2D-DIGE based proteome analysis of wheat-Thinopyrum intermedium 7XL/7DS translocation line under drought stress.

BACKGROUND: Drought stress is the most limiting factor for plant growth and crop production worldwide. As a major cereal crop, wheat is susceptible to drought. Thus, discovering and utilizing drought-tolerant gene resources from related species are highly important for improving wheat drought resistance. In this study, the drought tolerance of wheat Zhongmai 8601-Thinopyrum intermedium 7XL/7DS translocation line YW642 was estimated under drought stress, and then two-dimensional difference gel electrophoresis (2D-DIGE) based proteome analysis of the developing grains was performed to uncover the drought-resistant proteins. RESULTS: The results showed that 7XL/7DS translocation possessed a better drought-tolerance compared to Zhongmai 8601. 2D-DIGE identified 146 differential accumulation protein (DAP) spots corresponding to 113 unique proteins during five grain developmental stages of YW642 under drought stress. Among them, 55 DAP spots corresponding to 48 unique proteins displayed an upregulated expression, which were mainly involved in stress/defense, energy metabolism, starch metabolism, protein metabolism/folding and transport. The cis-acting element analysis revealed that abundant stress-related elements were present in the promoter regions of the drought-responsive protein genes, which could play important roles in drought defense. RNA-seq and RT-qPCR analyses revealed that some regulated DAP genes also showed a high expression level in response to drought stress. CONCLUSIONS: Our results indicated that Wheat-Th. intermedium 7XL/7DS translocation line carried abundant drought-resistant proteins that had potential application values for wheat drought tolerance improvement.

Genome-Wide Identification and Characterisation of Wheat MATE Genes Reveals Their Roles in Aluminium Tolerance.

The Multidrug and toxin efflux (MATE) gene family plays crucial roles in plant growth and development and response to adverse stresses. This work investigated the structural and evolutionary characteristics, expression profiling and potential functions involved in aluminium (Al) tolerance from a genome-wide level. In total, 211 wheat MATE genes were identified, which were classified into four subfamilies and unevenly distributed on chromosomes. Duplication analysis showed that fragments and tandem repeats played the main roles in the amplification of TaMATEs, and Type II functional disproportionation had a leading role in the differentiation of TaMATEs. TaMATEs had abundant Al resistance and environmental stress-related elements, and generally had a high expression level in roots and leaves and in response to Al stress. The 3D structure prediction by AlphaFold and molecular docking showed that six TaMATE proteins localised in the plasmalemma could combine with citrate via amino acids in the citrate exuding motif and other sites, and then transport citrate to soil to form citrate aluminium. Meanwhile, citrate aluminium formed in root cells might be transported to leaves by TaMATEs to deposit in vacuoles, thereby alleviating Al toxicity.

Comparative Transcriptome Analysis Reveals Key Insights into Fertility Conversion in the Thermo-Sensitive Cytoplasmic Male Sterile Wheat.

Thermo-sensitive cytoplasmic male sterility (TCMS) plays a crucial role in hybrid production and hybrid breeding; however, there are few studies on molecular mechanisms related to anther abortion in the wheat TCMS line. In this study, FA99, a new wheat thermo-sensitive cytoplasmic male sterility line, was investigated. Fertility conversion analysis showed that FA99 was mainly controlled by temperature, and the temperature-sensitive stage was pollen mother cell formation to a uninucleate stage. Further phenotypic identification and paraffin section showed that FA99 was characterized by indehiscent anthers and aborted pollen in a sterile environment and tapetum was degraded prematurely during the tetrad period, which was the critical abortion period of FA99. The contents of O2-, H2O2, MDA and POD were significantly changed in FA99 under a sterile environment by the determination of physiological indexes. Furthermore, through transcriptome analysis, 252 differentially expressed genes were identified, including 218 downregulated and 34 upregulated genes. Based on KOG function classification, GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic changes in FA99 under different fertility environments, and the major differences were "phenylalanine metabolism", "phenylpropanoid biosynthesis", "cutin, suberine and wax biosynthesis", "phenylalanine, tyrosine and tryptophan biosynthesis" and "citrate cycle (TCA cycle)". Finally, we proposed an intriguing transcriptome-mediated pollen abortion and male sterility network for FA99. These findings provided data on the molecular mechanism of fertility conversion in thermo-sensitive cytoplasmic male sterility wheat.

Comprehensive analysis of formin gene family highlights candidate genes related to pollen cytoskeleton and male fertility in wheat (Triticum aestivum L.).

BACKGROUND: Formin, a highly conserved multi-domain protein, interacts with microfilaments and microtubules. Although specifically expressed formin genes in anthers are potentially significant in research on male sterility and hybrid wheat breeding, similar reports in wheat, especially in thermo-sensitive genic male sterile (TGMS) wheat, remain elusive. RESULTS: Herein, we systematically characterized the formin genes in TGMS wheat line BS366 named TaFormins (TaFHs) and predicted their functions in inducing stress response. In total, 25 TaFH genes were uncovered, majorly localized in 2A, 2B, and 2D chromosomes. According to the neighbor-joining (NJ) method, all TaFH proteins from wheat and other plants clustered in 6 sub-groups (A-F). The modeled 3D structures of TaFH1-A/B, TaFH2-A/B, TaFH3-A/B and TaFH3-B/D were validated. And different numbers of stress and hormone-responsive regulatory elements in their 1500 base pair promoter regions were contained in the TaFH genes copies. TaFHs had specific temporal and spatial expression characteristics, whereby TaFH1, TaFH4, and TaFH5 were expressed highly in the stamen of BS366. Besides, the accumulation of TaFHs was remarkably lower in a low-temperature sterile condition (Nanyang) than fertile condition (Beijing), particularly at the early stamen development stage. The pollen cytoskeleton of BS366 was abnormal in the three stages under sterile and fertile environments. Furthermore, under different stress levels, TaFHs expression could be induced by drought, salt, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), indole-3-acetic acid (IAA), polyethylene glycol (PEG), and low temperature. Some miRNAs, including miR167, miR1120, and miR172, interacts with TaFH genes; thus, we constructed an interaction network between microRNAs, TaFHs, phytohormone responses, and distribution of cytoskeleton to reveal the regulatory association between upstream genes of TaFH family members and sterile. CONCLUSIONS: Collectively, this comprehensive analysis provides novel insights into TaFHs and miRNA resources for wheat breeding. These findings are, therefore, valuable in understanding the mechanism of TGMS fertility conversion in wheat.

Lysosome activable polymeric vorinostat encapsulating PD-L1KD for a combination of HDACi and immunotherapy.

PD-1/PD-L1 blocking therapy has become one of the most promising methods in the field of tumor treatment. However, it encounters the challenge of immune escape due to the exhaustion of T cells. Studies have shown that the epigenetic regulation drug histone deacetylase inhibitor (HDACi) may be able to reverse exhausted T cells by changing the epigenetic transcription program. Therefore, the combination of epigenetic therapy and PD-1/PD-L1 blockade therapy is expected to reverse the immune escape, whereas the overriding goal should aim at the spontaneous release and synergy of PD-1/PD-L1 blocking siRNA and HDACi. In this study, we develop PDDS{polyethylene glycol-b-asparaginate(diethylenetriamine-vorinostat), (PEG-b-P[Asp(DET-SAHA)n] PPDS)}encapsulating siRNA-PD-L1to provide micelles siRNA-PD-L1-loaded micelles (siRNA@PPDS). Transmission electron microscope (TEM) images demonstrate that siRNA@PPDS micelles presented spherical morphology with a size of about 120 nm; hydrodynamic data analysis indicates pH sensitivity of siRNA@PPDS micelles. The experiments reveal that siRNA@PPDS micelles could be well uptaken by the tumor cells to silence the expression of PD-L1 protein in a dose-dependent manner; compared with the free SAHA, the SAHA-loaded micelles PPDS show higher cytotoxicity to induce tumor cell apoptosis and block cell cycle in G1 phase on melanoma-bearing mice, siRNA@PPDS has shown outstanding inhibition of tumor growth and pulmonary metastasis. By comprehensively activating the immune system, lysosome activable polymeric vorinostat encapsulating PD-L1KD for the combination therapy of PD-L1-KD and HDACIs can be an effective strategy to reverse the unresponsiveness of immune checkpoint inhibitors and a promising treatment to inhibit tumor growth, recurrence, and metastasis in clinic.

Polymerized vorinostat mediated photodynamic therapy using lysosomal spatiotemporal synchronized drug release complex.

A combination of photodynamic therapy (PDT) and histone deacetylase inhibitor (HDACis) could potentiate single-mode anti-tumor activity of HDACis or PDT to inhibit tumor relapse and metastasis. However, poor solubility and heterogeneity in cellular uptake and tissue distribution hamper the dual mode antitumor effect. For a controlled drug release of photosensitizers and HDACis in cytoplasm, photosensitizer pyropheophorbide-a (Pyro) encapsulated in polymer polyethylene glycol-b-poly (asparaginyl-vorinostat) (simplified as Pyro@FPPS) are fabricated to achieve their lysosomal spatiotemporal synchronized release. With HDACis modeling PDT in vitro and in vivo, it seems that polymerized Vorinostat encapsulated photosensitizers significantly inhibited the tumor proliferation and metastasis by spatiotemporal synchronized drugs release, and Pyro@FPPS reported here reveals a promising prospect to exert drugs' synergistic effect in a spatiotemporal synchronized manner and can be an effective strategy to inhibit tumor growth, recurrence and metastasis in clinic.

Gelated Vorinostat with inner-lysosome triggered release for tumor-targeting chemotherapy.

Histonedeacetylase inhibitor (HDACi) has great potential in targeted antitumor therapy by inhibiting tumor migration, invasion, and metastasis. As one of the typical HDACis, vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) was approved as a therapeutic agent for cancer therapy, however, challenges remain due to their poor solubility, short half-life and low efficiency in cellular penetration. Considering the disadvantages of usual drug carriers, folate and vorinostat bound BSA nanogel (FVBN)was fabricated to implement higher solubility, stability, cellular uptake, and lipase-responsive release. With good dispersion and stability, FVBN significantly increased the cellular uptake of vorinostat through folate-mediated endocytosis. FVBN exhibited comparable cytotoxicity with free SAHA, and the growth of tumor cells was blocked in G1/G0 phase just like SAHA performed in cell cycle arrest tests. Moreover, FVBN not only effectively inhibited the growth of melanoma but also observably prevented pulmonary metastasis of melanoma. In the experiment against nude mice bearing solid ovarian cancer, FVBN showed excellent antitumor effect without liver damage, demonstrating the superiority of gelated and inner-lysosome triggered release strategies to the free SAHA, and it is promising to expand the scope of application of HDACi in clinical cancer therapy.

Folate-mediated and pH-responsive chidamide-bound micelles encapsulating photosensitizers for tumor-targeting photodynamic therapy.

Background: Nonspecific tumor targeting, potential relapse and metastasis of tumor after treatment are the main barriers in clinical photodynamic therapy (PDT) for cancer, hence, inhibiting relapse and metastasis of tumor is significant issues in clinic. Purpose: In this work, chidamide as a histone deacetylases inhibitor (HADCi) was bound onto a pH-responsive block polymer folate polyethylene glycol-b-poly(aspartic acid) (PEG-b-PAsp) grafted folate (FA-PEG-b-PAsp) to obtain the block polymer folate polyethylene glycol-b-poly(asparaginyl-chidamide) (FA-PEG-b-PAsp-chidamide, FPPC) as multimodal tumor-targeting drug-delivery carrier to inhibiting tumor cell proliferation and tumor metastasis in mice. Methods: Model photosensitizer pyropheophorbide-a (Pha) was encapsulated by FPPC in PBS to form the polymer micelles Pha@FPPC [folate polyethylene glycol-b-poly(asparaginyl-chidamide) micelles encapsulating Pha]. Pha@FPPC was characterized by transmission electron microscope and dynamic light scattering; also, antitumor activity in vivo and in vitro were investigated by determination of cellular ROS level, detection of cell apoptosis and cell cycle arrest, PDT antitumor activity in vivo and histological analysis. Results: With favorable and stable sphere morphology under transmission electron microscope (TEM) (~93.0 nm), Pha@FPPC greatly enhanced the cellular uptake due to its folate-mediated effective endocytosis by mouse melanoma B16-F10 cells and the yield of ROS in tumor cells induced by PDT, and mainly caused necrocytosis and blocked cell growth cycle not only in G2 phase but also in G1/G0 phase after PDT. Pha@FPPC exhibited lower dark cytotoxicity in vitro and a better therapeutic index because of its higher dark cytotoxicity/photocytotoxicity ratio. Moreover, Pha@FPPC not only significantly inhibited the growth of implanted tumor and prolonged the survival time of melanoma-bearing mice due to both its folate-mediated tumor-targeting and selectively accumulation at tumor site by EPR (enhanced permeability and retention)effect as micelle nanoparticles but also remarkably prevented pulmonary metastasis of mice melanoma after PDT compared to free Pha, demonstrating its dual antitumor characteristics of PDT and HDACi. Conclusion: As a folate-mediated and acid-activated chidamide-grafted drug-delivery carrier, FPPC may have great potential to inhibit tumor metastasis in clinical photodynamic treatment for cancer because of its effective and multimodal tumor-targeting performance as photosensitizer vehicle.