Cassava phosphatase PP2C1 modulates thermotolerance via fine-tuning dephosphorylation of antioxidant enzymes.

Global warming is an adverse environmental factor that threatens crop yields and food security. 2C-type protein phosphatases (PP2Cs), as core protein phosphatase components, play important roles in plant hormone signaling to cope with various environmental stresses. However, the function and underlying mechanism of PP2Cs in the heat stress response remain elusive in tropical crops. Here, we report that MePP2C1 negatively regulated thermotolerance in cassava (Manihot esculenta Crantz), accompanied by the modulation of reactive oxygen species (ROS) accumulation and the underlying antioxidant enzyme activities of catalase (CAT) and ascorbate peroxidase (APX). Further investigation found that MePP2C1 directly interacted with and dephosphorylated MeCAT1 and MeAPX2 at serine (S) 112 and S160 residues, respectively. Moreover, in vitro and in vivo assays showed that protein phosphorylation of MeCAT1S112 and MeAPX2S160 was essential for their enzyme activities, and MePP2C1 negatively regulated thermotolerance and redox homeostasis by dephosphorylating MeCAT1S112 and MeAPX2S160. Taken together, this study illustrates the direct relationship between MePP2C1-mediated protein dephosphorylation of MeCAT1 and MeAPX2 and ROS accumulation in thermotolerance to provide insights for adapting to global warming via fine-tuning thermotolerance of the tropical crop cassava.

Study on automatic ultrasound scanning of lumbar spine and visualization system for path planning in lumbar puncture surgery.

In lumbar puncture surgery, compared with the conventional methodologies like computed tomography and magnetic resonance imaging, ultrasound imaging offers the advantages of being low cost, no radiation and real-time image generation. However, the use of ultrasound equipment in lumbar puncture involves a cumbersome and time-consuming process for the subjective imaging of the overall structure of the lumbar spine in order to determine the exact puncture point and path. Meanwhile, the robotic arm puncture system has the advantages of high precision, good stability and simple and efficient operation. As a result, robotic-assisted ultrasound scanning is valuable for the assessment of a puncture path in spinal tap surgery. In this pursuit, based on the official URSDK development package for a robot arm and the Transmission Control Protocol/Internet Protocol, the system proposed in the present study involves a program to control the robot arm to clamp down onto an ultrasonic probe to enable automatic scanning and acquisition of images. A three-dimensional reconstruction program based on the visualization toolkit was designed, and a lumbar spine experiment was conducted with this system. A total of 136 two-dimensional ultrasound images were collected in the lumbar spine model experiment by enhancing contrast of and denoising the original ultrasound images, and a linear interpolation algorithm was used to perform the three-dimensional reconstruction of the lumbar spine model. The reconstructed structure was defective, but the location of the spinous process gap was determined with the sagittal and coronal images. The feasibility of the system was verified by the reconstruction results, which can provide a reference for determining the puncture point and path planning in the lumbar puncture surgery.

PP2C1 fine-tunes melatonin biosynthesis and phytomelatonin receptor PMTR1 binding to melatonin in cassava.

Melatonin is an important molecule in both animals and plants, regulating circadian rhythms and stress responses. Therefore, the improvement of melatonin accumulation not only strengthens the function of melatonin but also improves stress resistance in crops. Although melatonin biosynthetic enzymes have been identified through reverse genetics previously, an investigation of melatonin level-related genes through forward genetics in plants has yet to be performed. In this study, a genome-wide association study using cassava natural population of 298 genetic resources identified melatonin accumulation 1 (MA1), which regulates the natural variation of melatonin levels in cassava. We found that MA1 encodes type 2C protein phosphatase 1 (PP2C1), which serves as a negative regulator of melatonin levels in cassava. MePP2C1 physically interacts with MeRAV1/2 and MeWRKY20 and dephosphorylates them at serine (S) 35 residue, S34 residue, and S176 residue, respectively, thereby hindering their transcriptional activation on downstream melatonin biosynthetic genes. Notably, MePP2C1 interacts with phytomelatonin receptor MePMTR1 and dephosphorylates it at S11 residue, repressing its binding to melatonin. In summary, this study demonstrates that MePP2C1 as MA1 plays dual roles in negatively regulating both melatonin accumulation and signaling, extending the understanding of the molecular mechanism underlying melatonin accumulation and signaling through forward genetics in plants.

Fine-tuning of pathogenesis-related protein 1 (PR1) activity by the melatonin biosynthetic enzyme ASMT2 in defense response to cassava bacterial blight.

Melatonin is widely involved in plant disease resistance through modulation of immune responses. Pathogenesis-related (PR) proteins play important roles in plant immune responses. However, the direct association between melatonin biosynthetic enzyme and PR protein remains elusive in plants. In this study, we found that N-acetylserotonin O-methyltransferase 2 (MeASMT2) physically interacted with MePR1 in vitro and in vivo, thereby promoting the anti-bacterial activity of MePR1 against Xanthomonas axonopodis pv. manihotis (Xam). Consistently, MeASMT2 improved the effect of MePR1 on positively regulating cassava disease resistance. In addition, we found that type 2C protein phosphatase 1 (MePP2C1) interacted with MeASMT2 to interfere with MePR1-MeASMT2 interaction, so as to inhibiting the effect of MeASMT2 and MePR1 on positively regulating cassava disease resistance. In contrast to the increased transcripts of MeASMT2 and MePR1 in response to Xam infection, the transcript of MePP2C1 was decreased upon Xam infection. Therefore, disease activated MeASMT2 was released from disease inhibited MePP2C1, so as to improving the anti-bacterial activity of MePR1, resulting in improved immune response. In summary, this study illustrates the dynamic modulation of the MePP2C1-MeASMT2-MePR1 module on cassava defense response against cassava bacterial blight (CBB), extending the understanding of the correlation between melatonin biosynthetic enzyme and PR in plants.

Phosphorylation of RAV1/2 by KIN10 is essential for transcriptional activation of CAT6/7, which underlies oxidative stress response in cassava.

Related to ABI3/VP1 (RAV) transcription factors have important roles in plant stress responses; however, it is unclear whether RAVs regulates oxidative stress response in cassava (Manihot esculenta). In this study, we report that MeRAV1/2 positively regulate oxidative stress resistance and catalase (CAT) activity in cassava. Consistently, RNA sequencing (RNA-seq) identifies three MeCATs that are differentially expressed in MeRAV1/2-silenced cassava leaves. Interestingly, MeCAT6 and MeCAT7 are identified as direct transcriptional targets of MeRAV1/2 via binding to their promoters. In addition, protein kinase MeKIN10 directly interacts with MeRAV1/2 to phosphorylate them at Ser45 and Ser44 residues, respectively, to promote their direct transcriptional activation on MeCAT6 and MeCAT7. Site mutation of MeRAV1S45A or MeRAV2S44A has no significant effect on the activities of MeCAT6 and MeCAT7 promoters or on oxidative stress resistance. In summary, this study demonstrates that the phosphorylation of MeRAV1/2 by MeKIN10 is essential for its direct transcriptional activation of MeCAT6/7 in response to oxidative stress.

Human adipose tissue-derived stem cells inhibit the activity of keloid fibroblasts and fibrosis in a keloid model by paracrine signaling.

BACKGROUND: Human adipose tissue-derived mesenchymal stem cells (ASCs) have potential utility as modulators of the regeneration of tissue that is inflamed or scarred secondary to injuries such as burns or trauma. However, the effect of ASCs on one particular type of scarring, keloidal disease, remains unknown. The absence of an optimal model for investigation has hindered the development of an effective therapy using ASCs for keloids. OBJECTIVE: To investigate the influence of ASCs on angiogenesis, extracellular matrix deposition, and inflammatory cell influx in keloids. METHODS: We analyzed the proliferation, migration, and apoptosis of human keloid-derived fibroblasts treated with a starvation-induced, conditioned medium from ASCs (ASCs-CM). This was achieved by Brdu proliferation assay, a validated co-culture migration assay, and flow cytometry, respectively. To assess the change in phenotype to a pro-fibrotic state, fibroblasts were analyzed by real-time PCR and contraction assay. A keloid implantation animal model was used to assess the paracrine effect of ASCs histochemically and immunohistochemically on scar morphology, collagen deposition, inflammatory cell composition, and blood vessel density. In tandem, an antibody-based array was used to identify protein concentration in the presence of ASCs-CM at time point 0, 24, and 48h. RESULTS: ASCs-CM inhibited the proliferation and collagen synthesis of human keloid-derived fibroblasts. ASCs-CM was associated with reduced inflammation and fibrosis in the keloid implantation model. Thirty-four cytokines were differentially regulated by ASCs-CM at 24h. These included molecules associated with apoptosis, matrix metalloproteases, and their inhibitors. The same molecules were present at relatively higher concentrations at the 48h timepoint. CONCLUSION: These results suggest that ASCs are associated with the inhibition of fibrosis in keloids by a paracrine effect. This phenomenon may have utility as a therapeutic approach in the clinical environment.