Transcriptomic Analysis Reveals the Positive Role of Abscisic Acid in Endodormancy Maintenance of Leaf Buds of Magnolia wufengensis

Magnolia wufengensis (Magnoliaceae) is a deciduous landscape species, known for its ornamental value with uniquely shaped and coloured tepals. The species has been introduced to many cities in south China, but low temperatures limit the expansion of this species in cold regions. Bud dormancy is critical for plants to survive in cold environments during the winter. In this study, we performed transcriptomic analysis of leaf buds using RNA sequencing and compared their gene expression during endodormancy, endodormancy release, and ecodormancy. A total of 187,406 unigenes were generated with an average length of 621.82 bp (N50 = 895 bp). In the transcriptomic analysis, differentially expressed genes involved in metabolism and signal transduction of hormones especially abscisic acid (ABA) were substantially annotated during dormancy transition. Our results showed that ABA at a concentration of 100 μM promoted dormancy maintenance in buds of M. wufengensis. Furthermore, the expression of genes related to ABA biosynthesis, catabolism, and signalling pathway was analysed by qPCR. We found that the expression of MwCYP707A-1-2 was consistent with ABA content and the dormancy transition phase, indicating that MwCYP707A-1-2 played a role in endodormancy release. In addition, the upregulation of MwCBF1 during dormancy release highlighted the enhancement of cold resistance. This study provides new insights into the cold tolerance of M. wufengensis in the winter from bud dormancy based on RNA-sequencing and offers fundamental data for further research on breeding improvement of M. wufengensis.

The Dwarfing Effects of Different Plant Growth Retardants on Magnolia wufengensis L.Y. Ma et L. R. Wang

The effects of varieties, concentrations, and number of applications of plant growth retardants (PGRs) on the morphological, physiological, and endogenous hormones of Magnolia wufengensis L.Y. Ma et L. R. Wang were assessed to obtain the most suitable dwarfing protocol for M. wufengensis and to provide theoretical support and technical guidance for the cultivation and promotion of this species. One-year-old M. wufengensis ‘Jiaohong No. 2’ grafted seedlings served as the experimental materials. In the first part of the experiment, three PGRs (uniconazole, paclobutrazol, prohexadione calcium), three concentrations (500, 1000, 1500 ppm), and three applications (one, three, and five applications) were applied in dwarfing experiments to perform L9 (34) orthogonal tests. In the second part of the study, dwarfing experiments were supplemented with different high uniconazole concentrations (0, 1500, 2000, 2500 ppm). Spraying 1500 ppm uniconazole five times achieved the best M. wufengensis dwarfing effect, related indicators of M. wufengensis under this treatment were better than other treatment combinations. Here, M. wufengensis plant height, internode length, scion diameter, and node number were significantly reduced by 56.9%, 62.6%, 72.8%, and 74.4%, respectively, compared with the control group. This treatment increased superoxide dismutase (SOD) activity by 66.0%, peroxidase (POD) activity by 85.0%, soluble protein contents by 43.3%, and soluble sugar contents by 27.6%, and reduced malondialdehyde (MDA) contents by 32.1% in leaves of M. wufengensis compared with the control. The stress resistance of M. wufengensis was enhanced. The treatment also reduced gibberellin (GA3) levels by 73.0%, auxin (IAA) by 58.0%, and zeatin (ZT) by 70.6%, and increased (abscisic acid) ABA by 98.1% in the leaves of M. wufengensis. The uniconazole supplementation experiment also showed that 1500 ppm was the optimal uniconazole concentration. The leaves exhibited abnormalities such as crinkling or adhesion when 2000 or 2500 ppm was applied. Given the importance of morphological indicators and dwarfing for the ornamental value of M. wufengensis, the optimal dwarfing treatment for M. wufengensis was spraying 1500 ppm uniconazole five times.

MawuAP1 promotes flowering and fruit development in the basal angiosperm Magnolia wufengensis (Magnoliaceae)

The APETALA1/SQUAMOSA (AP1/SQUA)-like genes of flowering plants play crucial roles in the development processes of floral meristems, sepals, petals and fruits. Although many of the AP1/SQUA-like genes have been characterized in angiosperms, few have been identified in basal angiosperm taxa. Therefore, the functional evolution of the AP1/SQUA subfamily is still unclear. We characterized an AP1 homolog, MawuAP1, from Magnolia wufengensis that is an ornamental woody plant belonging to the basal angiosperms. Gene sequence and phylogenetic analyses suggested that MawuAP1 was clustered with the FUL-like homologous genes of basal angiosperms and had FUL motif and paleoAP1 motif domain, but it did not have the euAP1 motif domain of core eudicots. Expression pattern analysis showed that MawuAP1 was highly expressed in vegetative and floral organs, particularly in the early stage of flower bud development and pre-anthesis. Protein–protein interaction pattern analysis revealed that MawuAP1 has interaction with an A-class gene (MawuAP1), C-class gene (MawuAG-1) and E-class gene (MawuAGL9) of the MADS-box family genes. Ectopic expression in Arabidopsis thaliana indicated that MawuAP1 could significantly promote flowering and fruit development, but it could not restore the sepal and petal formation of ap1 mutants. These results demonstrated that there are functional differences in the specification of sepal and petal floral organs and development of fruits among the AP1/SQUA-like genes, and functional conservation in the regulation of floral meristem. These findings provide strong evidence for the important functions of MawuAP1 in floral meristem determination, promoting flowering and fruit development, and further highlight the importance of AP1/SQUA subfamily in biological evolution and diversity.

Fall Ethephon Application Enhances the Freezing Tolerance of Magnolia wufengensis During Overwintering

Magnolia wufengensis L.Y. Ma et L. R. Wang, a rare species which has been introduced and cultivated from southern China to northern China, frequently suffers from freezing injuries. To figure out the influence of ethephon (ETH) application on the cold tolerance during the natural overwintering of M. wufengensis, one-year shoots subjected to ethephon application at four concentrations (0, 700, 1000, and 1500 mg·L−1) were collected to measure the physiological and biochemical changes from September 2017 to 1 April 2018. The fall ETH application increased the freezing tolerance of M. wufengensis, and the optimum concentration for M. wufengensis was 1000 mg·L−1 (T2), which not only improved the shoot freezing tolerance by 1.4 times, but also led to a 25.0% faster cold acclimation rate and 13.7% slower de-acclimation rate. Moreover, 1000 mg·L−1 ETH delayed bud-burst in the spring by 10. 7 d, which was helpful for effectively avoiding cold spells in the spring, and improved the rates of bud survival by 47.1%. The improved freezing tolerance under exogenous ETH application was associated with an increased dehydration and accumulation of proline content. It seems that exogenous ETH application may be used on M. wufengensis grown in northern China to protect against freezing tolerance during the overwintering period.

Conservation and divergence of ancestral AGAMOUS/SEEDSTICK subfamily genes from the basal angiosperm Magnolia wufengensis

AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon–intron structures, expression patterns, protein–protein interaction patterns and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis (Magnoliaceae). MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of the STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of the SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves differ from those found in monocots and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon–intron structural changes of the AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon–intron structures, expression and interaction patterns, and functions, and provide strong evidence for the significance of gene duplication in the expansion and evolution of the AG/STK subfamily.