ß-Cyclodextrin Polymer-Based Fluorescence Enhancement Strategy via Host-Guest Interaction for Sensitive Assay of SARS-CoV-2.

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that ß-cyclodextrin polymer (ß-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host-guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host-guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3' terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host ß-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of ß-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019.

Platinum Nanoparticles Loaded Graphitic Carbon Nitride Nanosheets with Enhanced Peroxidase-like Activity for H2O2 and Oxidase-Based Sensing.

Platinum nanoparticles (PtNPs) are classical peroxidase-like nanozyme; self-agglomeration of nanoparticles leads to the undesirable reduction in stability and catalytic activity. Herein, a hybrid peroxidase-like nanocatalyst consisting of PtNPs in situ growing on g-C3N4 nanosheets with enhanced peroxidase-mimic catalytic activity (PtNP@g-C3N4 nanosheets) was prepared for H2O2 and oxidase-based colorimetric assay. g-C3N4 nanosheets can be used as carriers to solve the problem of poor stability of PtNPs. We observed that the catalytic ability could be maintained for more than 90 days. PtNP@g-C3N4 nanosheets could quickly catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), and the absorbance of blue color oxidized TMB (oxTMB) showed a robust linear relationship with the concentration of H2O2 (the detection limit (LOD): 3.33 µM). By utilizing H2O2 as a mediator, this strategy can be applied to oxidase-based biomolecules (glucose, organophosphorus, and so on, that generate or consume hydrogen peroxide) sensing. As a proof of concept, a sensitive assay of cholesterol that combined PtNP@g-C3N4 nanosheets with cholesterol oxidase (ChOx) cascade catalytic reaction was constructed with an LOD of 9.35 µM in a widespread range from 10 to 800 µM (R2 = 0.9981). In addition, we also verified its ability to detect cholesterol in fetal bovine serum. These results showed application prospect of PtNP@g-C3N4 nanosheets-based colorimetry in sensing and clinical medical detection.

TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana.

How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.

The ubiquitin receptors DA1, DAR1, and DAR2 redundantly regulate endoreduplication by modulating the stability of TCP14/15 in Arabidopsis.

Organ growth involves the coordination of cell proliferation and cell growth with differentiation. Endoreduplication is correlated with the onset of cell differentiation and with cell and organ size, but little is known about the molecular mechanisms linking cell and organ growth with endoreduplication. We have previously demonstrated that the ubiquitin receptor DA1 influences organ growth by restricting cell proliferation. Here, we show that DA1 and its close family members DAR1 and DAR2 are redundantly required for endoreduplication during leaf development. DA1, DAR1, and DAR2 physically interact with the transcription factors TCP14 and TCP15, which repress endoreduplication by directly regulating the expression of cell-cycle genes. We also show that DA1, DAR1, and DAR2 modulate the stability of TCP14 and TCP15 proteins in Arabidopsis thaliana. Genetic analyses demonstrate that DA1, DAR1, and DAR2 function in a common pathway with TCP14/15 to regulate endoreduplication. Thus, our findings define an important genetic and molecular mechanism involving the ubiquitin receptors DA1, DAR1, and DAR2 and the transcription factors TCP14 and TCP15 that links endoreduplication with cell and organ growth.

High level of microsynteny and purifying selection affect the evolution of WRKY family in Gramineae.

The WRKY gene family, which encodes proteins in the regulation processes of diverse developmental stages, is one of the largest families of transcription factors in higher plants. In this study, by searching for interspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found 35 chromosomal segments of subgroup I genes of WRKY family (WRKY I) in four Gramineae species (Brachypodium, rice, sorghum, and maize) formed eight orthologous groups. After a stepwise gene-by-gene reciprocal comparison of all the protein sequences in the WRKY I gene flanking areas, highly conserved regions of microsynteny were found in the four Gramineae species. Most gene pairs showed conserved orientation within syntenic genome regions. Furthermore, tandem duplication events played the leading role in gene expansion. Eventually, environmental selection pressure analysis indicated strong purifying selection for the WRKY I genes in Gramineae, which may have been followed by gene loss and rearrangement. The results presented in this study provide basic information of Gramineae WRKY I genes and form the foundation for future functional studies of these genes. High level of microsynteny in the four grass species provides further evidence that a large-scale genome duplication event predated speciation.

Control of root meristem size by DA1-RELATED PROTEIN2 in Arabidopsis.

The control of organ growth by coordinating cell proliferation and differentiation is a fundamental developmental process. In plants, postembryonic root growth is sustained by the root meristem. For maintenance of root meristem size, the rate of cell differentiation must equal the rate of cell division. Cytokinin and auxin interact to affect the cell proliferation and differentiation balance and thus control root meristem size. However, the genetic and molecular mechanisms that determine root meristem size still remain largely unknown. Here, we report that da1-related protein2 (dar2) mutants produce small root meristems due to decreased cell division and early cell differentiation in the root meristem of Arabidopsis (Arabidopsis thaliana). dar2 mutants also exhibit reduced stem cell niche activity in the root meristem. DAR2 encodes a Lin-11, Isl-1, and Mec-3 domain-containing protein and shows an expression peak in the border between the transition zone and the elongation zone. Genetic analyses show that DAR2 functions downstream of cytokinin and SHORT HYPOCOTYL2 to maintain normal auxin distribution by influencing auxin transport. Further results indicate that DAR2 acts through the PLETHORA pathway to influence root stem cell niche activity and therefore control root meristem size. Collectively, our findings identify the role of DAR2 in root meristem size control and provide a novel link between several key regulators influencing root meristem size.

Control of lateral root initiation by DA3 in Arabidopsis.

Lateral root (LR) initiation is controlled by the pericycle and the neighboring endodermis in Arabidopsis. Here, we demonstrate that UBIQUITIN-SPECIFIC PROTEASE14/DA3 regulates LR initiation by modulating auxin signaling in the pericycle and endodermis. DA3 negatively affects the mRNA and protein levels of AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 in the pericycle and endodermis but positively regulates the protein stability of SHORT HYPOCOTYL 2 (SHY2/IAA3), an auxin signaling repressor, in the endodermis. We show that DA3 interacts with ARF7 and ARF19, inhibiting their binding to the locus of LATERAL ORGAN BOUNDARY DOMAIN16 (LBD16) to repress its expression in the pericycle. SHY2 also interacts with ARF7 and ARF19 in the endodermis and enhances the DA3 repressive effect on ARF7 and ARF19, thus modulating LBD16 expression in the pericycle. Overall, our findings show that DA3 acts with SHY2, ARF7, and ARF19 to coordinate auxin signaling in the pericycle and endodermis to control LR initiation in Arabidopsis.

Maize factors ZmUBP15, ZmUBP16 and ZmUBP19 play important roles for plants to tolerance the cadmium stress and salt stress.

Ubiquitin-Specific Protease16 (UBP16) has been described involved in cadmium stress and salt stress in Arabidopsis, however nothing is known about the functions of its homologs in maize. In this study, we investigate the functions of ZmUBP15, ZmUBP16 and ZmUBP19, three Arabidopsis UBP16 homologs in maize. Our results indicate that ZmUBP15, ZmUBP16 and ZmUBP19 are ubiquitously expressed throughout plant development, and ZmUBP15, ZmUBP16 and ZmUBP19 proteins are mainly localized in plasma membrane. Complementation analyses show that over-expression of ZmUBP15 or ZmUBP16 can rescue the defective phenotype of ubp16-1 in cadmium stress. In addition, over-expression of ZmUBP15, ZmUBP16 or ZmUBP19 can increase the plant tolerance to cadmium stress. These results indicate that ZmUBP15, ZmUBP16 and ZmUBP19 are required for plant to tolerance the cadmium stress. Consistent with this point, cadmium-related genes are markedly up-regulated in seedlings over-expressing ZmUBP15, ZmUBP16 or ZmUBP19. Furthermore, our data indicate that ZmUBP15, ZmUBP16 and ZmUBP19 partially rescue the salt-stress phenotype of ubp16-1. Thus, our research uncover the functions of three novel maize proteins, ZmUBP15, ZmUBP16 and ZmUBP19, which are required for plants in response to cadmium stress and salt stress.

BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis.

Sugars function as signal molecules to regulate growth, development, and gene expression in plants, yeasts, and animals. A coordination of sugar availability with phytohormone signals is crucial for plant growth and development. The molecular link between sugar availability and hormone-dependent plant growth are largely unknown. Here we report that BRI1 and BAK1 are involved in sugar-responsive growth and development. Glucose influences the physical interactions and phosphorylations of BRI1 and BAK1 in a concentration-dependent manner. BRI1 and BAK1 physically interact with G proteins that are essential for mediating sugar signaling. Biochemical data show that BRI1 can phosphorylate G protein ß subunit and γ subunits, and BAK1 can phosphorylate G protein γ subunits. Genetic analyses suggest that BRI1 and BAK1 function in a common pathway with G-protein subunits to regulate sugar responses. Thus, our findings reveal an important genetic and molecular mechanism by which BR receptors associate with G proteins to regulate sugar-responsive growth and development.

DAR2 acts as an important node connecting cytokinin, auxin, SHY2 and PLT1/2 in root meristem size control.

Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.