Intrinsic Protein Research Articles

Two aquaporins, PIP1;1 and PIP2;1, mediate the uptake of neonicotinoid pesticides in plants

Neonicotinoids (NEOs), a large class of organic compounds, are a type of commonly used pesticide for crop protection. Their uptake and accumulation in plants are prerequisites for their intra- and intercellular movements, transformation, and function. Understanding the molecular mechanisms that underpin NEO uptake by plants is crucial for effective application, which remains elusive. Here, we demonstrate that NEOs enter plant cells primarily through the transmembrane symplastic pathway and accumulate mainly in the cytosol. Two plasma membrane intrinsic proteins discovered in Brassica rapa, BraPIP1;1 and BraPIP2;1, were found to encode aquaporins (AQPs) that are highly permeable to NEOs in different plant species and facilitate NEO subcellular diffusion and accumulation. Their conserved transport function was further demonstrated in Xenopus laevis oocyte and yeast assays. BraPIP1;1 and BraPIP2;1 gene knockouts and interaction assays suggested that their proteins can form functional heterotetramers. Assessment of the potential of mean force indicated a negative correlation between NEO uptake and the energy barrier of BraPIP1;1 channels. This study shows that AQPs transport organic compounds with greater osmolarity than previously thought, providing new insight into the molecular mechanisms of organic compound uptake and facilitating innovations in systemic pesticides.

Analysis of genes specific to the early maturation stage of Sesamum indicum seeds by subtraction method*,*.

The mechanisms regulating the content ratio of unsaturated fatty acid in sesame oil need to be clarified in order to breed novel varieties with high contents of unsaturated fatty acids. Full-length cDNA libraries prepared from sesame seeds 1 to 3 weeks after flowering were subtracted with cDNAs from plantlets of 4 weeks after germination. A total of 1545 cDNA clones was sequenced. The functions of novel genes expressed specifically during the early maturation of sesame seeds were investigated by the transformation of Arabidopsis thaliana. Thirteen genes for a transcription factor were identified, four of which were involved in ethylene signaling. Fifty-nine genes, including those for the aquaporin-like protein and ethylene response factor, were analyzed by overexpression in A. thaliana. The overexpression of novel genes and the aquaporin-like protein gene in A. thaliana increased the content of unsaturated fatty acids. The localization of these products was investigated by the induction of the expression vectors for the GFP fusion protein into onion epidermal cells and sesame root cells with a particle gun. As a result, two cDNA clones were identified as good candidate genes to clarify the regulation in the yield and the ratio of unsaturated fatty acids in sesame seeds. Sein60414 (Accession No. LC603128), an intrinsic membrane protein, may be involved in the increase of unsaturated fatty acids, and Sein61074 (Accession No. LC709278) MAP3K δ-1 protein kinase in the regulation of the total ratio of unsaturated fatty acids in sesame seeds.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Integrating network pharmacology and in silico analysis deciphers Withaferin-A's anti-breast cancer potential via hedgehog pathway and target network interplay.

This study examines the remarkable effectiveness of Withaferin-A (WA), a withanolide obtained from Withania somnifera (Ashwagandha), in encountering the mortiferous breast malignancy, a global peril. The predominant objective is to investigate WA's intrinsic target proteins and hedgehog (Hh) pathway proteins in breast cancer targeting through the application of in silico computational techniques and network pharmacology predictions. The databases and webtools like Swiss target prediction, GeneCards, DisGeNet and Online Mendelian Inheritance in Man were exploited to identify the common target proteins. The culmination of the WA network and protein-protein interaction network were devised using Stitch and String web tools, through which the drug-target network of 30 common proteins was constructed employing Cytoscape-version 3.9. Enrichment analysis was performed by incorporating Gprofiler, Metascape and Cytoscape plugins. David compounded the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, and enrichment was computed through bioinformatics tools. The 20 pivotal proteins were docked harnessing Glide, Schrodinger Suite 2023-2. The investigation was governed by docking scores and affinity. The shared target proteins underscored the precise Hh and WA network roles with the affirmation enrichment P-value of <0.025. The implications for hedgehog and cancer pathways were profound with enrichment (P < 0.01). Further, the ADMET and drug-likeness assessments assisted the claim. Robust interactions were noticed with docking studies, authenticated through molecular dynamics, molecular mechanics generalized born surface area scores and bonds. The computational investigation emphasized WA's credible anti-breast activity, specifically with Hh proteins, implying stem-cell-level checkpoint restraints. Rigorous testament is imperative through in vitro and in vivo studies.

Genome-wide analysis and expression of the aquaporin gene family in Avena sativa L.

Oat (Avena sativa L.) belongs to the early maturity grass subfamily of the Gramineae subfamily oats (Avena) and has excellent characteristics, such as tolerance to barrenness, salt, cold, and drought. Aquaporin (AQP) proteins belong to the major intrinsic protein (MIP) superfamily, are widely involved in plant growth and development, and play an important role in abiotic stress responses. To date, previous studies have not identified or analyzed the AsAQP gene family system, and functional studies of oat AQP genes in response to drought, cold, and salt stress have not been performed. In this study, AQP genes (AsAQP) were identified from the oat genome, and various bioinformatics data on the AQP gene family, gene structure, gene replication, promoters and regulatory networks were analyzed. Quantitative real-time PCR technology was used to verify the expression patterns of the AQP gene family in different oat tissues under different abiotic stresses. In this study, a total of 45 AQP genes (AsAQP) were identified from the oat reference genome. According to a phylogenetic analysis, 45 AsAQP were divided into 4 subfamilies (PIP, SIP, NIP, and TIP). Among the 45 AsAQP, 23 proteins had interactions, and among these, 5AG0000633.1 had the largest number of interacting proteins. The 20 AsAQP genes were expressed in all tissues, and their expression varied greatly among different tissues and organs. All 20 AsAQP genes responded to salt, drought and cold stress. The NIP subfamily 6Ag0000836.1 gene was significantly upregulated under different abiotic stresses and could be further verified as a key candidate gene. The findings of this study provide a comprehensive list of members and their sequence characteristics of the AsAQP protein family, laying a solid theoretical foundation for further functional analysis of AsAQP in oats. This research also offers valuable reference for the creation of stress-tolerant oat varieties through genetic engineering techniques.

Primary, seminal and lateral roots of maize show type-specific growth and hydraulic responses to water deficit.

The water uptake capacity of a root system is determined by its architecture and hydraulic properties, which together shape the root hydraulic architecture. Here, we investigated root responses to water deficit (WD) in seedlings of a maize (Zea mays) hybrid line (B73H) grown in hydroponic conditions, taking into account the primary root (PR), the seminal roots (SR), and their respective lateral roots. WD was induced by various polyethylene glycol concentrations and resulted in dose-dependent inhibitions of axial and lateral root growth, lateral root formation, and hydraulic conductivity (Lpr), with slightly distinct sensitivities to WD between PR and SR. Inhibition of Lpr by WD showed a half-time of 5 to 6 min and was fully (SR) or partially (PR) reversible within 40 min. In the two root types, WD resulted in reduced aquaporin expression and activity, as monitored by mRNA abundance of 13 plasma membrane intrinsic protein (ZmPIP) isoforms and inhibition of Lpr by sodium azide, respectively. An enhanced suberization/lignification of the epi- and exodermis was observed under WD in axial roots and in lateral roots of the PR but not in those of SR. Inverse modeling revealed a steep increase in axial conductance in root tips of PR and SR grown under WD that may be due to the decreased growth rate of axial roots in these conditions. Overall, our work reveals that these root types show quantitative differences in their anatomical, architectural, and hydraulic responses to WD, in terms of sensitivity, amplitude and reversibility. This distinct functionalization may contribute to integrative acclimation responses of whole root systems to soil WD.

Rapid clearing and imaging of Mohs and melanoma surgical margins using a low-cost tissue processor.

Tissue clearing methods render biological tissues transparent while maintaining tissue structure, enabling visualization of entire tissues. Recent developments in tissue clearing have predominantly emphasized preserving intrinsic fluorescent proteins or aqueous-based tissue clearing and so typically involve complex procedures and long processing times. The utilization of tissue clearing protocols in standard of care histology settings has been less well explored, and protocols for rapid clearing of human tissue specimens are limited. This study presents a novel rapid clearing protocol and demonstrates a low-cost tissue processor for high volume rapid tissue clearing that can be intergraded into standard histology workflow. We demonstrate rapid clearing in dermatological specimens, including both nonmelanoma and melanoma excisions.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03–10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

Boron homeostasis affects Longan yield: a study of NIP and BOR boron transporter of two cultivars

BackgroundEssential micronutrient Boron (B) plays crucial roles in plant survival and reproduction but becomes toxic in higher quantities. Although plant cells have different B transport systems, B homeostasis is mainly maintained by two transporter protein families: B exporters (BOR) and nodulin-26-like intrinsic proteins (NIP). Their diversity and differential expression are responsible for varied B tolerance among plant varieties and species. Longan is a highly admired subtropical fruit with a rising market in China and beyond. In the present study, we cultured Shixia (SX) and Yiduo (YD), two differently characterized Longan cultivars, with foliar B spray. We analyzed their leaf physiology, fruit setting, B content, and boron transporter gene expression of various tissue samples. We also traced some of these genes' subcellular localization and overexpression effects.ResultsYD and SX foliage share similar microstructures, except the mesophyll cell wall thickness is double in YD. The B spray differently influenced their cellular constituents and growth regulators. Gene expression analysis showed reduced BOR genes expression and NIP genes differential spatiotemporal expression. Using green fluorescent protein, two high-expressing NIPs, NIP1 and NIP19, were found to translocate in the transformed tobacco leaves' cell membrane. NIPs transformation of SX pollen was confirmed using magnetic beads and quantified using a fluorescence microscope and polymerase chain reaction. An increased seed-setting rate was observed when YD was pollinated using these pollens. Between the DlNIP1 and DlNIP19 transformed SX pollen, the former germinated better with increasing B concentrations and, compared to naturally pollinated plants, had a better seed-setting rate in YD♀ × SX♂.ConclusionSX and YD Longan have different cell wall structures and react differently to foliar B spray, indicating distinct B tolerance and management. Two B transporter NIP genes were traced to localize in the plasma membrane. However, under high B concentrations, their differential expression resulted in differences in Jasmonic acid content, leading to differences in germination rate. Pollination of YD using these NIPs transformed SX pollen also showed NIP1 overexpression might overcome the unilateral cross incompatibility between YD♀ × SX♂ and can be used to increase Longan production.

Overproduction of mycotoxin biosynthetic enzymes triggers Fusarium toxisome-shaped structure formation via endoplasmic reticulum remodeling.

Mycotoxin deoxynivalenol (DON) produced by the Fusarium graminearum complex is highly toxic to animal and human health. During DON synthesis, the endoplasmic reticulum (ER) of F. graminearum is intensively reorganized, from thin reticular structure to thickened spherical and crescent structure, which was referred to as "DON toxisome". However, the underlying mechanism of how the ER is reorganized into toxisome remains unknown. In this study, we discovered that overproduction of ER-localized DON biosynthetic enzyme Tri4 or Tri1, or intrinsic ER-resident membrane proteins FgHmr1 and FgCnx was sufficient to induce toxisome-shaped structure (TSS) formation under non-toxin-inducing conditions. Moreover, heterologous overexpression of Tri1 and Tri4 proteins in non-DON-producing fungi F. oxysporum f. sp. lycopersici and F. fujikuroi also led to TSS formation. In addition, we found that the high osmolarity glycerol (HOG), but not the unfolded protein response (UPR) signaling pathway was involved in the assembly of ER into TSS. By using toxisome as a biomarker, we screened and identified a novel chemical which exhibited high inhibitory activity against toxisome formation and DON biosynthesis, and inhibited Fusarium growth species-specifically. Taken together, this study demonstrated that the essence of ER remodeling into toxisome structure is a response to the overproduction of ER-localized DON biosynthetic enzymes, providing a novel pathway for management of mycotoxin contamination.

An intrinsically semi-permeable PDMS nanosheet encapsulating adipose tissue-derived stem cells for enhanced angiogenesis.

Cell encapsulation devices are expected to be promising tools that can control the release of therapeutic proteins secreted from transplanted cells. The protein permeability of the device membrane is important because it allows the isolation of transplanted cells while enabling the effectiveness of the device. In this study, we investigated free-standing polymeric ultra-thin films (nanosheets) as an intrinsically semi-permeable membrane made from polydimethylsiloxane (PDMS). The PDMS nanosheet with a thickness of 600 nm showed intrinsic protein permeability, and the device fabricated with the PDMS nanosheet showed that VEGF secreted from implanted adipose tissue-derived stem cells (ASCs) could be released for at least 5 days. The ASC encapsulation device promoted angiogenesis and the development of granulation tissue 1 week after transplantation to the subcutaneous area of a mouse. This cell encapsulation device consisting of PDMS nanosheets provides a new method for pre-vascularization of the subcutaneous area in cell transplantation therapy.

Populus euphratica CPK21 interacts with heavy metal stress-associated proteins to mediate Cd tolerance in Arabidopsis

High cadmium (Cd, 50 µM) caused transcriptional upregulation of Ca2+-dependent protein kinase 21 (CPK21) in roots and leaves of Populus euphratica. The promoter region (1803 bp) of PeCPK21 was engineered to fuse PeCPK21-pro::GUS and transformed in Arabidopsis. The activity of GUS significantly increased in roots, cotyledons, and hypocotyls under 100 μM CdCl2 treatment. The 1599 bp coding sequence of PeCPK21 was transferred into Arabidopsis, and overexpression of PeCPK21 increased Cd tolerance as evidenced by less reduction in root length and plant growth under CdCl2 stress conditions (100 µM, 7 d). PeCPK21-interacting proteins were enriched by HaloTag pull-down and corresponding expression profiles after Cd exposure indicate that PeCPK21 interacts with heavy metal stress-associated proteins (HMAPs) to mediate Cd tolerance in transgenic Arabidopsis. PeCPK21 interacted with the Cd-regulated cation/heavy metal transport proteins annexin, OPT3, COPT5, and PDF2.2 to limit Cd uptake and accumulation in transgenic plants. PeCPK21 may also interact with the V-type proton ATPase subunits, VHA-B1, VHA-C, and AVA-P2, which were transcriptionally upregulated by Cd to accelerate vacuolar Cd compartmentalization. Cd-triggered H2O2 production was limited in plants overexpressing PeCPK21, which was due to the activated antioxidant enzymes. The interaction of PeCPK21 with the antioxidant enzymes TAPX, APX1, APX2, TRXM4, GPX3, CDSP32, and PRXQ, which showed transcriptional upregulation by Cd, could enhance antioxidant defense in transgenic Arabidopsis. It is also possible that PeCPK21 interacts with plasma membrane intrinsic proteins, PIP1–1, PIP2A, and PIP2–7, which had an Cd-enhanced transcript, to improve water status in transgenic Arabidopsis. In summary, we hypothesize that the calcium sensor PeCPK21 can attenuate Cd toxicity by interacting with HMAPs to effectively limit Cd accumulation, increase ROS scavenging, and improve water status in transgenic Arabidopsis.

Combined Soil Microorganism Amendments and Foliar Micronutrient Nanofertilization Increased the Production of Allium cepa L. through Aquaporin Gene Regulation.

The aim of this study was to investigate the impact of changes in aquaporin expression on the growth of onion (Allium cepa L.) plants when subjected to dual applications of microorganism-based soil amendments and foliar nanoencapsulated mineral nutrients. Multiple physiological parameters related to water, gas exchange, and nutrient content in leaf, root, and bulb tissues were determined. Additionally, the gene expression of aquaporins, specifically PIP1, PIP2 (aquaporin subfamily plasma membrane intrinsic protein), and TIP2 (aquaporin subfamily tonoplast intrinsic protein), was analyzed. The findings revealed that the foliar application of nutrients in a nanoencapsulated form significantly enhanced nutrient penetration, mobilization, and overall plant growth to a greater extent than free-form fertilizers. Amendments with microorganisms alone did not promote growth but influenced the production of secondary metabolites in the bulbs. The combination of microorganisms and nanoencapsulated mineral nutrients demonstrated synergistic effects, increasing dry matter, mineral content, and aquaporin gene expression. This suggests that aquaporins play a pivotal role in the transport of nutrients from leaves to storage organs, resulting in the overexpression of PIP2 aquaporins in bulbs, improved water uptake, and enhanced cell growth. Therefore, the combined treatment with microorganisms and nanoencapsulated mineral nutrients may be an optimal approach for enhancing onion productivity by regulating aquaporins under field conditions.

A bioengineered anti-VEGF protein with high affinity and high concentration for intravitreal treatment of wet age-related macular degeneration.

Intravitreal (IVT) injection of anti-vascular endothelial growth factor (anti-VEGF) has greatly improved the treatment of many retinal disorders, including wet age-related macular degeneration (wAMD), which is the third leading cause of blindness. However, frequent injections can be difficult for patients and may lead to various risks such as elevated intraocular pressure, infection, and retinal detachment. To address this issue, researchers have found that IVT injection of anti-VEGF proteins at their maximally viable concentration and dose can be an effective strategy. However, the intrinsic protein structure can limit the maximum concentration due to stability and solution viscosity. To overcome this challenge, we developed a novel anti-VEGF protein called nanoFc by fusing anti-VEGF nanobodies with a crystallizable fragment (Fc). NanoFc has demonstrated high binding affinity to VEGF165 through multivalency and potent bioactivity in various bioassays. Furthermore, nanoFc maintains satisfactory chemical and physical stability at 4°C over 1 month and is easily injectable at concentrations up to 200 mg/mL due to its unique architecture that yields a smaller shape factor. The design of nanoFc offers a bioengineering strategy to ensure both strong anti-VEGF binding affinity and high protein concentration, with the goal of reducing the frequency of IV injections.

Global characterization of OsPIP aquaporins reveals that the H2O2 transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins (PIPs) are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress. To elucidate their roles in plant immunity to pathogen attack, we characterized the expression patterns, subcellular localizations, and H2O2-transport ability of 11 OsPIPs in rice (Oryza sativa), and identified OsPIP2;6 as necessary for rice disease resistance. OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H2O2. Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae, indicating a positive function in plant immunity. OsPIP2;6 interacts with OsPIP2;2, which has been reported to increase rice resistance to pathogens via H2O2 transport. Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Disparate viral pandemics from COVID19 to monkeypox and beyond: a simple, effective and universal therapeutic approach hiding in plain sight.

The field of antiviral therapeutics is fixated on COVID19 and rightly so as the fatalities at the height of the pandemic in the United States were almost 1,000,000 in a twelve month period spanning parts of 2020/2021.A coronavirus called SARS-CoV2 is the causative virus. Development of a vaccine through molecular biology approaches with mRNA as the inducer of virus spike protein has played a major role in driving down mortality and morbidity. Antivirals have been of marginal value in established infections at the level of hospitalization. Thus, the current focus is on early symptomatic infection of about the first five days. The Pfizer drug paxlovid which is composed of nirmatrelvir, a peptidomimetic protease inhibitor of SARS-CoV2 Mpro enzyme, and ritonavir to retard degradation of nirmatrelvir, is the current FDA recommended treatment of early COVID19. There is no evidence of broad antiviral activity of paxlovid against other diverse viruses such as the influenza virus, poxviruses, as well as a host of respiratory viruses. Although type I interferons (IFNs) are effective against SARS-CoV2 in cell cultures and in early COVID19 infections, they have not been broadly recommended as therapeutics for COVID19. We have developed stable peptidomimetics of both types I and II IFNs based on our noncanonical model of IFN signaling involving the C-terminus of the IFNs. We have also identified two members of intracellular checkpoint inhibitors called suppressors of cytokine signaling (SOCS), SOCS1 and SOCS3 (SOCS1/3), and shown that they are virus induced intrinsic virulence proteins with activity against IFN signaling enzymes JAK2 and TYK2. We developed a peptidomimetic antagonist, based on JAK2 activation loop, against SOCS1/3 and showed that it synergizes with the IFN mimetics for potent broad spectrum antiviral activity without the toxicity of intact IFN molecules. IFN mimetics and the SOCS1/3 antagonist should have an advantage over currently used antivirals in terms of safety and potency against a broad spectrum of viruses.

Rice tonoplast intrinsic protein member OsTIP1;2 confers tolerance to arsenite stress

The International Agency for Research on Cancer categorizes arsenic (As) as a group I carcinogen. Arsenic exposure significantly reduces growth, development, metabolism, and crop yield. Tonoplast intrinsic proteins (TIPs) belong to the major intrinsic protein (MIP) superfamily and transport various substrates, including metals/metalloids. Our study aimed to characterize rice OsTIP1;2 in As[III] stress response. The gene expression analysis showed that the OsTIP1;2 expression was enhanced in roots on exposure to As[III] treatment. The heterologous expression of OsTIP1;2 in S. cerevisiae mutant lacking YCF1 (ycf1∆) complemented the As[III] transport function of the YCF1 transporter but not for boron (B) and arsenate As[V], indicating its substrate selective nature. The ycf1∆ mutant expressing OsTIP1;2 accumulated more As than the wild type (W303–1A) and ycf1∆ mutant strain carrying the pYES2.1 vector. OsTIP1;2 activity was partially inhibited in the presence of the aquaporin (AQP) inhibitors. The subcellular localization studies confirmed that OsTIP1;2 is localized to the tonoplast. The transient overexpression of OsTIP1;2 in Nicotiana benthamiana leaves resulted in increased activities of enzymatic and non-enzymatic antioxidants, suggesting a potential role in mitigating oxidative stress induced by As[III]. The transgenic N. tabacum overexpressing OsTIP1;2 displayed an As[III]-tolerant phenotype, with increased fresh weight and root length than the wild-type (WT) and empty vector (EV line). The As translocation factor (TF) for WT and EV was around 0.8, while that of OE lines was around 0.4. Moreover, the OE line bioconcentration factor (BCF) was more than 1. Notably, the reduced TF and increased BCF in the OE line imply the potential of OsTIP1;2 for phytostabilization.

Analysis of Carica papaya Informs Lineage-Specific Evolution of the Aquaporin (AQP) Family in Brassicales.

Aquaporins (AQPs), a type of intrinsic membrane proteins that transport water and small solutes across biological membranes, play crucial roles in plant growth and development. This study presents a first genome-wide identification and comparative analysis of the AQP gene family in papaya (Carica papaya L.), an economically and nutritionally important fruit tree of tropical and subtropical regions. A total of 29 CpAQP genes were identified, which represent five subfamilies, i.e., nine plasma intrinsic membrane proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), seven NOD26-like intrinsic proteins (NIPs), two X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). Although the family is smaller than the 35 members reported in Arabidopsis, it is highly diverse, and the presence of CpXIP genes as well as orthologs in Moringa oleifera and Bretschneidera sinensis implies that the complete loss of the XIP subfamily in Arabidopsis is lineage-specific, sometime after its split with papaya but before Brassicaceae-Cleomaceae divergence. Reciprocal best hit-based sequence comparison of 530 AQPs and synteny analyses revealed that CpAQP genes belong to 29 out of 61 identified orthogroups, and lineage-specific evolution was frequently observed in Brassicales. Significantly, the well-characterized NIP3 group was completely lost; lineage-specific loss of the NIP8 group in Brassicaceae occurred sometime before the divergence with Cleomaceae, and lineage-specific loss of NIP2 and SIP3 groups in Brassicaceae occurred sometime after the split with Cleomaceae. In contrast to a predominant role of recent whole-genome duplications (WGDs) on the family expansion in B. sinensis, Tarenaya hassleriana, and Brassicaceae plants, no recent AQP repeats were identified in papaya, and ancient WGD repeats are mainly confined to the PIP subfamily. Subfamily even group-specific evolution was uncovered via comparing exon-intron structures, conserved motifs, the aromatic/arginine selectivity filter, and gene expression profiles. Moreover, down-regulation during fruit ripening and expression divergence of duplicated CpAQP genes were frequently observed in papaya. These findings will not only improve our knowledge on lineage-specific family evolution in Brassicales, but also provide valuable information for further studies of AQP genes in papaya and species beyond.

Mechanistic Basis of Silicon Mediated Cold Stress Tolerance in Alfalfa (Medicago sativa L.)

AbstractCold stress (CS) impact on crops is one of the critical constraints for sustainable and smart agricultural production. CS adversely affects plants leading to growth retardation, necrosis, chlorosis, and significant yield loss. The objective of this study was to explore the mechanistic basis of silicon (Si) in enhancing CS tolerance in alfalfa plants. The fluorescence staining indicated that Si-reduced the intensity of CS-induced superoxide radical (O2•–) and hydrogen peroxide (H2O2) generation in plants that improved plant photosynthesis, cellular integrity, and alfalfa biomass production under CS. The exogenous supplementation of Si significantly restored the endogenous Si status accompanied by the upregulation of NIP (nodulin 26-like intrinsic protein) genes NIP2, NIP5;1, and NIP6;1 in alfalfa. The elemental concentration analysis revealed that exogenous silicon (E-Si) triggers the increase of calcium (Ca), magnesium (Mg), and sulfur (S) in plants subjected to Si-supplementation compared to the plants cultivated without Si under CS. The application of Si significantly increased the activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Furthermore, Si significantly enhanced the expression of CS-responsive candidate genes including ICE1, CBF1/DREB1C, CBF2/DREB1B, CBF3/DREB1A, COR15A, COR47, and KIN1 in alfalfa. These findings together provide mechanistic insights into Si-involving CS tolerance in alfalfa. This eco-friendly SC management strategy using Si treatment can be useful to plant breeders and farmers for developing CS-resilient smart alfalfa production through breeding program.