The TIFY family is a novel plant-specific protein family, and is characterized by a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current study, we focused on the regulatory roles and possible physiological and molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We demonstrated the positive function of TIFY10s in alkaline responses by using the AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively lower germination rates of attify10a and attify10b mutant seeds under alkaline stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress. Furthermore, expression levels of the stress responsive genes including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression increased the jasmonate content of the transgenic alfalfa. In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize with each other in both yeast and plant cells. Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress.
Tau-class glutathione S-transferases (GSTUs) are ubiquitous proteins encoded by a large gene family in plants, which play important roles in combating different environmental stresses. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three GSTUs (GsGSTU13, GsGSTU14 and GsGSTU19) as potential salt-alkaline stress-responsive genes. Two of them, GsGSTU14 and GsGSTU19, have been shown to positively regulate plant salt-alkaline tolerance. In this study, we further demonstrated the positive function of GsGSTU13 in plant salt-alkaline stress responses by overexpressing it in Medicago sativa. Stress tolerance tests suggested that GsGSTU13 transgenic lines showed better growth and physiological indicators than wild alfalfa (cv. Zhaodong) under alkaline stress. Considering the shortage of methionine in alfalfa, we then co-transformed GsGSTU13 into two main alfalfa cultivars in Heilongjiang Province (cv. Zhaodong and cv. Nongjing No. 1) together with SCMRP, a synthesized gene that could improve the methionine content. We found that GsGSTU13/SCMRP transgenic alfalfa displayed not only higher methionine content but also higher tolerance to alkaline and salt stresses, respectively. Taken together, our results demonstrate that GsGSTU13 acts as a positive regulator in plant responses to salt and alkaline stresses, and can be used as a good candidate for generation of salt-alkaline tolerant crops.
Flowering time of rice (Oryza sativa L.) is among the most important agronomic traits for region adaptation and grain yield. In the process of rice breeding, efficient and slightly modulating the flowering time of an elite cultivar would be more popular with breeder. Hence, we are interested in slightly increasing the expression of flowering repressors by CRISPR/Cas9 genome editing system. It was predicated there were three uORFs in 5’ leader sequence of Hd2. In this study, through editing Hd2 uORFs, we got four homozygous mutant lines. Phenotypic analysis showed that the hd2 urf edited lines flowered later by 4.6–11.2 days relative to wild type SJ2. Supporting the later flowering phenotype, the expression of Ehd1, Hd3a, and RFT1 is significantly decreased in hd2 urf than that in wild type. Moreover, we found that the transcription level of Hd2 is not affected, whereas the Hd2 protein level was increased in hd2 urf compared with wild type, which indicated that Hd2 uORFs indeed affect the translation of a downstream Hd2 pORF. In summary, we developed a efficient approach for delaying rice heading date based on editing uORF region of flowering repressor, which is time and labor saving compared to traditional breeding. In future, uORF of other flowering time related genes, including flowering promoter and flowering repressor genes, can also be used as targets to fine-tune the flowering time of varieties.
Apomixis is a means of asexual reproduction by which plants produce embryos without fertilization and meiosis, therefore the embryo is of clonal and maternal origin. Interspecific hybrids between diploid B. vulgaris (2n=2x=18) and tetraploid B. corolliflora (2n=4x=36) were established, and then back-crossed with B. vulgaris. Among their offspring, monosomic addition line M14 (2n=2x=18+1) was selected because of the apomictic phenotype. We documented chromosome transmission from B. corolliflora into M14 by using genomic in situ hybridization (GISH). Suppression of cross-hybridization by blocking DNA was not necessary, indicating that the investigated Beta genome contains sufficient species-specific DNA, thus enabling the determination of genomic composition of the hybrids. We analyzed BAC microarrays of B. corolliflora chromosome 9 by using fluorescence-specific mRNA of B. vulgaris and Beta M14. BAC clones 16-M11 and 26-L15 were detected as fluorescence-specifics of BAC DNA of Beta M14. Then both BAC clones 16-M11 and 26-L15 were in situ hybridized to M14 chromosomes. The two hybridized BAC clones were located close to the telomere region of the long arm of a single chromosome 9, and showed hemizygosity. The results of BAC microarrays showed that these developments of embryo and endosperm have conservative expression patterns, indicating that sexual reproduction and apomixis have an interrelated pathway with common regulatory components and that the induction of a modified sexual reproduction program may enable the manifestation of apomixis in Beta species. It would be sufficient for the expression of apomixes with those apomictic-specific genes on chromosome 9 of B. corolliflora.
Nanomedicine exhibits emerging potentials to deliver advanced therapeutic strategies in the fight against triple-negative breast cancer (TNBC). Nevertheless, it is still difficult to develop a precise codelivery system that integrates highly effective photosensitizers, low toxicity, and hydrophobicity. In this study, PCN-224 is selected as the carrier to enable effective cancer therapy through light-activated reactive oxygen species (ROS) formation, and the PCN-224@Mn 3 O 4 @HA is created in a simple one-step process by coating Mn 3 O 4 nanoshells on the PCN-224 template, which can then be used as an "ROS activator" to exert catalase-and glutathione peroxidase-like activities to alleviate tumor hypoxia while reducing tumor reducibility, leading to improved photodynamic therapeutic (PDT) effect of PCN-224. Meanwhile, Mn 2+ produced cytotoxic hydroxyl radicals (•OH) via the Fenton-like reaction, thus producing a promising spontaneous chemodynamic therapeutic (CDT) effect. Importantly, by remodeling the tumor microenvironment (TME), Mn 3 O 4 nanoshells downregulated hypoxia-inducible factor 1𝜶 expression, inhibiting tumor growth and preventing tumor revival. Thus, the developed nanoshells, via light-controlled ROS formation and multimodality imaging abilities, can effectively inhibit tumor proliferation through synergistic PDT/CDT, and prevent tumor resurgence by remodeling TME.
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