Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

Discovery of Five New Ethylene-Forming Enzymes for Clean Production of Ethylene in E. coli.

Ethylene is an essential platform chemical with a conjugated double bond, which can produce many secondary chemical products through copolymerisation. At present, ethylene production is mainly from petroleum fractionation and cracking, which are unsustainable in the long term, and harmful to our environment. Therefore, a hot research field is seeking a cleaner method for ethylene production. Based on the model ethylene-forming enzyme (Efe) AAD16440.1 (6vp4.1.A) from Pseudomonas syringae pv. phaseolicol, we evaluated five putative Efe protein sequences using the data derived from phylogenetic analyses and the conservation of their catalytic structures. Then, pBAD expression frameworks were constructed, and relevant enzymes were expressed in E. coli BL21. Finally, enzymatic activity in vitro and in vivo was detected to demonstrate their catalytic activity. Our results show that the activity in vitro measured by the conversion of α-ketoglutarate was from 0.21-0.72 µmol ethylene/mg/min, which varied across the temperatures. In cells, the activity of the new Efes was 12.28-147.43 µmol/gDCW/h (DCW, dry cellular weight). Both results prove that all the five putative Efes could produce ethylene.

Identification and Characterization of Malate Dehydrogenases in Tomato (Solanum lycopersicum L.).

Malate dehydrogenase, which facilitates the reversible conversion of malate to oxaloacetate, is essential for energy balance, plant growth, and cold and salt tolerance. However, the genome-wide study of the MDH family has not yet been carried out in tomato (Solanum lycopersicum L.). In this study, 12 MDH genes were identified from the S. lycopersicum genome and renamed according to their chromosomal location. The tomato MDH genes were split into five groups based on phylogenetic analysis and the genes that clustered together showed similar lengths, and structures, and conserved motifs in the encoded proteins. From the 12 tomato MDH genes on the chromosomes, three pairs of segmental duplication events involving four genes were found. Each pair of genes had a Ka/Ks ratio < 1, indicating that the MDH gene family of tomato was purified during evolution. Gene expression analysis exhibited that tomato MDHs were differentially expressed in different tissues, at various stages of fruit development, and differentially regulated in response to abiotic stresses. Molecular docking of four highly expressed MDHs revealed their substrate and co-factor specificity in the reversible conversion process of malate to oxaloacetate. Further, co-localization of tomato MDH genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. This study lays the foundation for functional analysis of MDH genes and genetic improvement in tomato.

Histone Deacetylase (HDAC) Gene Family in Allotetraploid Cotton and Its Diploid Progenitors: In Silico Identification, Molecular Characterization, and Gene Expression Analysis under Multiple Abiotic Stresses, DNA Damage and Phytohormone Treatments.

Histone deacetylases (HDACs) play a significant role in a plant's development and response to various environmental stimuli by regulating the gene transcription. However, HDACs remain unidentified in cotton. In this study, a total of 29 HDACs were identified in allotetraploid Gossypium hirsutum, while 15 and 13 HDACs were identified in Gossypium arboretum and Gossypium raimondii, respectively. Gossypium HDACs were classified into three groups (reduced potassium dependency 3 (RPD3)/HDA1, HD2-like, and Sir2-like (SRT) based on their sequences, and Gossypium HDACs within each subgroup shared a similar gene structure, conserved catalytic domains and motifs. Further analysis revealed that Gossypium HDACs were under a strong purifying selection and were unevenly distributed on their chromosomes. Gene expression data revealed that G. hirsutum HDACs were differentially expressed in various vegetative and reproductive tissues, as well as at different developmental stages of cotton fiber. Furthermore, some G. hirsutum HDACs were co-localized with quantitative trait loci (QTLs) and single-nucleotide polymorphism (SNPs) of fiber-related traits, indicating their function in fiber-related traits. We also showed that G. hirsutum HDACs were differentially regulated in response to plant hormones (abscisic acid (ABA) and auxin), DNA damage agent (methyl methanesulfonate (MMS)), and abiotic stresses (cold, salt, heavy metals and drought), indicating the functional diversity and specification of HDACs in response to developmental and environmental cues. In brief, our results provide fundamental information regarding G. hirsutum HDACs and highlight their potential functions in cotton growth, fiber development and stress adaptations, which will be helpful for devising innovative strategies for the improvement of cotton fiber and stress tolerance.

Healthcare professionals' understanding and perception of drug-related issues linked to off-label pharmacological medications used for COVID-19.

BACKGROUND: To evaluate the opinions of healthcare workers regarding drug therapy problems linked to anti-infective medicines used for the treatment of COVID-19 infection in Pakistan. RESEARCH METHODOLOGY: This cross-sectional study was conducted from January to October 2022 using a self-administered questionnaire developed by the authors, having three sections: demographics, knowledge, and perception. The study was validated by research experts and pilot-tested on 30 subjects. The study included medical doctors, nurses, pharmacists from Punjab's government and private medical institutes. RESULTS: In this study, 382 of 400 participants replied. The mean knowledge score was 9.52 (SD 2.97), showing that participants had adequate knowledge of COVID-19 pharmacological interventions. The knowledge scores were significantly higher of those who treated COVID-19 patients and were from Lahore. Many doctors considered that elderly patients and those with blood disorders were at greater risk of experiencing drug-related problems. Most pharmacists support electronic prescription systems. Many doctors thought the lack of unified treatment guidelines, multiple prescribers, and self-medication were key obstacles in managing COVID-19 patients. CONCLUSION: Most respondents had adequate knowledge. Older patients with comorbidities are at risk of adverse effects. Self-medication, polypharmacy, and multiple prescriptions can lead to misdiagnosis and complications. Electric prescriptions, team effort, and training programs can decrease these issues.

Genome-wide analysis of Glutathione peroxidase (GPX) gene family in Chickpea (Cicer arietinum L.) under salinity stress.

Chickpea is the second most widely grown legume in the world. Its cultivation is highly affected by saline soils. Salt stress damages its all growth stages from germination to maturity. It has a huge genetic diversity containing adaptation loci that can help produce salt-tolerant cultivars. The glutathione peroxidase (GPX) gene family plays an important role in regulating plant response to abiotic stimuli and protects cells from oxidative damage. In current research, the role of GPX genes is studied for inducing salt tolerance in chickpea. This study identifies the GPX gene family in Cicer arietinum. In response to the NaCl stress, the gene expression profiles of CaGPX3 were examined using real-time qRT-PCR. The results of phylogenetic analysis show that CaGPX genes have an evolutionary relationship with monocots, dicots, chlorophytes, and angiosperms. Gene structure analysis showed that CaGPX3, CaGPX4, and CaGPX5 have six, CaGPX2 has five, and CaGPX1 contains 9 exons. According to the Ka and Ks analysis chickpea has one pair of duplicated genes of GPX and the duplication was tandem with negative (purifying) selection Ka < Ks (<1). In-silico gene expression analysis revealed that CaGPX3 is a salt stress-responsive gene among all other five GPX members in chickpea. The qRT-PCR results showed that the CaGPX3 gene expression was co-ordinately regulated under salt stress conditions, confirming CaGPX3's key involvement in salt tolerance.

Unraveling two decades of phyllosphere endophytes: tracing research trends and insights through visualized knowledge maps, with emphasis on microbial interactions as emerging frontiers.

Phyllosphere endophytes play a critical role in a myriad of biological functions, such as maintaining plant health and overall fitness. They play a determinative role in crop yield and quality by regulating vital processes, such as leaf functionality and longevity, seed mass, apical growth, flowering, and fruit development. This study conducted a comprehensive bibliometric analysis aiming to review the prevailing research trajectories in phyllosphere endophytes and harness both primary areas of interest and emerging challenges. A total of 156 research articles on phyllosphere endophytes, published between 2002 and 2022, were retrieved from the Web of Science Core Collection (WoSCC). A systematic analysis was conducted using CiteSpace to visualize the evolution of publication frequency, the collaboration network, the co-citation network, and keywords co-occurrence. The findings indicated that initially, there were few publications on the topic of phyllosphere endophytes. However, from 2011 onwards, there was a notable increase in the number of publications on phyllosphere endophytes, gaining worldwide attention. Among authors, Arnold, A Elizabeth is widely recognized as a leading author in this research area. In terms of countries, the USA and China hold the highest rankings. As for institutional ranking, the University of Arizona is the most prevalent and leading institute in this particular subject. Collaborative efforts among the authors and institutions tend to be confined to small groups, and a large-scale collaborative network needs to be established. This study identified the influential journals, literature, and hot research topics. These findings also highlight the interconnected nature of key themes, e.g., phyllosphere endophyte research revolves around the four pillars: diversity, fungal endophytes, growth, and endophytic fungi. This study provides an in-depth perspective on phyllosphere endophytes studies, revealing the identification of biodiversity and microbial interaction of phyllosphere endophytes as the principal research frontiers. These analytical findings not only elucidate the recent trajectory of phyllosphere endophyte research but also provide invaluable insights for similar studies and their potential applications on a global scale.

Clinical Disease Characteristics and Treatment Trajectories Associated with Mortality among COVID-19 Patients in Punjab, Pakistan.

BACKGROUND: Data on Pakistani COVID-19 patient mortality predictors is limited. It is essential to comprehend the relationship between disease characteristics, medications used, and mortality for better patient outcomes. METHODS: The medical records of confirmed cases in the Lahore and Sargodha districts were examined using a two-stage cluster sampling from March 2021 to March 2022. Demographics, signs and symptoms, laboratory findings, and pharmacological medications as mortality indicators were noted and analyzed. RESULTS: A total of 288 deaths occurred out of the 1000 cases. Death rates were higher for males and people over 40. Most of those who were mechanically ventilated perished (OR: 124.2). Dyspnea, fever, and cough were common symptoms, with a significant association amid SpO2 < 95% (OR: 3.2), RR > 20 breaths/min (OR: 2.5), and mortality. Patients with renal (OR: 2.3) or liver failure (OR: 1.5) were at risk. Raised C-reactive protein (OR: 2.9) and D-dimer levels were the indicators of mortality (OR: 1.6). The most prescribed drugs were antibiotics, (77.9%), corticosteroids (54.8%), anticoagulants (34%), tocilizumab (20.3%), and ivermectin (9.2%). CONCLUSIONS: Older males having breathing difficulties or signs of organ failure with raised C-reactive protein or D-dimer levels had high mortality. Antivirals, corticosteroids, tocilizumab, and ivermectin had better outcomes; antivirals were associated with lower mortality risk.

Transcriptome Profiling Reveals Role of MicroRNAs and Their Targeted Genes during Adventitious Root Formation in Dark-Pretreated Micro-Shoot Cuttings of Tetraploid Robinia pseudoacacia L.

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L-1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.

Differential response of bacterial diversity and community composition to different tree ages of pomelo under red and paddy soils.

Rhizosphere soil microbial communities substantially impact plant growth by regulating the nutrient cycle. However, dynamic changes in soil microbiota under different tree ages have received little attention. In this study, changes in soil physicochemical properties, as well as bacterial diversity and community structures (by high-throughput Illumina MiSeq sequencing), were explored in pomelo trees of different ages (i.e., 10, 20, and 30 years) under red and paddy soils cultivated by farmers with high fertilizer input. Moreover, soil factors that shape the bacterial community, such as soil pH, AP (available phosphorous), AK (available potassium), and AN (available nitrogen), were also investigated. Results showed that pH significantly decreased, while AP, AK, and AN increased with increasing tree age under red soil. For paddy soil, pH was not changed, while AP was significantly lower under 10-year-old pomelo trees, and AK and AN contents were minimum under 30-year-old pomelo trees. Both soil types were dominated by Proteobacteria, Acidobacteria, and Actinobacteria and showed contrasting patterns of relative abundance under different tree age groups. Bacterial richness and diversity decreased with increasing tree age in both soil types. Overall, bacterial community composition was different under different tree ages. RDA analysis showed that soil pH, AP, and AN in red soil, and pH and AP in paddy soil showed the most significant effects in changing the bacterial community structure. A random forest model showed Sinomonas and Streptacidiphilus in red soil, while Actinoallomurus and Microbacterium in paddy soil were the most important genera explaining the differences among different age groups. The ternary plot further revealed that genera enrichment for Age_30 was higher than that for Age_10 and Age_20 in red soil, whereas specific genera enrichment decreased with increasing tree age under paddy soil. Co-occurrence network revealed that bacterial species formed a complex network structure with increasing tree age, indicating a more stable microbial association under 20 and 30 years than 10-year-old pomelo trees. Hence, contrasting patterns of changes in soil physicochemical properties and soil microbial communities were recorded under different tree ages, and tree ages significantly affected the bacterial community structure and richness. These findings provide valuable information regarding the importance of microbes for the sustainable management of pomelo orchards by optimizing fertilizer input for different ages of trees.

Response of Fungal Diversity, Community Composition, and Functions to Nutrients Management in Red Soil.

Soil fungi play a critical role in plant performance and soil nutrient cycling. However, the understanding of soil fungal community composition and functions in response to different nutrients management practices in red soils remains largely unknown. Here, we investigated the responses of soil fungal communities and functions under conventional farmer fertilization practice (FFP) and different nutrient management practices, i.e., optimization of NPK fertilizer (O) with soil conditioner (O + C), with lime and mushroom residue (O + L + M), and with lime and magnesium fertilizer (O + L + Mg). Illumina high-throughput sequencing was used for fungal identification, while the functional groups were inferred with FUNGuild. Nutrient management practices significantly raised the soil pH to 4.79-5.31 compared with FFP (3.69), and soil pH had the most significant effect (0.989 ***) on fungal communities. Predominant phyla, including Ascomycota, Basidiomycota, and Mortierellomycota were identified in all treatments and accounted for 94% of all fungal communities. The alpha diversity indices significantly increased under nutrients management practices compared with FFP. Co-occurrence network analysis revealed the keystone fungal species in the red soil, i.e., Ascomycota (54.04%), Basidiomycota (7.58%), Rozellomycota (4.55%), and Chytridiomycota (4.04%). FUNGuild showed that the relative abundance of arbuscular mycorrhizal fungi and ectomycorrhizal fungi was higher, while pathogenic fungi were lower under nutrient management practices compared with FFP. Our findings have important implications for the understanding of improvement of acidic soils that could significantly improve the soil fungal diversity and functioning in acidic soils.

Formation of Common Mycorrhizal Networks Significantly Affect Plant Biomass and Soil Properties of the Neighboring Plants under Various Nitrogen Levels.

Common mycorrhizal networks (CMNs) allow the transfer of nutrients between plants, influencing the growth of the neighboring plants and soil properties. Cleistogene squarrosa (C. squarrosa) is one of the most common grass species in the steppe ecosystem of Inner Mongolia, where nitrogen (N) is often a key limiting nutrient for plant growth, but little is known about whether CMNs exist between neighboring individuals of C. squarrosa or play any roles in the N acquisition of the C. squarrosa population. In this study, two C. squarrosa individuals, one as a donor plant and the other as a recipient plant, were planted in separate compartments in a partitioned root-box. Adjacent compartments were separated by 37 µm nylon mesh, in which mycorrhizal hyphae can go through but not roots. The donor plant was inoculated with arbuscular mycorrhizal (AM) fungi, and their hyphae potentially passed through nylon mesh to colonize the roots of the recipient plant, resulting in the establishment of CMNs. The formation of CMNs was verified by microscopic examination and 15N tracer techniques. Moreover, different levels of N fertilization (N0 = 0, N1 = 7.06, N2 = 14.15, N3 = 21.19 mg/kg) were applied to evaluate the CMNs' functioning under different soil nutrient conditions. Our results showed that when C. squarrosa-C. squarrosa was the association, the extraradical mycelium transferred the 15N in the range of 45-55% at different N levels. Moreover, AM fungal colonization of the recipient plant by the extraradical hyphae from the donor plant significantly increased the plant biomass and the chlorophyll content in the recipient plant. The extraradical hyphae released the highest content of glomalin-related soil protein into the rhizosphere upon N2 treatment, and a significant positive correlation was found between hyphal length and glomalin-related soil proteins (GRSPs). GRSPs and soil organic carbon (SOC) were significantly correlated with mean weight diameter (MWD) and helped in the aggregation of soil particles, resulting in improved soil structure. In short, the formation of CMNs in this root-box experiment supposes the existence of CMNs in the typical steppe plants, and CMNs-mediated N transfer and root colonization increased the plant growth and soil properties of the recipient plant.