Links between the pandemic and urban green spaces, a perspective on spatial indices of landscape garden cities in China.

The COVID-19 pandemic has inevitably changed people's lifestyles and demands for urban green space and public open space. The National Landscape Garden Cities in China (NLGCC) policy is one of the key development models in China aimed at building sustainable cities and society. In this paper, the development of the study's selection criteria and the significance and benefits of the NLGCC policy are first summarised. 391 cities were chosen from the NLGCC list to analyse the spatial distribution and construction of driving factors. The results show that the NLGCC's selection criteria have shifted from a focus on quantity to overall habitat quality. During the COVID-19 pandemic, city resilience has been examined more closely. The NLGCC policies have boosted to address ecological and environmental crises and enhanced urban disaster preparedness. The spatial distribution analysis shows that the NLGCC is spatially unevenly distributed and has a clustering trend. A total of 54.96% of the NLGCC is concentrated in China's eastern and central regions. The natural environment and socioeconomics are two main categories of driving factors. This study provides significant value to the understanding of the spatial pattern of the NLGCC offers a reference for decision-making about the construction of urban environments worldwide.

Genome-Wide Identification and Low-Temperature Expression Analysis of bHLH Genes in Prunus mume.

Prunus mume is an illustrious ornamental woody plant with colorful flowers, delicate fragrances, and graceful tree forms. Low temperature limits its geographical distribution. The basic helix-loop-helix (bHLH) proteins exist in most eukaryotes as a transcription factor superfamily, which play a crucial role in metabolism, physiology, development, and response to various stresses of higher organisms. However, the characteristics of the bHLH gene family and low-temperature response remain unknown in P. mume. In the present study, we distinguished 95 PmbHLH genes in the P. mume whole-genome and analyzed their features. PmbHLHs were divided into 23 subfamilies and one orphan by phylogenetic analysis. Similar gene structures and conserved motifs appeared in the same subfamily. These genes were situated in eight chromosomes and scaffolds. Gene duplication events performed a close relationship to P. mume, P. persica, and P. avium. Tandem duplications probably promoted the expansion of PmbHLHs. According to predicted binding activities, the PmbHLHs were defined as the Non-DNA-binding proteins and DNA-binding proteins. Furthermore, PmbHLHs exhibited tissue-specific and low-temperature induced expression patterns. By analyzing transcriptome data, 10 PmbHLHs which are responsive to low-temperature stress were selected. The qRT-PCR results showed that the ten PmbHLH genes could respond to low-temperature stress at different degrees. There were differences in multiple variations among different varieties. This study provides a basis to research the evolution and low-temperature tolerance of PmbHLHs, and might enhance breeding programs of P. mume by improving low-temperature tolerance.

Metabolic, Enzymatic Activity, and Transcriptomic Analysis Reveals the Mechanism Underlying the Lack of Characteristic Floral Scent in Apricot Mei Varieties.

Apricot mei, a hybrid of Prunus mume and Prunus sibirica, usually has greater cold resistance than P. mume; however, most varieties of Apricot mei lack the characteristic floral scent of P. mume. The volatile and intracellular metabolites, activity levels of key enzymes, and transcriptomes of blooming flowers were comprehensively investigated in five varieties of P. mume. Benzyl acetate and eugenol were determined to be the main components of the P. mume floral scent. However, benzyl benzoate and benzyl alcohol benzoyltransferase activity was detected in only the low-fragrance varieties "Dan Fenghou" and "Yanxing." No benzyl alcohol or benzaldehyde reductase (BAR) activity was detected in the non-fragrant variety "Fenghou." PmBAR1 and PmBAR3 were identified as the key genes responsible for BAR activity. The lack of benzyl alcohol synthesis in the "Fenghou" variety was caused by low activity of PmBAR1-Fen and low expression of PmBAR3. The 60-aa segment at the N-terminus of PmBAR3 was found to play an important role in its enzymatic activity. Correlation tests between floral scent metabolites and the transcriptomes of the five different scented varieties showed that some transcripts associated with hormones, stresses, posttranslational modifications and transporters may also play important regulatory roles in floral scent metabolism in the different varieties.

Screening of optimal reference genes for qRT-PCR and preliminary exploration of cold resistance mechanisms in Prunus mume and Prunus sibirica varieties.

Prunus sibirica and Prunus mume are closely related plant species that differ in cold tolerance. Hybrids of P. sibirica and true mume, belonging to the apricot mei group, inherited strong cold resistance from P. sibirica. These materials are favourable for research on the molecular mechanisms of cold resistance. However, no suitable reference genes have been identified for analysing gene expression patterns between P. sibirica and P. mume. Ten candidate reference genes were assessed, namely, actins (ACT2-1, ACT2-2, ACT2-3, ACT2-4), protein phosphatase 2A-1 (PP2A-1), ubiquitins (UBQ2, UBQ3), ubiquitin extension protein (UBQ1) and tubulins (TUB1, TUB2), with four distinct algorithms (geNorm, NormFinder, BestKeeper and RefFinder). UBQ2 was recognized as the best reference gene in stems and buds across materials (P. sibirica; 'Xiaohong Zhusha', 'Beijing Yudie', and 'Xiao Lve' for true mume; and 'Dan Fenghou', 'Fenghou', and 'Yanxing' for apricot mei) under cold stress. In addition, the temporal and spatial expression patterns of PmCBF6 and PmLEA10 among seven varieties during winter periods were analysed using UBQ2 as a reference gene. The expression differed significantly among cultivars, which may contribute to their differences in cold tolerance. This paper confirmed the strong cold tolerance of apricot mei. And the best internal reference gene suitable for seven varieties was selected: UBQ2. Based on the above results, the expression of PmCBF6 and PmLEA10 genes during wintering in seven varieties was analysed. The molecular mechanisms of cold resistance were found to be possibly different in different varieties of P. sibirica and P. mume.

Genome-Wide Analysis of Members of the WRKY Gene Family and Their Cold Stress Response in Prunus mume.

Prunus mume, which is a rosaceous arbor with very high ornamental, edible and medical values, has a distribution that is mainly restricted by low temperature. WRKY transcription factor genes play crucial roles in the growth, development, and stress responses of plants. However, the WRKY gene family has not been characterised in P. mume. There were 58 PmWRKYs identified from genome of P. mume. They were anchored onto eight link groups and categorised into three broad groups. The gene structure and motif composition were reasonably conservative in each group. Investigation of gene duplication indicated that nine and seven PmWRKYs were arranged in tandem and segmental duplications, respectively. PmWRKYs were discriminately expressed in different tissues (i.e., roots, stems, leaves, flowers and fruits) in P. mume. The 17 cold-related candidate genes were selected based on RNA-seq data. Further, to investigate the function of PmWRKYs in low temperatures, the expression patterns under artificial cold treatments were analysed. The results showed that the expression levels of the 12 PmWRKYs genes significantly and 5 genes slightly changed in stems. In particular, the expression level of PmWRKY18 was up-regulated after ABA treatment. In addition, the spatiotemporal expression patterns of 17 PmWRKYs were analysed in winter. These results indicated that 17 PmWRKYs were potential transcription factors regulating cold resistance in P. mume.

Genome-wide identification, characterization, expression and enzyme activity analysis of coniferyl alcohol acetyltransferase genes involved in eugenol biosynthesis in Prunus mume.

Prunus mume, a traditional Chinese flower, is the only species of Prunus known to produce a strong floral fragrance, of which eugenol is one of the principal components. To explore the molecular mechanism of eugenol biosynthesis in P. mume, patterns of dynamic, spatial and temporal variation in eugenol were analysed using GC-MS. Coniferyl alcohol acetyltransferase (CFAT), a member of the BAHD acyltransferase family, catalyses the substrate of coniferyl alcohol to coniferyl acetate, which is an important substrate for synthesizing eugenol. In a genome-wide analysis, we found 90 PmBAHD genes that were phylogenetically clustered into five major groups with motif compositions relatively conserved in each cluster. The phylogenetic tree showed that the PmBAHD67-70 proteins were close to the functional CFATs identified in other species, indicating that these four proteins might function as CFATs. In this work, 2 PmCFAT genes, named PmCFAT1 and PmCFAT2, were cloned from P. mume 'Sanlunyudie', which has a strong fragrance. Multiple sequences indicated that PmCFAT1 contained two conserved domains, HxxxD and DFGWG, whereas DFGWG in PmCFAT2 was changed to DFGFG. The expression levels of PmCFAT1 and PmCFAT2 were examined in different flower organs and during the flowering stages of P. mume 'Sanlunyudie'. The results showed that PmCFAT1 was highly expressed in petals and stamens, and this expression increased from the budding stage to the full bloom stage and decreased in the withering stage, consistent with the patterns of eugenol synthesis and emission. However, the peak of gene expression appeared earlier than those of eugenol synthesis and emission. In addition, the expression level of PmCFAT2 was higher in pistils and sepals than in other organs and decreased from the budding stage to the blooming stage and then increased in the withering stage, which was not consistent with eugenol synthesis. Subcellular localization analysis indicated that PmCFAT1 and PmCFAT2 were located in the cytoplasm and nucleus, while enzyme activity assays showed that PmCFAT1 is involved in eugenol biosynthesis in vitro. Overall, the results suggested that PmCFAT1, but not PmCFAT2, contributed to eugenol synthesis in P. mume.

Expansion of PmBEAT genes in the Prunus mume genome induces characteristic floral scent production.

Prunus mume is the only plant in the genus Prunus of the Rosaceae family with a characteristic floral scent, and the main component of this scent is benzyl acetate. By contrast, benzyl acetate is not synthesized in Prunus persica flowers. Here, we searched for benzyl alcohol acetyltransferase (BEAT) genes based on genomic data from P. mume and P. persica and found 44 unique PmBEATs in P. mume. These genes, which were mainly detected in clusters on chromosomes, originated from gene duplication events during the species evolution of P. mume, and retroduplication and tandem duplication were the two dominant duplication patterns. The genes PmBEAT34, PmBEAT36 and PmBEAT37, which were generated by tandem duplication, were highly expressed in flowers, and their highest levels were detected during the blooming stage. In vitro, PmBEAT34, PmBEAT3, and PmBEAT37 all had benzyl alcohol acetyltransferase activity that was localized in the cytoplasm. Overexpression of the PmBEAT36 or PmBEAT37 genes increased benzyl acetate production in the petal protoplasts of P. mume, and interference in the expression of these genes slightly decreased the benzyl acetate content. In addition, light and temperature regulated the expression of the PmBEAT34, PmBEAT36 and PmBEAT37 genes. According to these results, we hypothesize that the expansion of the PmBEAT genes in the genome induce the characteristic floral scent of P. mume.

A Comparative Analysis of Floral Scent Compounds in Intraspecific Cultivars of Prunus mume with Different Corolla Colours.

Prunus mume is the only fragrant flowering species of Prunus. According to the previous studies, benzyl acetate and eugenol dominate its floral scent. However, the diversity of its floral scents remains to be elucidated. In this work, the floral volatiles emitted from eight intraspecific cultivars of P. mume with white, pink and red flowers, were collected and analyzed using headspace solid-phase microextraction combined with gas chromatograms-mass spectrometry (HS-SPME-GC-MS). In total, 31 volatile compounds were identified, in which phenylpropanoids/benzenoids accounted for over 95% of the total emission amounts. Surprisingly, except for benzyl acetate and eugenol, several novel components, such as benzyl alcohol, cinnamyl acohol, cinnamy acetate, and benzyl benzoate were found in some cultivars. The composition of floral volatiles in cultivars with white flowers was similar, in which benzyl acetate was dominant, while within pink flowers, there were differences of floral volatile compositions. Principal component analysis (PCA) showed that the emissions of benzyl alcohol, cinnamyl alcohol, benzyl acetate, eugenol, cinnamyl acetate, and benzyl benzoate could make these intraspecific cultivars distinguishable from each other. Further, hierarchical cluster analysis indicated that cultivars with similar a category and amount of floral compounds were grouped together. Our findings lay a theoretical basis for fragrant plant breeding in P. mume.