A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

PpZAT5 suppresses the expression of a B-box gene PpBBX18 to inhibit anthocyanin biosynthesis in the fruit peel of red pear.

BBX (B-box) proteins play a vital role in light-induced anthocyanin biosynthesis. PpBBX18 was an indispensable regulator for the induction of anthocyanin biosynthesis in the peel of red pear fruit (Pyrus pyrifolia Nakai.). However, the upstream regulation of BBX genes has not been well characterized. In this study, PpZAT5, a cysteine2/histidine2-type transcription factor, was discovered as the upstream negative regulator of PpBBX18. The results showed that PpZAT5 functions as a transcriptional repressor and directly binds to the CAAT motif of PpBBX18 and inhibits its expression. PpZAT5 expression was inhibited by light, which is converse to the expression pattern of anthocyanin-related structural genes. In addition, less anthocyanin accumulated in the PpZAT5-overexpressing pear calli than in the wild-type pear calli; on the contrary, more anthocyanin accumulated in PpZAT5-RNAi pear calli. Moreover, the crucial genes involved in light-induced anthocyanin biosynthesis were markedly down-regulated in the transcriptome of PpZAT5 overexpression pear calli compared to wild-type. In conclusion, our study indicates that PpBBX18 is negatively regulated by a C2H2-type transcriptional repressor, PpZAT5, which reduces anthocyanin content in pear. The present results demonstrate an upstream molecular mechanism of PpBBX18 and provide insights into light-induced anthocyanin biosynthesis.

A Novel Method for Non-invasive Estimation of Primary Productivity in Aquatic Ecosystems Using a Chlorophyll Fluorescence-Induced Dynamic Curve.

Photosynthetic microalgae are a major contributor to primary productivity in aquatic ecosystems, but typical measurements of their biomass and productivity are costly and relatively inefficient. The chlorophyll fluorescence induced dynamic (OJIP) curve can reflect the original photochemical reaction and the changes to the function and structure of photosystems as well as the effects of environmental factors on photosynthetic systems. Here, we present a novel method for estimating the Chl a content and photosynthetic microalgal cell density in water samples using the integral area of the OJIP curve. We identify strong linear relationships between OJIP curve integrals and both Chl a contents and cell densities for a variety of microalgal cultures and natural communities. Based on these findings, we present a non-invasive method to estimate primary productivity in aquatic ecosystems and monitor microalgal populations. We believe that this technique will allow for widespread, rapid, and inexpensive estimating of water primary productivity and monitoring of microalgal populations in natural water. This method is potentially useful in health assessment of natural water and as an early warning indicator for algal blooms.

d-/l-Isothymidine incorporation in the core sequence of aptamer BC15 enhanced its binding affinity to the hnRNP A1 protein.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to participate in the development of a variety of tumors. BC15 is a DNA aptamer targeting hnRNP A1. Firstly, through sequence truncation, we identified 31-mer sequence BC15-31 as the core sequence of BC15 with a strong binding affinity and high selectivity to the hnRNP A1 protein. Isothymidine (isoT) modification was then applied for the structural optimization of BC15-31, systematic modification and biological evaluation were carried out. Incorporation of isoT in the 1,3 sites at the 5'-end of BC15-31 can significantly enhance the protein affinity. Chemical modifications close to the 3'-end can greatly improve the stability of the aptamer. Furthermore, BC15-31 modified with isoT at both the 5'-end and 3'-end displayed an additive effect with enhanced bioactivity and stability at the same time. Our study strategy on BC15 provides a useful guideline for chemical modification and optimization of the aptamer for further clinical application.

The Bioactivity of D-/L-Isonucleoside- and 2'-Deoxyinosine-Incorporated Aptamer AS1411s Including DNA Replication/MicroRNA Expression.

In this study, chemical modification of 2'-deoxyinosine (2'-dI) and D-/L-isothymidine (D-/L-isoT) was performed on AS1411. They could promote the nucleotide-protein interaction by changing the local conformation. Twenty modified sequences were obtained, FCL-I and FCL-II showed the most noticeable activity improvement. They stabilized the G-quadruplex, remained highly resistant to serum degradation and specificity for nucleolin, further inhibited tumor cell growth, exhibited a stronger ability to influence the different phases of the tumor cell cycle, induced S-phase arrest, promoted the inhibition of DNA replication, and suppressed the unwound function of a large T antigen as powerful as AS1411. The microarray analysis and TaqMan PCR results showed that FCL-II can upregulate the expression of four breast-cancer-related, lowly expressed miRNAs and downregulate the expression of three breast-cancer-related, highly expressed miRNAs (>2.5-fold). FCL-II resulted in enhanced treatment effects greater than AS1411 in animal experiments (p < 0.01). The computational results further proved that FCL-II exhibits more structural advantages than AS1411 for binding to the target protein nucleolin, indicating its great potential in antitumor therapy.

Chemical modification improves the stability of the DNA aptamer GBI-10 and its affinity towards tenascin-C.

Aptamers are useful tools in molecular imaging due to their numerous attractive properties, such as excellent affinity and selectivity to diverse types of target molecules and biocompatibility. We carried out structure-activity relationship studies with the tenascin-C (TN-C) binding aptamer GBI-10, which is a promising candidate in tumor imaging. To increase the tumor targeting ability and nuclease resistance under physiological conditions, systematic modifications of GBI-10 with single and multiple 2'-deoxyinosine (2'-dI) or d-/l-isonucleoside (d-/l-isoNA) were performed. Results indicated that sector 3 of the proposed secondary structure is the most important region for specific binding with TN-C. By correlating the affinity of eighty-four GBI-10 derivatives with their predicted secondary structure by Zuker Mfold, we first validated the preferred secondary structure at 37 °C. We found that d-/l-isoNA modified GBI-10 derivatives exhibited improved affinity to the target as well as plasma stability. Affinity measurement and confocal imaging analysis highlighted one potent compound: 4AL/26TL/32TL, which possessed a significantly increased targeting ability to tumor cells. These results revealed the types of modified nucleotides, and the position and number of substituents in GBI-10 that were critical to the TN-C binding ability. Stabilized TN-C-binding DNA aptamers were prepared and they could be further developed for tumor imaging. Our strategy to introduce 2'-dI and d-/l-isoNA modifications after the selection process is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding ability.

Preparation and In Vitro Evaluation of a MRI Contrast Agent Based on Aptamer-Modified Gadolinium-Loaded Liposomes for Tumor Targeting.

The aim of this study was to prepare aptamer-modified liposomes loaded with gadolinium (Gd) to enhance the effective diagnosis for tumor by MRI. A modified GBI-10 (GBI-10m) was used to prepare targeted liposomes (GmLs). Liposomes with GBI-10 aptamer (GLs) and without aptamer (non-targeted liposomes (NLs)) were also prepared as controls. The particle size and zeta potential of GmLs, GLs, and NLs were all assayed. A clinical 3.0 T MR scanner was employed to assess the imaging efficiency and measure the longitudinal relaxivity (r 1) of the above liposomes. Confocal laser scanning microscopy and flow cytometry were used to analyze and compare the targeting effects of GmLs, GLs, and NLs to MDA-MB-435s cells at 37°C. The particle size of the prepared liposomes was scattered at 100-200 nm, and their values of r 1 were ∼4 mM-1 s-1. The images of confocal laser scanning microscopy showed that GmLs in the cytoplasm were significantly more than GLs and both GmLs and GLs were more than NLs. The fluorescence intensity of GmLs was increased by about two times than that of GLs and three times than that of NLs by flow cytometry. Therefore, the GmLs in this initial study were suggested to be a potential MRI contrast agent at 37°C for diagnosing tumors with the protein of tenascin-C over-expressed.

Characterization and Comparative Expression Profiling of Browning Response in Medinilla formosana after Cutting.

Plant browning is a recalcitrant problem for in vitro culture and often leads to poor growth of explants and even failure of tissue culture. However, the molecular mechanisms underlying browning-induced physiological processes remain unclear. Medinilla is considered one of the most difficult genera for tissue culture owning to its severe browning. In the present study, intact aseptic plantlets of Medinilla formosana Hayata previously obtained by ovary culture, were used to explore the characteristics and molecular mechanism of the browning response. Successive morphological and anatomical observations after cutting showed that the browning of M. formosana was not lethal but adaptive. De novo transcriptome and digital gene expression (DGE) profiling using Illumina high-throughput sequencing were then used to explore molecular regulation after cutting. About 7.5 million tags of de novo transcriptome were obtained and 58,073 unigenes were assembled and annotated. A total of 6,431 differentially expressed genes (DEGs) at three stages after cutting were identified, and the expression patterns of these browning-related genes were clustered and analyzed. A number of putative DEGs involved in signal transduction and secondary metabolism were particularly studied and the potential roles of these cutting-responsive mRNAs in plant defense to diverse abiotic stresses are discussed. The DGE profiling data were also validated by quantitative RT-PCR analysis. The data obtained in this study provide an excellent resource for unraveling the molecular mechanisms of browning processes during in vitro tissue culture, and lay a foundation for future studies to inhibit and eliminate browning damage.

In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging.

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N'-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor.

Heat shock protein 70 is necessary for Rice stripe virus infection in plants.

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive-stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative-stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull-down assays, the N-terminus of RSV RNA-dependent RNA polymerase (RdRp) interacted and co-localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N-terminus of RdRp when expressed alone was not obviously different from when it was co-expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.