A NAC transcription factor ZaNAC93 confers floral initiation, fruit development, and prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is a dioecious prickly plant which developed apomictic reproduction. The increases in male flowers and prickle density in female plants lead to low yield and picking efficiency. However, little is known concerning the mechanisms of floral development and prickle formation. NAC is a well-known transcription factor that participates in multiple aspects of plant growth and development. Herein, we characterize the functions and regulatory mechanisms of candidate NACs controlling both traits in Z. armatum. A total of 159 ZaNACs were identified, and 16 of these were male-biased, represented by the NAP subfamily members ZaNAC93 and ZaNAC34, orthologs of AtNAC025 and AtNARS1/NAC2 respectively. Overexpression of ZaNAC93 in tomato led to modifications in flower and fruit development, including earlier flowering, increased numbers of lateral shoots and flowers, accelerated plant senescence, and reduced size and weight of fruits and seeds. In addition, the trichome density in leaves and inflorescences was dramatically reduced in ZaNAC93-OX lines. Overexpression of ZaNAC93 resulted in the up-/downregulation of genes associated with GA, ABA and JA signaling pathways, such as GAI, PYL and JAZ, as well as several TFs, including bZIP2, AGL11, FBP24 and MYB52. Yeast two-hybrid analysis revealed that ZaNAC93 protein could interact with AP1, GAI, bZIP2 and AGL11 in Z. armatum, which might contribute to floral induction, fruit growth, and trichome initiation. This work provides new insights into the molecular mechanisms of ZaNAC93 in reproductive development and prickle formation in Z. armatum.

Comparative transcriptome analysis reveals hormone, transcriptional and epigenetic regulation involved in prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is an evergreen plant with high economical and medicinal values. The presence of prickles on stems and leaves is undesirable for them make picking difficult. To date, little is known of prickle formation in Z. armatum. Herein, the morphological and molecular features of prickle initiation in prickless (WC) and three types of prickly Z. armatum were characterized. Compared to WC, the levels of cytokinin and auxin were increased, while GA and JA declined in prickly Z. armatum. Transcriptome analysis identified 6258 differentially expressed genes (DEGs) between prickless and prickly Z. armatum. Among them, several DEGs related to hormone metabolism and signaling, including LOG7, CKX3, AHK1, three DELLAs, six JAZs and TIR1, were candidate genes involved in prickle formation. Transcription factors associated with prickle formation were screened, including MYB6-1/MYB6-2, WER, GL3-2, SPL4/5, SOC1, and SCL32. Of them, MYB6-1 and WER might negatively regulate prickles initiation via interacting with GL3-2. Additionally, the histone acetylation and DNA methylation levels, the transcripts of histone acetyltransferase/deacetylase and DNA methyltransferases showed significant differences between prickless and prickly plants, indicating their involvements in prickle initiation. These findings illustrate the regulation of prickle formation might be mediated by phytohormones (especially cytokinin), transcription factors and epigenetic modifications in Z. armatum.

25-hydroxyvitamin D3 inhibits oxidative stress and ferroptosis in retinal microvascular endothelial cells induced by high glucose through down-regulation of miR-93.

BACKGROUND: The decrease of vitamin D plays a critical role in diabetes mellitus (DM)-induced oxidative stress and vascular endothelial injury. Therefore, we investigated the effect and mechanism of 25-hydroxyvitamin D3 (25 (OH) D3) on oxidative stress and ferroptosis induced by high glucose in human retinal microvascular endothelial cells (hRMVECs). And the objective of this paper was to propose a new strategy for the prevention and treatment of diabetic retinopathy (DR). METHODS: First, hRMVECs were transfected with mimics NC or miR-93. After that, cells were treated with 100 nM / 500 nM 25 (OH) D3 and then cultured in a high glucose (30 mM) environment. Subsequently, qRT-PCR was employed to detect the expression level of miR-93; CCK-8 for the proliferation of cells in each group; biochemical tests for the level of intracellular reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH) and ferrous ion (Fe2+); and Western blot for the expression of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and SLC7A11). RESULTS: Under a high glucose environment, 25 (OH) D3 at 100 nM/500 nM could significantly promote the proliferation of hRMVECs, remarkably decrease the level of intracellular ROS/MDA, and up-regulate the level of GSH. Besides, 25 (OH) D3 greatly reduced Fe2+ level in the cells while increased protein level of GPX4 and SLC7A11. Subsequently, we found that high glucose induced miR-93 expression, while 25 (OH) D3 markedly decreased high glucose-induced miR-93 overexpression. Furthermore, overexpression of miR-93 inhibited the functions of 25 (OH) D3 by activating ROS (ROS and MDA were up-regulated while GSH was down-regulated) and inducing Fe2+ (Fe2+ level was up-regulated while GPX4 and SLC7A11 level was down-regulated) in cells. CONCLUSION: 25 (OH) D3 may inhibit oxidative stress and ferroptosis in hRMVECs induced by high glucose via down-regulation of miR-93.

Genome-wide identification, interaction of the MADS-box proteins in Zanthoxylum armatum and functional characterization of ZaMADS80 in floral development.

As a typical dioecious species, Zanthoxylum armatum establishes apomictic reproduction, hence only female trees are cultivated. However, male and hermaphrodite flowers have recently appeared in female plants, resulting in a dramatic yield reduction. To date, the genetic basis underlying sex determination and apomixis in Z. armatum has been largely unknown. Here, we observed abortion of the stamen or carpel prior to primordium initiation, thus corroborating the potential regulation of MADS-box in sex determination. In Z. armatum, a total of 105 MADS-box genes were identified, harboring 86 MIKC-type MADSs with lack of FLC orthologues. Transcriptome analysis revealed candidate MADSs involved in floral organ identity, including ten male-biased MADSs, represented by ZaMADS92/81/75(AP3/PI-like), and twenty-six female-specified, represented by ZaMADS80/49 (STK/AGL11-like) and ZaMADS42 (AG-like). Overexpressing ZaMADS92 resulted in earlier flowering, while ZaMADS80 overexpression triggered precocious fruit set and parthenocarpy as well as dramatic modifications in floral organs. To characterize their regulatory mechanisms, a comprehensive protein-protein interaction network of the represented MADSs was constructed based on yeast two-hybrid and bimolecular fluorescence complementation assays. Compared with model plants, the protein interaction patterns in Z. armatum exhibited both conservation and divergence. ZaMADS70 (SEP3-like) interacted with ZaMADS42 and ZaMADS48 (AP3-like) but not ZaMADS40 (AP1-like), facilitating the loss of petals in Z. armatum. The ZaMADS92/ZaMADS40 heterodimer could be responsible for accelerating flowering in ZaMADS92-OX lines. Moreover, the interactions between ZaMADS80 and ZaMADS67(AGL32-like) might contribute to apomixis. This work provides new insight into the molecular mechanisms of MADS-boxes in sex organ identity in Z. armatum.

Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases.

The CRISPR/Cas system is a protective adaptive immune system against attacks from foreign mobile genetic elements. Since the discovery of the excellent target-specific sequence recognition ability of the CRISPR/Cas system, the CRISPR/Cas system has shown excellent performance in the development of pathogen nucleic-acid-detection technology. In combination with various biosensing technologies, researchers have made many rapid, convenient, and feasible innovations in pathogen nucleic-acid-detection technology. With an in-depth understanding and development of the CRISPR/Cas system, it is no longer limited to CRISPR/Cas9, CRISPR/Cas12, and other systems that had been widely used in the past; other CRISPR/Cas families are designed for nucleic acid detection. We summarized the application of CRISPR/Cas-related technology in infectious-disease detection and its development in SARS-CoV-2 detection.

The CRISPR-Cas system as a tool for diagnosing and treating infectious diseases.

Emerging and relapsing infectious diseases pose a huge health threat to human health and a new challenge to global public health. Rapid, sensitive and simple diagnostic tools are keys to successful management of infectious patients and containment of disease transmission. In recent years, international research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related proteins (Cas) has revolutionized our understanding of biology. The CRISPR-Cas system has the advantages of high specificity, high sensitivity, simple, rapid, low cost, and has begun to be used for molecular diagnosis and treatment of infectious diseases. In this paper, we described the biological principles, application fields and prospects of CRISPR-Cas system in the molecular diagnosis and treatment of infectious diseases, and compared it with existing molecular diagnosis methods, the advantages and disadvantages were summarized.

Effect of the meiotic inhibitor cilostamide on resumption of meiosis and cytoskeletal distribution in buffalo oocytes.

Improving the quality of in vitro maturated buffalo oocytes is essential for embryo production. We report here the effects on microtubules and microfilaments in oocytes and embryo development that result from treating buffalo oocytes with the phosphodiesterase 3 (PDE3) inhibitor cilostamide. Addition of 20µM or 50µM cilostamide for 24h during in vitro maturation showed no differences in the percentage of oocytes arrested at the germinal vesicle (GV) stage. When 20µM cilostamide was added to the pre-maturation culture for 6h, 12h or 24h and continued for another 24h without cilostamide, oocytes resumed meiosis, but with significantly lower (P<0.01) maturation rates in the 24h group than that in the other two groups. During oocyte maturation in vitro, no microtubules were detected before GV breakdown (GVBD). After GVBD, microtubules combined with condensed chromatin to form the meiotic metaphase spindle. Microfilaments covered a thick area around the cellular cortex and overlying chromosomes. Cilostamide had no effects on microtubules and microfilaments in metaphase II oocytes, and there were no significant differences in the rates of cleavage, blastocyst formation and number of blastocyst cells between oocytes treated pre-maturation with inhibitor for 6h and those of the control group (P>0.05). In summary, cilostamide reversibly arrested the resumption of meiosis without any adverse impact on the dynamic changes in microtubules and microfilaments in buffalo oocytes and their in vitro developmental capacity.