AtRKD5 inhibits the parthenogenic potential mediated by AtBBM.

Parthenogenesis, the development of unfertilized egg cells into embryos, is a key component of apomixis. AtBBM (BABY BOOM), a crucial regulator of embryogenesis in Arabidopsis, possesses the capacity to shift nutritional growth toward reproductive growth. However, the mechanisms underlying AtBBM-induced parthenogenesis remain largely unexplored in dicot plants. Our findings revealed that in order to uphold the order of sexual reproduction, the embryo-specific promoter activity of AtBBM as well as repressors that inhibit its expression in egg cells combine to limiting its ability to induce parthenogenesis. Notably, AtRKD5, a RWP-RK domain-containing (RKD) transcription factor, binds to the 3' end of AtBBM and is identified as one of the inhibitory factors for AtBBM expression in the egg cell. In the atrkd5 mutant, we successfully achieved enhanced ectopic expression of AtBBM in egg cells, resulting in the generation of haploid offspring via parthenogenesis at a rate of 0.28%. Furthermore, by introducing chimeric Arabidopsis and rice BBM genes into the egg cell, we achieved a significant 4.6-fold enhancement in haploid induction through the atdmp8/9 mutant. These findings lay a strong foundation for further exploration of the BBM-mediated parthenogenesis mechanism and the improvement of haploid breeding efficiency mediated by the dmp8/9 mutant.

Viromics unveils extraordinary genetic diversity of the family Closteroviridae in wild citrus.

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production.

Complete genome sequence of Edgeworthia chrysantha mosaic-associated virus, a tentative new member of the genus Coguvirus (family Phenuiviridae).

A new negative-strand RNA (nsRNA) virus genome was discovered in Edgeworthia chrysantha Lindl. This virus, tentatively named "Edgeworthia chrysantha mosaic-associated virus" (ECMaV), has a bipartite genome that comprises (i) a nsRNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the open reading frames separated by a long AU-rich intergenic region (IR). Sequence comparisons and phylogenetic analysis showed that the RdRp is closely related to those of other recently discovered plant-infecting nsRNA viruses in the new genus Coguvirus and that ECMaV can be classified as a member of a novel species.

Genome wide analysis of kinesin gene family in Citrullus lanatus reveals an essential role in early fruit development.

BACKGROUND: Kinesin (KIN) as a motor protein is a versatile nano-machine and involved in diverse essential processes in plant growth and development. However, the kinesin gene family has not been identified in watermelon, a valued and nutritious fruit, and yet their functions have not been characterized. Especially, their involvement in early fruit development, which directly determines the size, shape, yield and quality of the watermelon fruit, remains unclear. RESULTS: In this study, we performed a whole-genome investigation and comprehensive analysis of kinesin genes in C. lanatus. In total, 48 kinesins were identified and categorized into 10 kinesin subfamilies groups based on phylogenetic analysis. Their uneven distribution on 11 chromosomes was revealed by distribution analysis. Conserved motif analysis showed that the ATP-binding motif of kinesins was conserved within all subfamilies, but not the microtubule-binding motif. 10 segmental duplication pairs genes were detected by the syntenic and phylogenetic approaches, which showed the expansion of the kinesin gene family in C. lanatus genome during evolution. Moreover, 5 ClKINs genes are specifically and abundantly expressed in early fruit developmental stages according to comprehensive expression profile analysis, implying their critical regulatory roles during early fruit development. Our data also demonstrated that the majority of kinesin genes were responsive to plant hormones, revealing their potential involvement in the signaling pathways of plant hormones. CONCLUSIONS: Kinesin gene family in watermelon was comprehensively analyzed in this study, which establishes a foundation for further functional investigation of C. lanatus kinesin genes and provides novel insights into their biological functions. In addition, these results also provide useful information for understanding the relationship between plant hormone and kinesin genes in C. lanatus.

Molecular characterization of a novel cytorhabdovirus associated with chrysanthemum yellow dwarf disease.

Deep-sequencing analysis of a chrysanthemum plant with yellow dwarf symptoms led to the discovery of a novel putative cytorhabdovirus, here tentatively named "chrysanthemum yellow dwarf associated virus" (CYDaV). Its negative-sense single-stranded RNA genome comprises 14,086 nucleotides and contains eight open reading frames in the order 3' leader-N-P'-P-P3-M-G-P6-L-5' trailer. CYDaV shares moderate sequence similarity (< 54.2% nucleotide and 51% amino acid sequence identity) with its cytorhabdovirus counterparts in cognate genes. Phylogenetic analysis showed that CYDaV clustered with strong support with alfalfa dwarf virus, raspberry vein chlorosis virus, and strawberry crinkle virus. These findings suggest that CYDaV should be considered a novel member of the genus Cytorhabdovirus, family Rhabdoviridae.

Complete genome sequence of a novel citrus virus with characteristics of members of the family Tymoviridae.

A novel positive-stranded RNA virus provisionally named "citrus virus C" (CVC) was discovered in citrus trees displaying mottling symptoms. Its genome comprises 7,215 nucleotides (nt), excluding the 3' poly(A) tail, and contains two open reading frames (ORFs) that encode a replication-associated polyprotein (RP) and a putative coat protein (CP). The CVC genome contains a 16-nt 'marafibox', which is highly conserved in most viruses belonging to the genus Marafivirus of the same family. Sequence analysis suggested that the virus is most closely related to grapevine Red Globe virus (GRGV), which is yet to be officially classified in the family Tymoviridae. The sequence identities between CVC and GRGV in the whole genome (50.7%, nt) and CP (49.4% for amino acid, and 53.9% for nt) are lower than the thresholds (80% in the genome and 90% in the CP) for species demarcation in the family. Therefore, it is legitimate to propose that CVC is a member of new species in the family Tymoviridae.

Complete nucleotide sequence of loquat virus A, a member of the family Betaflexiviridae with a novel genome organization.

Analysis of a loquat tree with leaf curl symptoms by deep sequencing revealed a novel virus with a single-stranded RNA genome, for which the name "loquat virus A" (LoVA) was proposed. The complete genome sequence comprised 7553 nucleotides (nt) and an additional poly(A) tail at the 3' terminus. Sequence comparisons of LoVA showed moderate similarity to cherry virus A (CVA), currant virus A (CuVA), and mume virus A (MuVA), which are members of the genus Capillovirus in the family Betaflexiviridae. Phylogenetic analysis of the full-genome nt sequence and replicase-like protein supported the placement of LoVA within the genus Capillovirus. However, it has a distinct genome organization, differing from recognized capilloviruses, as it contains three open reading frames (ORFs), with the coat protein (CP) expressed separately from the replication-associated protein (RP) rather than being encoded in the same ORF. This indicates that LoVA is a novel member of the genus Capillovirus in the family Betaflexiviridae with a distinct genomic organization.

Synthesis, characterization and biological activity of novel copper complexes containing a ß-carboline derivative and amino acids.

Copper complexes have long been considered as a promising class of anticancer or antibacterial therapeutics. In this paper, two novel copper(II) complexes containing a ß-carboline derivative and amino acids, namely [Cu(1-Im-ßc)(L-Val)]ClO4·0.5H2O (Cu1) and [Cu(1-Im-ßc)(L-Phe)]ClO4·0.5H2O (Cu2), where 1-Im-ßc = 1-(2-imidazolyl)-ß-carboline, L-Val = L-valine, and L-Phe = L-phenylalanine, were designed and synthesized. The complexes were characterized by elemental analysis, infrared spectroscopy, molar conductivity measurements, and mass spectrometry to determine their spatial structures and compositions. Both complexes bind to DNA by insertion. The complexes also show a good affinity for human serum albumin (HSA). In addition, the antitumor activity of the two complexes against lung cancer cells (A549), cervical cancer cells (HeLa), and breast cancer cells (MBA-MD-231) is significantly superior to that of the traditional antitumor drug, cisplatin. Finally, the anticancer mechanism results show that the complexes can induce apoptosis in HeLa cells, which is associated with mitochondrial damage, oxidative stress caused by reactive oxygen species (ROS) production, and activation of the caspase protein family. This study demonstrates that the introduction of aromatic heterocyclic alkaloid ligands with a broad spectrum of biological activities and water-soluble amino acid ligands into copper complexes can regulate their amphiphilic properties and biological activity, so as to obtain highly efficient copper-based therapeutics.

A novel water-soluble Cu(II) gluconate complex inhibits cancer cell growth by triggering apoptosis and ferroptosis related mechanisms.

Metal copper complexes have attracted extensive attention as potential alternatives to platinum-based anticancer drugs due to their possible different modes of action. Herein, a new copper(II) gluconate complex, namely [Cu(DPQ)(Gluc)]·2H2O (CuGluc, DPQ = pyrazino[2,3-f][1,10]phenanthroline), with good water-solubility and high anticancer activity was synthesized by using D-gluconic acid (Gluc-2H) as an auxiliary ligand. The complex was well characterized by single-crystal X-ray diffraction analysis, elemental analysis, molar conductivity, and Fourier transform infrared spectroscopy (FTIR). The DNA-binding experiments revealed that CuGluc was bound to DNA by intercalation with end-stacking binding. CuGluc could oxidatively cleave DNA, in which 1O2 and H2O2 were involved. In addition, CuGluc was bound to the IIA subdomain of human serum albumin (HSA) through hydrophobic interaction and hydrogen bonding, showing a good affinity for HSA. The complex showed superior anticancer activity toward several cancer cells than cisplatin in vitro. Further studies indicated that CuGluc caused apoptotic cell death in human liver cancer (HepG2) cells through elevated intracellular reactive oxygen species (ROS) levels, mitochondrial dysfunction, cell cycle arrest, and caspase activation. Interestingly, CuGluc also triggered the ferroptosis mechanism through lipid peroxide accumulation and inhibition of glutathione peroxidase 4 (GPX4) activity. More importantly, CuGluc significantly inhibited tumor growth in vivo, which may benefit from the combined effects of apoptosis and ferroptosis. This work provides a promising strategy to develop highly effective antitumor copper complexes by coordinating with the glucose metabolite D-gluconic acid and exploiting the synergistic effects of apoptosis and ferroptosis mechanisms.

Effects of Oregano Essential Oil on IgA+, IgG+, and IgM+ Cells in the Jejunum of Castrated Holstein Bulls.

The aim of this study was to investigate the effect of oregano essential oil on IgA+, IgG+, and IgM+ cells in the jejunum of castrated Holstein bulls. Twelve castrated Holstein bulls were randomly divided into control (YCK) and oregano essential oil (YEO) groups. Pathological changes in the jejunum were observed by HE staining, and the expression levels of IgA, IgG, and IgM in the jejunum were detected by ELISA. The distributions of IgA+, IgG+, and IgM+ cells in the jejunum were analysed by multiplex immunofluorescence and immunohistochemistry. The results showed that the jejunal villi were detached in the YCK group, which may have been related to inflammation, while the intestinal epithelium was clear and intact in the YEO group. The expressions of IgA, IgG, and IgM were significantly reduced by 40.75%, 30.76%, and 50.87%. The IgA+, IgG+, and IgM+ cells were diffusely distributed in the lamina propria of the jejunum, and were reduced by 17.07%, 6.44%, and 6.15%, respectively. Oregano essential oil did not alter the distribution characteristics of IgA+, IgG+, or IgM+ cells in the jejunum, but it suppressed inflammatory response, decreased immunoglobulin content, and significantly enhanced the formation of an immune barrier in the gastrointestinal mucosa.

Synthesis, structural studies, interaction with DNA/HSA and antitumor evaluation of new Cu(II) complexes containing 2-(1H-imidazol-2-yl)pyridine and amino acids.

Copper complexes are considered as potential candidates for anticancer therapy and medical applications. In this paper, three new Cu(II) complexes, [Cu(IPY)2](ClO4)2·H2O (CuI1), [Cu(IPY)(L-Phe)H2O]ClO4·0.5H2O (CuI2) and [Cu(IPY)(L-Val)H2O]ClO4 (CuI3) (where IPY = 2-(1H-imidazol-2-yl)pyridine, L-Phe = L-phenylalanine, and L-Val = L-valine), with good amphipathic properties were synthesized and characterized. Their single crystal X-ray diffraction results revealed that CuI1 was four-coordinated, while CuI2 and CuI3 both adopted a five-coordinated tetragonal pyramidal configuration. Multi-spectral methods, viscosity experiment and molecular docking technique showed that the three complexes interacted with DNA through insertion. The results of the gel electrophoresis experiments indicated that DNA was oxidatively cleaved by all the complexes in a concentration-dependent manner. Moreover, singlet oxygen (1O2), hydrogen peroxide (H2O2) and superoxide anion radicals (˙O2-) were associated with the oxidative cleavage of DNA. All the complexes also had good binding affinity with human serum albumin (HSA). The MB degradation assay revealed that all complexes could react with H2O2 to form ˙OH through Fenton-like processes. The complexes displayed good antiproliferative activity against the tested human cancer cells in vitro, including cervical carcinoma cells (HeLa), liver cancer cells (HepG2 and BEL-7402) and gastric adenocarcinoma cells (SGC-7901), but showed lower toxicity to normal liver cells (LO2). The anticancer mechanism research revealed that CuI1, CuI2 and CuI3 arrested the cell cycle at the S phase, elevated intracellular reactive oxygen species (ROS) levels and induced loss of mitochondrial membrane potential (MMP). The results indicated that these Cu(II) complexes could induce DNA damage and ROS-mediated mitochondrial dysfunction, leading to cancer cell apoptosis. Our work provides a theoretical basis for the design of new low-toxicity and highly efficient anticancer Cu(II) complexes by incorporating biological metabolites and aromatic heterocyclic ligands.

Sparfloxacin - Cu(II) - aromatic heterocyclic complexes: synthesis, characterization and in vitro anticancer evaluation.

Two new copper(II) complexes of sparfloxacin (sf), [Cu(Hsf)(HPB)(H2O)](ClO4)2 (1) and [Cu(Hsf)(PBT)(H2O)](ClO4)2 (2) (where HPB = 2-(2'-pyridyl)benzimidazole and PBT = 2-(4'-pyridyl) benzothiazole), have been synthesized and characterized by physicochemical and spectroscopic techniques. The oil-water partition coefficient (log P) values of complexes 1 and 2 were 1.47 and 1.71, respectively. By studying the interaction between the complexes and DNA, it was found that the complexes could bind to DNA through an intercalation mode. Moreover, both complexes were evaluated for antitumor activity, revealing that the complexes displayed good inhibitory activity toward the tested cancer cell lines (human lung carcinoma A549 cells, human hepatocellular carcinoma Bel-7402 cells and human esophageal carcinoma Eca-109 cells), but showed relatively low toxicity against normal human hepatic LO2 cells. In particular, the antitumor mechanism of the complexes on Eca-109 cells was investigated by morphological analysis, apoptosis analysis and determination of cell cycle arrest, mitochondrial membrane potential, reactive oxygen species (ROS) levels, and release of cytochrome c and Ca2+. The results demonstrated that the complexes could induce loss of intracellular mitochondrial functions and increase of ROS levels, which led to an increase of Ca2+ levels and the release of cytochrome c into the cytoplasm. In addition, the cell cycle was arrested in the G2/M phase, and western blot analysis showed that the caspase family was activated. These results fully proved that the complexes could induce apoptosis through DNA damage and loss of mitochondrial functions, accompanied by the regulation of endogenous proteins.

Synthesis, DNA binding, antibacterial and anticancer properties of two novel water-soluble copper(II) complexes containing gluconate.

In this paper, two new Cu(II) complexes, [Cu(Gluc)(HPB)(H2O)]Gluc (CuG1) and [Cu(Gluc)(HPBC)(H2O)]Gluc (CuG2) (where HPB = 2-(2'-pyridyl)benzimidazole, HPBC = 5-chloro-2-(2'-pyridyl)benzimidazole, Gluc = d-Gluconic acid), with good water solubility were synthesized and characterized. These complexes exhibited a five-coordinated tetragonal pyramidal geometry. The DNA binding and cleavage properties of the complexes were investigated using multi-spectroscopy, viscosity measurement, molecular docking and gel electrophoresis analysis methods. The results showed that the complexes could interact with DNA by insertion and groove binding, and cleave CT-DNA through a singlet oxygen-dependent pathway in the presence of ascorbic acid. The studies on antibacterial and anticancer activities in vitro demonstrated that both complexes had good inhibitory activity against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes) and one Gram-negative bacterium (Escherichia coli) and good cytotoxic activity toward the tested cancer cells (A549, HeLa and SGC-7901). CuG2 showed higher antimicrobial and cytotoxic activities than CuG1, which was consistent with their binding strength and cleavage ability to DNA, indicating that their antimicrobial and cytotoxic activities may be related to the DNA interaction. Moreover, the cell-based mechanism studies have indicated that CuG1 and CuG2 could arrest the cell cycle at G2/M phase, elevate the levels of intracellular reactive oxygen species (ROS) and decrease the mitochondrial membrane potential (MMP). The results showed that the complexes could induce apoptosis through DNA-damaged and ROS-mediated mitochondrial dysfunction pathways. Finally, the in vivo antitumor study revealed that CuG2 inhibited tumor growth by 50.44%, which is better than that of cisplatin (40.94%).

A 2.09 Mb fragment translocation on chromosome 6 causes abnormalities during meiosis and leads to less seed watermelon.

Seedlessness is a valuable agronomic trait in watermelon (Citrullus lanatus) breeding. Conventional less seed watermelons are mainly triploid, which has many disadvantages due to unbalanced genome content. Less seed watermelon can be achieved at the diploid level when certain reproductive genes are mutated or by chromosome translocation, which leads to defects during meiosis. However, the formation mechanism of diploid less seed watermelons remains largely unknown. Here, we identified a spontaneous mutant line, watermelon line "148", which can set seeds normally when self-pollinated. A total of 148 × JM F1 hybrid plants exhibited seed number reductions to 50.3% and 47.3% of those of the two parental lines, respectively, which are considered to be less seed. Examination of pollen viability and hybridization experiments revealed that F1 hybrids produce semisterile pollen and ovules. Further cytological observations indicated that semisterility was a result of a reciprocal translocation of chromosomes, which exhibited one quadrivalent ring of four chromosomes at prometaphase I during meiosis. RT-qPCR analysis indirectly confirmed that the semisterile phenotype is caused by chromosome translocation rather than disruption of specific meiotic gene expression. F2 population genetic analysis indicated that the "148" watermelon line is a homozygous translocation and that the less seed phenotype of the F1 hybrid is prompted by one chromosome fragment translocation. The translocated fragment was further fine mapped to a 2.09 Mb region on chromosome 6 by whole-genome resequencing and genetic map cloning procedures. Our work revealed that a 2.09 Mb chromosome fragment translocation on chromosome 6, causing meiotic defects at metaphase I during meiosis, leads to diploid less seed watermelon. Our findings provide a new promising method for less seed watermelon breeding at the diploid level, as well as a fragment size reference for breeding less seed watermelon through artificially induced chromosome translocation.

Genetic mapping of the LOBED LEAF 1 (ClLL1) gene to a 127.6-kb region in watermelon (Citrullus lanatus L.).

The lobed leaf character is a unique morphologic trait in crops, featuring many potential advantages for agricultural productivity. Although the majority of watermelon varieties feature lobed leaves, the genetic factors responsible for lobed leaf formation remain elusive. The F2:3 leaf shape segregating population offers the opportunity to study the underlying mechanism of lobed leaf formation in watermelon. Genetic analysis revealed that a single dominant allele (designated ClLL1) controlled the lobed leaf trait. A large-sized F3:4 population derived from F2:3 individuals was used to map ClLL1. A total of 5,966 reliable SNPs and indels were identified genome-wide via a combination of BSA and RNA-seq. Using the validated SNP and indel markers, the location of ClLL1 was narrowed down to a 127.6-kb region between markers W08314 and W07061, containing 23 putative ORFs. Expression analysis via qRT-PCR revealed differential expression patterns (fold-changes above 2-fold or below 0.5-fold) of three ORFs (ORF3, ORF11, and ORF18) between lobed and non-lobed leaf plants. Based on gene annotation and expression analysis, ORF18 (encoding an uncharacterized protein) and ORF22 (encoding a homeobox-leucine zipper-like protein) were considered as most likely candidate genes. Furthermore, sequence analysis revealed no polymorphisms in cDNA sequences of ORF18; however, two notable deletions were identified in ORF22. This study is the first report to map a leaf shape gene in watermelon and will facilitate cloning and functional characterization of ClLL1 in future studies.

Local melatonin application induces cold tolerance in distant organs of Citrullus lanatus L. via long distance transport.

Melatonin is a ubiquitous chemical substance that regulates plant growth and responses to stress. Several recent studies show that exogenous melatonin confers cold tolerance to plants; however, the underlying mechanisms remain largely unknown. Here, we report that melatonin application at optimal dose, either on the leaves or the roots, not only induced cold stress tolerance in the site of application, but also systemically induced cold tolerance in untreated distant parts. Foliar or rhizospheric treatment with melatonin increased the melatonin levels in untreated roots or leaves, respectively, under both normal and cold stress conditions, whereas rhizospheric melatonin treatment increased the melatonin exudation rates from the xylem. An increased accumulation of melatonin accompanied with an induction in antioxidant enzyme activity in distant untreated tissues alleviated cold-induced oxidative stress. In addition, RNA-seq analysis revealed that an abundance of cold defense-related genes involved in signal sensing and transduction, transcriptional regulation, protection and detoxification, and hormone signaling might mediate melatonin-induced cold tolerance. Taken together, our results suggest that melatonin can induce cold tolerance via long distance signaling, and such induction is associated with an enhanced antioxidant capacity and optimized defense gene expression. Such a mechanism can be greatly exploited to benefit the agricultural production.

High-Throughput MicroRNA and mRNA Sequencing Reveals That MicroRNAs May Be Involved in Melatonin-Mediated Cold Tolerance in Citrullus lanatus L.

Transcriptional regulation of cold-responsive genes is crucial for exogenous melatonin-mediated cold tolerance in plants. Nonetheless, how melatonin regulates cold-responsive genes is largely unknown. In this study, we found that exogenous melatonin improved cold tolerance in watermelon by regulating expression of microRNAs (miRNAs). We identified a set of miRNAs that were regulated by melatonin under unstressed or cold conditions. Importantly, mRNA-seq analysis revealed that melatonin-induced downregulation of some miRNAs, such as miR159-5p, miR858, miR8029-3p, and novel-m0048-3p correlated with the upregulation of target genes involved in signal transduction (CDPK, BHLH, WRKY, MYB, and DREB) and protection/detoxification (LEA and MDAR) under cold stress. These results suggest that miRNAs may be involved in melatonin-mediated cold tolerance in watermelon by negatively regulating the expression of target mRNAs.