study indicate that sugar unloading from the phloem occurs through an apoplastic pathway. Post-phloem transport of sugar then follows a symplastic pathway in strawberry fruit.

Chinese chestnuts (Castanea mollissima Blume.) contain 12–18 ovules in one ovary, but only one ovule develops into a seed, indicating a high ovule abortion rate. In this study, the Chinese chestnut ‘Huaihuang’ was used to explore the possible mechanisms of ovule abortion with respect to morphology and proteomics. The morphology and microstructure of abortive ovules were found to be considerably different from those of fertile ovules at 20 days after anthesis (20 DAA). The fertile ovules had completely formed tissues, such as the embryo sac, embryo and endosperm. By contrast, in the abortive ovules, there were no embryo sacs, and wide spaces between the integuments were observed, with few nucelli. Fluorescence labelling of the nuclei and transmission electron microscopy (TEM) observations showed that cells of abortive ovules were abnormally shaped and had thickened cell walls, folded cell membranes, condensed cytoplasm, ruptured nuclear membranes, degraded nucleoli and reduced mitochondria. The iTRAQ (isobaric tag for relative and absolute quantitation) results showed that in the abortive ovules, low levels of soluble protein with small molecular weights were found, and most of differently expressed proteins (DEPs) were related to protein synthesis, accumulation of active oxygen free radical, energy synthesis and so on. These DEPs might be associated with abnormal ovules formation.

There are many factors that affect the yield of Chinese chestnut (Castanea mollissima), with single nut weight (SNW) being one of the most important. Leaf length is also related to Chinese chestnut yield. However, the genetic architecture and gene function associated with Chinese chestnut nut yield have not been fully explored. In this study, we performed genotyping by sequencing 151 Chinese chestnut cultivars, followed by a genome-wide association study on 6 horticultural traits. First, we analyzed the phylogeny of the Chinese chestnut and found that the Chinese chestnut cultivars divided into two ecotypes, a northern and southern cultivar group. Differences between the cultivated populations were found in the pathways of plant growth and adaptation to the environment. In the selected regions, we also found interesting tandemly arrayed genes that may influence Chinese chestnut traits and environmental adaptability. To further investigate which horticultural traits were selected, we performed a genome-wide association study using 6 horticultural traits from 151 cultivars. Forty-five loci that strongly associated with horticultural traits were identified, and 6 genes highly associated with these traits were screened. In addition, a candidate gene associated with SNW, APETALA2 (CmAP2), and another candidate gene associated with leaf length (LL), CRYPTOCHROME INTERACTING BASIC HELIX-LOOP-HELIX 1 (CmCIB1), were verified in Chinese chestnut and Arabidopsis (Arabidopsis thaliana). Our results showed that CmAP2 affected SNW by negatively regulating cell size. CmCIB1 regulated the elongation of new shoots and leaves by inducing cell elongation, potentially affecting photosynthesis. This study provided valuable information and insights for Chinese chestnut breeding research.