Chromosome-level assembly of Lindenbergia philippensis and comparative genomic analyses shed light on genome evolution in Lamiales.

Lamiales, comprising over 23,755 species across 24 families, stands as a highly diverse and prolific plant group, playing a significant role in the cultivation of horticultural, ornamental, and medicinal plant varieties. Whole-genome duplication (WGD) and its subsequent post-polyploid diploidization (PPD) process represent the most drastic type of karyotype evolution, injecting significant potential for promoting the diversity of this lineage. However, polyploidization histories, as well as genome and subgenome fractionation following WGD events in Lamiales species, are still not well investigated. In this study, we constructed a chromosome-level genome assembly of Lindenbergia philippensis (Orobanchaceae) and conducted comparative genomic analyses with 14 other Lamiales species. L. philippensis is positioned closest to the parasitic lineage within Orobanchaceae and has a conserved karyotype. Through a combination of Ks analysis and syntenic depth analysis, we reconstructed and validated polyploidization histories of Lamiales species. Our results indicated that Primulina huaijiensis underwent three rounds of diploidization events following the γ-WGT event, rather than two rounds as reported. Besides, we reconfirmed that most Lamiales species shared a common diploidization event (L-WGD). Subsequently, we constructed the Lamiales Ancestral Karyotype (LAK), comprising 11 proto-chromosomes, and elucidated its evolutionary trajectory, highlighting the highly flexible reshuffling of the Lamiales paleogenome. We identified biased fractionation of subgenomes following the L-WGD event across eight species, and highlighted the positive impacts of non-WGD genes on gene family expansion. This study provides novel genomic resources and insights into polyploidy and karyotype remodeling of Lamiales species, essential for advancing our understanding of species diversification and genome evolution.

Unveiling the effect of gibberellin-induced iron oxide nanoparticles on bud dormancy release in sweet cherry (Prunus avium L.).

Hydrogen cyanide has been extensively used worldwide for bud dormancy break in fruit trees, consequently enhancing fruit production via expedited cultivation, especially in areas with controlled environments or warmer regions. A novel and safety nanotechnology was developed since the hazard of hydrogen cyanide for the operators and environments, there is an urgent need for the development of novel and safety approaches to replace it to break bud dormancy for fruit trees. In current study, we have systematically explored the potential of iron oxide nanoparticles, specifically α-Fe2O3, to modulate bud dormancy in sweet cherry (Prunus avium). The synthesized iron oxide nanoparticles underwent meticulous characterization and assessment using various techniques, including Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), and ultraviolet-visible infrared (UV-Vis) spectroscopy. Remarkably, when applied at a concentration of 10 mg L-1 alongside gibberellin (GA4+7), these iron oxide nanoparticles exhibited a substantial 57% enhancement in bud dormancy release compared to control groups, all achieved within a remarkably short time span of 4 days. Our RNA-seq analyses further unveiled that 2757 genes within the sweet cherry buds were significantly up-regulated when treated with 10 mg L-1 α-Fe2O3 nanoparticles in combination with GA, while 4748 genes related to dormancy regulation were downregulated in comparison to the control. Moreover, we discovered an array of 58 transcription factor families among the crucial differentially expressed genes (DEGs). Through hormonal quantification, we established that the increased bud burst was accompanied by a reduced concentration of abscisic acid (ABA) at 761.3 ng/g fresh weight in the iron oxide treatment group, coupled with higher levels of gibberellins (GAs) in comparison to the control. Comprehensive transcriptomic and metabolomic analyses unveiled significant alterations in hormone contents and gene expression during the bud dormancy-breaking process when α-Fe2O3 nanoparticles were combined with GA. In conclusion, our findings provide valuable insights into the intricate molecular mechanisms underlying the impact of iron oxide nanoparticles on achieving uniform bud dormancy break in sweet cherry trees.

Evolutionary and Integrative Analysis of Gibberellin-Dioxygenase Gene Family and Their Expression Profile in Three Rosaceae Genomes (F. vesca, P. mume, and P. avium) Under Phytohormone Stress.

The gibberellin-dioxygenase (GAox) gene family plays a crucial role in regulating plant growth and development. GAoxs, which are encoded by many gene subfamilies, are extremely critical in regulating bioactive GA levels by catalyzing the subsequent stages in the biosynthesis process. Moreover, GAoxs are important enzymes in the GA synthesis pathway, and the GAox gene family has not yet been identified in Rosaceae species (Prunus avium L., F. vesca, and P. mume), especially in response to gibberellin and PCa (prohexadione calcium; reduce biologically active GAs). In the current investigation, 399 GAox members were identified in sweet cherry, Japanese apricot, and strawberry. Moreover, they were further classified into six (A-F) subgroups based on phylogeny. According to motif analysis and gene structure, the majority of the PavGAox genes have a remarkably well-maintained exon-intron and motif arrangement within the same subgroup, which may lead to functional divergence. In the systematic investigation, PavGAox genes have several duplication events, but segmental duplication occurs frequently. A calculative analysis of orthologous gene pairs in Prunus avium L., F. vesca, and P. mume revealed that GAox genes are subjected to purifying selection during the evolutionary process, resulting in functional divergence. The analysis of cis-regulatory elements in the upstream region of the 140 PavGAox members suggests a possible relationship between genes and specific functions of hormone response-related elements. Moreover, the PavGAox genes display a variety of tissue expression patterns in diverse tissues, with most of the PavGAox genes displaying tissue-specific expression patterns. Furthermore, most of the PavGAox genes express significant expression in buds under phytohormonal stresses. Phytohormones stress analysis demonstrated that some of PavGAox genes are responsible for maintaining the GA level in plant-like Pav co4017001.1 g010.1.br, Pav sc0000024.1 g340.1.br, and Pav sc0000024.1 g270.1.mk. The subcellular localization of PavGAox protein utilizing a tobacco transient transformation system into the tobacco epidermal cells predicted that GFP signals were mostly found in the cytoplasm. These findings will contribute to a better understanding of the GAox gene family's interaction with prohexadione calcium and GA, as well as provide a strong framework for future functional characterization of GAox genes in sweet cherry.

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

The role of VvMYBA2r and VvMYBA2w alleles of the MYBA2 locus in the regulation of anthocyanin biosynthesis for molecular breeding of grape (Vitis spp.) skin coloration.

In grape, MYBA1 and MYBA2 at the colour locus are the major genetic determinants of grape skin colour, and the mutation of two functional genes (VvMYBA1 and VvMYBA2) from these loci leads to white skin colour. This study aimed to elucidate the regulation of grape berry coloration by isolating and characterizing VvMYBA2w and VvMYBA2r alleles. The overexpression of VvMYBA2r up-regulated the expression of anthocyanin biosynthetic genes and resulted in higher anthocyanin accumulation in transgenic tobacco than wild-type (WT) plants, especially in flowers. However, the ectopic expression of VvMYBA2w inactivated the expression of anthocyanin biosynthetic genes and could not cause obvious phenotypic modulation in transgenic tobacco. Unlike in VvMYBA2r, CA dinucleotide deletion shortened the C-terminal transactivation region and disrupted the transcriptional activation activity of VvMYBA2w. The results indicated that VvMYBA2r positively regulated anthocyanin biosynthesis by forming the VvMYBA2r-VvMYCA1-VvWDR1 complex, and VvWDR1 enhanced anthocyanin accumulation by interacting with the VvMYBA2r-VvMYCA1 complex; however, R44 L substitution abolished the interaction of VvMYBA2w with VvMYCA1. Meanwhile, both R44 L substitution and CA dinucleotide deletion seriously affected the efficacy of VvMYBA2w to regulate anthocyanin biosynthesis, and the two non-synonymous mutations were additive in their effects. Investigation of the colour density and MYB haplotypes of 213 grape germplasms revealed that dark-skinned varieties tended to contain HapC-N and HapE2, whereas red-skinned varieties contained high frequencies of HapB and HapC-Rs. Regarding ploidy, the higher the number of functional alleles present in a variety, the darker was the skin colour. In summary, this study provides insight into the roles of VvMYBA2r and VvMYBA2w alleles and lays the foundation for the molecular breeding of grape varieties with different skin colour.

Combined Application of Bacteriophages and Carvacrol in the Control of Pseudomonas syringae pv. actinidiae Planktonic and Biofilm Forms.

Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of the bacterial canker of kiwifruit (Actinidia spp.). Phage therapy has been suggested as a viable alternative approach to controlling this disease, but its efficacy is limited by the emergence of phage-resistant mutants. Carvacrol is an essential oil that may be useful for the control of Psa. Combination therapies can be used to overcome resistance development. Here, the combination of phages (single phage suspensions of phages PN05 and PN09, and a cocktail of both phages) and carvacrol was investigated in controlling Psa planktonic and biofilm forms in vitro. The phage therapy alone (with phages PN05 and PN09), and the carvacrol alone (minimum inhibitory concentration 2.0 mg/mL), inhibited Psa growth, but the combined effect of both therapies was more effective. The phages alone effectively inhibited Psa growth for 24 h, but Psa regrowth was observed after this time. The carvacrol (2.0 mg/mL) alone prevented the biofilm formation for 48 h, but did not destroy the pre-formed biofilms. The combined treatment, phages and carvacrol (2.0 mg/mL), showed a higher efficacy, preventing Psa regrowth for more than 40 h. In conclusion, the combined treatment with phages and carvacrol may be a promising, environment-friendly and cost-effective approach to controlling Psa in the kiwifruit industry.

VvmiR160s/VvARFs interaction and their spatio-temporal expression/cleavage products during GA-induced grape parthenocarpy.

BACKGROUND: Grape (Vitis vinifera) is highly sensitive to gibberellin (GA), which effectively induce grape parthenocarpy. Studies showed that miR160s and their target AUXIN RESPONSIVE FACTOR (ARF) responding hormones are indispensable for various aspects of plant growth and development, but their functions in GA-induced grape parthenocarpy remain elusive. RESULTS: In this study, the morphological changes during flower development in response to GA treatments were examined in the 'Rosario Bianco' cultivar. The precise sequences of VvmiR160a/b/c/d/e and their VvARF10/16/17 target genes were cloned, sequenced and characterized. The phylogenetic relationship and intron-exon structure of VvARFs and other ARF family members derived from different species were investigated. All VvmiR160s (except VvmiR160b) and VvARF10/16/17 had the common cis-elements responsive to GA, which support their function in GA-mediated grape parthenocarpy. The cleavage role of VvmiR160s-mediated VvARF10/16/17 was verified in grape flowers. Moreover, spatio-temporal expression analysis demonstrated that among VvmiR160 family, VvmiR160a/b/c highly expressed at late stage of flower/berry development, while VvARF10/16/17showed a reverse expression trend. Interestingly, GA exhibited a long-term effect through inducing the expression of VvmiR160a/b/c/e to increase their cleavage product accumulations from 5 to 9 days after treatment, but GA enhanced the expressions of VvARF10/16/17 only at short term. Pearson correlation analysis based on expression data revealed a negative correlation between VvmiR160a/b/c and VvARF10/16/17 in flowers not berries during GA-induced grape parthenocarpy. CONCLUSIONS: This work demonstrated that the negative regulation of VvARF10/16/17 expression by VvmiR160a/b/c as key regulatory factors is critical for GA-mediated grape parthenocarpy, and provide significant implications for molecular breeding of high-quality seedless berry.

Genome-Wide Identification, Characterization, and Transcript Analysis of the TCP Transcription Factors in Vitis vinifera.

The TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) protein, belonging to a plant-specific transcription factors (TFs) family, participates in the control of plant growth and development by regulating cell proliferation. Until now, a comprehensive study of concerning the TCP gene family and their roles in grapevine (Vitis vinifera L.) has not been completed. Using bioinformatics approaches, 17 VvTCP genes were identified and further classified into two classes, designated class I (PCF subclass) and class II (CIN and CYC/TB1 subclass), which was further supported by exon-intron organizations and conserved motif analysis. Promoter analysis demonstrated that VvTCPs have numerous cis-acting elements related to plant growth and development, phytohormone, and abiotic/biotic stress responses. The singleton duplication of grapevine TCP genes contributed to this gene family expansion. The syntenic analyses among Vitis vinifera, Arabidopsis, and Oryza sativa showed that these genes located in corresponding syntenic blocks arose before the divergence of V. vinifera, Arabidopsis, and O. sativa. The expression levels of 17 VvTCPs were determined in different tissues and fruit developmental stages, and abscisic acid (ABA) treatment. Seventeen VvTCPs exhibited distinct tissue-specific expression patterns, potentially illustrating the functional divergence of VvTCPs in all tested tissues. Eleven VvTCPs were down-regulated in five berry developmental stages, while three VvTCPs were up-regulated. Additionally, many members were strongly modulated by ABA treatment, suggesting these VvTCPs have important and diverse regulatory roles in ABA treatment. Our results provide valuable information on the evolution and functions of the VvTCPs, pave the way for further functional verification of these VvTCPs in grapevine.

Characterization of VvPAL-like promoter from grapevine using transgenic tobacco plants.

A 2000-bp 5'-flanking region of VvPAL-like was isolated from 'Summer Black' grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the ß-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5' progressive deletions of the promoter revealed the presence of a negative regulatory region (-424 to -292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) (N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the -1461/-930 fragment, while the element(s) for the MeJA-responsive expression may be present in the -424/-292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.