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The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyBY276H (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHBG767R variant in transgenic Arabidopsis seedlings. We show that YHBG767R elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHBG767R. However, as expected for a cytoplasm-tethered phyB, YHBG767R elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHBG767R also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell‐type‐specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time‐course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single‐molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots. Summary statement: Transcriptome analysis of nuclear and cytoplasmic fractions in response to nitrate revealed transcripts with specific subcellular localization, revealing a novel layer of gene regulation. The dynamic interplay between mRNA localization, synthesis, and decay is crucial for the adaptive response of Arabidopsis roots to nitrate. [ABSTRACT FROM AUTHOR]

Main conclusion: MiR171d and SCL6 are induced by the plant hormone auxin. MiR171d negatively regulates the expression of SCL6, thereby regulating the growth and development of plant adventitious roots. Under natural conditions, it is difficult to induce rooting in the process of propagating Acer rubrum L. via branches, which seriously limits its wide application in landscaping construction. In this study, the expression of Ar-miR171d was downregulated and the expression of ArSCL6 was upregulated after 300 mg/L indole-3-butyric acid (IBA) treatment. The transient interaction of Ar-miR171d and ArSCL6 in tobacco cells further confirmed their cleavage activity. Transgenic function verification confirmed that OE-Ar-miR171d inhibited adventitious root (AR) development, while OE-ArSCL6 promoted AR development. Tissue-specific expression verification of the ArSCL6 promoter demonstrated that it was specifically expressed in the plant root and leaf organs. Subcellular localization and transcriptional activation assays revealed that both ArSCL6 and ArbHLH089 were located in the nucleus and exhibited transcriptional activation activity. The interaction between the two was verified by bimolecular fluorescence complementarity (BIFC) experiments. These results help elucidate the regulatory mechanisms of the Ar-miR171d-ArSCL6 module during the propagation of A. rubrum and provide a molecular basis for the rooting of branches. [ABSTRACT FROM AUTHOR]

Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle. [ABSTRACT FROM AUTHOR]

Background: MYB transcription factors regulate anthocyanin biosynthesis across numerous plant species. However, comprehensive genome-wide investigations regarding the R2R3-MYB gene family and its involvement in regulating anthocyanin biosynthesis in the red and white fruit color morphs of Fragaria pentaphylla remain scarce. Results: A total of 101 FpR2R3-MYB genes were identified from the F. pentaphylla genome and were divided into 34 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were particularly conserved among the FpR2R3-MYB genes, especially members within the same subgroup. The FpR2R3-MYB genes were distributed over seven F. pentaphylla chromosomes. Analysis of gene duplication events revealed five pairs of tandem duplication genes and 16 pairs of segmental duplication genes, suggesting that segmental duplications are the major pattern for expansion of the FpR2R3-MYB gene family expansion in F. pentaphylla. Cis-regulatory elements of the FpR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponse. Based on the analysis of the FpR2R3-MYB gene family and transcriptome sequencing (RNA-seq) data, FpMYB9 was identified as a key transcription factor involved in the regulation of anthocyanin synthesis in F. pentaphylla fruits. The expression of FpMYB9 increases significantly during the ripening stage of red fruits, as confirmed by reverse transcription quantitative real-time PCR. In addition, subcellular localization experiments further confirmed the nuclear presence of FpMYB9, supporting its role as a transcription factor involved in anthocyanin biosynthesis. Conclusion: Our results showed that the FpR2R3-MYB genes are highly conserved and play important roles in the anthocyanin biosynthesis in F. pentaphylla fruits. Our results also provide a compelling basis for further understanding of the regulatory mechanism underlying the role of FpMYB9 in anthocyanin formation in F. pentaphylla fruits. [ABSTRACT FROM AUTHOR]

Pear chlorotic leaf spot‐associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single‐stranded, negative‐sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens‐mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self‐interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell‐to‐cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N‐terminal 1–89 amino acids of the P5 were responsible for the self‐interaction ability and RSS activity, for which the signal peptide at positions 1–19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement. [ABSTRACT FROM AUTHOR]

Protein S‐acylation or palmitoylation is a reversible post‐translational modification that influences many proteins encoded in plant genomes. Exciting progress in the past 3 years demonstrates that S‐acylation modulates subcellular localization, interacting profiles, activity, or turnover of substrate proteins in plants, participating in developmental processes and responses to abiotic or biotic stresses. In this review, we summarize and discuss the role of S‐acylation in the targeting of substrate proteins. We highlight complex roles of S‐acylation in receptor signaling. We also point out that feedbacks of protein S‐acyl transferase by signaling initiated from their substrate proteins may be a recurring theme. Finally, the reversibility of S‐acylation makes it a rapid and efficient way to respond to environmental cues. Future efforts on exploring these important aspects of S‐acylation will give a better understanding of how plants enhance their fitness under ever changing and often harsh environments. [ABSTRACT FROM AUTHOR]

Plant U-box genes play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance; however, their functions in coffee (Coffea canephora L.) remain largely unexplored. In this study, we identified 47 CcPUB genes in the C. canephora L. genome, clustering them into nine groups via phylogenetic tree. The CcPUB genes were unevenly distributed across the 11 chromosomes of C. canephora L., with the majority (11) on chromosome 2 and none on chromosome 8. The cis-acting elements analysis showed that CcPUB genes were involved in abiotic and biotic stresses, phytohormone responsive, and plant growth and development. RNA-seq data revealed diverse expression patterns of CcPUB genes across leaves, stems, and fruits tissues. qRT-PCR analyses under dehydration, low temperature, SA, and Colletotrichum stresses showed significant up-regulation of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 in leaves. Furthermore, subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. This study provides valuable insights into the roles of PUB genes in stress responses and phytohormone signaling in C. canephora L., and provided basis for functional characterization of PUB genes in C. canephora L. [ABSTRACT FROM AUTHOR]

Strawberry is frequently attacked by mites, which directly affects the yield and quality of this fruit species. The WRKY Group III transcription factors (TFs) play an important role in plant tolerance to biotic sources of stress, such as pathogens and insect pests. In this study, six Group III WRKY TFs (FaWRKY25, FaWRKY31, FaWRKY32, FaWRKY43, FaWRKY44, and FaWRKY45) were identified in strawberry. A phylogenetic analysis showed that the six WRKY III TFs were divided into two clades and all had a conserved WRKYGQK domain and the C-X7-C-X23-H-T-C zinc finger motif. An interaction network analysis revealed that FaWRKY44 was co-expressing with FaWRKY25 and FaWRKY45. The expression patterns showed that the WRKY Group III genes responded to plant hormones and mite infestation in strawberry. To further verify the role of FaWRKY25 in plant resistance to mites, we cloned the FaWRKY25 gene and overexpressed it in transgenic plants. An in vivo subcellular localization analysis indicated that the FaWRKY25 protein was localized in the nucleus. Fewer mites were also detected on the wild-type plants than on FaWRKY25-overexpressing transgenic plants, suggesting that FaWRKY25 negatively regulates the resistance of strawberry to mites. The present study advances our understanding on a potential target that mites use to manipulate host plant defenses. [ABSTRACT FROM AUTHOR]

Dryopteris fragrans (L.) Schott has anti-inflammatory and antioxidant properties, and terpenoids are important components of its active constituents. The methyl-D-erythritol 4-phosphate (MEP) pathway is one of the major pathways for the synthesis of terpene precursors in plants, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in this pathway. DXS has been shown to be associated with increased stress tolerance in plants. In this experiment, two DXS genes were extracted from the D. fragrans transcriptome and named DfDXS1 and DfDXS2. Based on phylogenetic tree and conserved motif analyses, DXS was shown to be highly conserved evolutionarily and its localization to chloroplasts was determined by subcellular localization. Prokaryotic expression results showed that the number and growth status of recombinant colonies were better than the control under 400 mM NaCl salt stress and 800 mM mannitol-simulated drought stress. In addition, the DfDXS1 and DfDXS2 transgenic tobacco plants showed improved resistance to drought and salt stress. DfDXS1 and DfDXS2 responded strongly to methyl jasmonate (MeJA) and PEG-mimicked drought stress following exogenous hormone and abiotic stress treatments of D. fragrans. The transcriptional active sites were investigated by dual luciferase and GUS staining assays, and the results showed that the STRE element (AGGGG), the ABRE element (ACGTGGC), and the MYC element (CATTTG) were the important transcriptional active sites in the promoters of the two DXS genes, which were closely associated with hormone response and abiotic stress. These results suggest that the DfDXS gene of D. fragrans plays an important role in hormone signaling and response to stress. This study provides a reference for analyzing the molecular mechanisms of stress tolerance in D. fragrans. [ABSTRACT FROM AUTHOR]

Background: The Flavonoid 3'-hydroxylase gene(F3'H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. Results: In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3'H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3'H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. Conclusions: The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke. [ABSTRACT FROM AUTHOR]

【Objective】 The DHNS (1,4-dihydroxy-2-naphthoyl-CoA synthase) gene in potatoes is a potentially important in the biosynthesis of glycoalkaloid metabolism in Solanaceae plants. Researching and verifying of the function of the potato DHNS gene can provide a source of genes and materials for the selection of low-glycoalkaloid potato varieties (lines) . 【Method】 The full-length sequence of ScDHNS cDNA was cloned from the wild potato species Solanum chacoense using the RACE technique. This sequence was analyzed for bioinformatics and subcellular localization, and its function was verified by constructing an overexpression vector pBWA (V) HS-DHNS to transform cultivated potato. 【Result】 The read open frame of ScDHNS cDNA sequence is 1 023 bp, encodes 340 amino acids, has a molecular weight of 37.34 kD and an isoelectric point of 8.592, and contains a typical ECH domain. It belongs to the enoyl hydratase/acetyl-CoA superfamily and is present in the genomes of plants such as Brachypodium distachyon and Medicago truncatula with homologous genes, showing gene expansion and contraction events. After the overexpression of the ScDHNS gene, it was found that the expressions of the transformed plants ScDHNS and SGT1 were significantly upregulated, and the expressions were notably higher than those in wild-type (WT) potato plants. Additionally, the total glycoalkaloid content in the corresponding transformed plants was significantly higher than that in WT potato plants, reaching a maximum of 364.3 mg/kg, which is 2.4 times that of the control. Subcellular localization results indicates that ScDHNS is localized in the peroxisome. 【Conclusion】 The ScDHNS gene in potato may regulate the expression of the key gene SGT1, which is involved in glycoalkaloid synthesis. It influences glycoalkaloid synthesis through the β-oxidation and mevalonate pathways. This gene is significantly associated with the subcellular compartmentalization of glycoalkaloids and has considerable application value in the cultivation of potato varieties with reduced glycoalkaloid content. [ABSTRACT FROM AUTHOR]

The subcellular localization of messenger RNA (mRNA) not only helps us to understand the localization regulation of gene expression but also helps to understand the relationship between RNA localization pattern and human disease mechanism, which has profound biological and medical significance. Several predictors have been proposed for predicting the subcellular localization of mRNA. However, there is still considerable room for improvement in their predictive performance, especially regarding multi-label prediction. This study proposes a novel multi-label predictor, DRpred, for mRNA subcellular localization prediction. This predictor first utilizes Bayesian networks to capture the dependencies among labels. Subsequently, it combines these dependencies with features extracted from mRNA sequences using Word2vec, forming the input for the predictor. Finally, it employs a neural network combining BiLSTM and an attention mechanism to capture the internal relationships of the input features for mRNA subcellular localization. The experimental validation on an independent test set demonstrated that DRpred obtained a competitive predictive performance in multi-label prediction and outperformed state-of-the-art predictors in predicting single subcellular localizations, obtaining accuracies of 82.14%, 93.02%, 80.37%, 94.00%, 90.58%, 84.53%, 82.01%, 79.71%, and 85.67% for the chromatin, cytoplasm, cytosol, exosome, membrane, nucleolus, nucleoplasm, nucleus, and ribosome, respectively. It is anticipated to offer profound insights for biological and medical research. [ABSTRACT FROM AUTHOR]

The microRNAs (miRNAs) play crucial roles in several biological processes. It is essential for a deeper insight into their functions and mechanisms by detecting their subcellular localizations. The traditional methods for determining miRNAs subcellular localizations are expensive. The computational methods are alternative ways to quickly predict miRNAs subcellular localizations. Although several computational methods have been proposed in this regard, the incomplete representations of miRNAs in these methods left the room for improvement. In this study, a novel computational method for predicting miRNA subcellular localizations, named PMiSLocMF, was developed. As lots of miRNAs have multiple subcellular localizations, this method was a multi-label classifier. Several properties of miRNA, such as miRNA sequences, miRNA functional similarity, miRNA-disease, miRNA-drug, and miRNA–mRNA associations were adopted for generating informative miRNA features. To this end, powerful algorithms [node2vec and graph attention auto-encoder (GATE)] and one newly designed scheme were adopted to process above properties, producing five feature types. All features were poured into self-attention and fully connected layers to make predictions. The cross-validation results indicated the high performance of PMiSLocMF with accuracy higher than 0.83, average area under the receiver operating characteristic curve (AUC) and area under the precision-recall curve (AUPR) exceeding 0.90 and 0.77, respectively. Such performance was better than all previous methods based on the same dataset. Further tests proved that using all feature types can improve the performance of PMiSLocMF, and GATE and self-attention layer can help enhance the performance. Finally, we deeply analyzed the influence of miRNA associations with diseases, drugs, and mRNAs on PMiSLocMF. The dataset and codes are available at https://github.com/Gu20201017/PMiSLocMF. [ABSTRACT FROM AUTHOR]

The cucumber mosaic virus (CMV) 2b protein is a potent counter-defense factor and symptom determinant that inhibits antiviral silencing by titrating short double-stranded RNAs. Expression of the CMV subgroup IA strain Fny-CMV 2b protein in transgenic Arabidopsis thaliana plants disrupts microRNA-mediated cleavage of host mRNAs by binding Argonaute 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated antiviral resistance and resistance to CMV’s aphid vectors. However, in authentic viral infections, the Fny-CMV 1a protein modulates 2b-AGO1 interactions, inhibiting induction of AGO2-mediated virus resistance and aphid resistance. Contrastingly, 2b proteins encoded by the subgroup II strain LS-CMV and the recently discovered subgroup IA strain Ho-CMV induce no symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation showed that Fny-CMV and Ho-CMV 2b proteins interact with Fny-CMV and LS-CMV 1a proteins, while the CMV-LS 2b protein cannot. However, Fny-CMV, Ho-CMV, and LS-CMV 2b proteins, all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the nucleus and cytoplasm, corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei. AGO2 transcript accumulation was used to assess the inhibition of AGO1-mediated mRNA degradation. Fny-CMV 2b induced a fivefold increase in AGO2 accumulation, but LS-CMV and Ho-CMV 2b proteins induced only twofold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization of 2b-AGO1 complexes influences the degree to which a 2b protein inhibits microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover. IMPORTANCE The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of “severe” CMV strains interacted with AGO1 and inhibited its microRNA-mediated “slicing” of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their in vivo localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm. [ABSTRACT FROM AUTHOR]

Simple Summary: Mammary serine protease inhibitor (maspin) is a tumor suppressor protein, and its nuclear localization is essential for its tumor suppressive activity. We previously reported that cytoplasmic-only maspin expression is an independent unfavorable prognostic indicator in patients with lung squamous cell carcinoma (LUSC). Taken together, we hypothesized that maspin has opposing roles in LUSC depending on its subcellular localization. Maspin was re-expressed in both the nucleus and cytoplasm of LK-2 cells, resulting in significantly decreased cell invasion and migration. In contrast, re-expressed maspin in RERF-LC-AI cells was detected only in the cytoplasm (cytMaspin) and significantly promoted cell invasion and migration. Increased cytMaspin expression downregulates the genes relevant to cell adhesion and activates PYK2 and SRC. This study suggests that the cytoplasm-to-nuclear translocation of maspin is dysregulated in RERF-LC-AI cells, and cytMaspin enhances the invasive potential by activating PYK2 and SRC in LUSC. Mammary serine protease inhibitor (maspin) is a tumor suppressor protein downregulated during carcinogenesis and cancer progression; cytoplasmic-only maspin expression is an independent, unfavorable prognostic indicator in patients with lung squamous cell carcinoma (LUSC). We hypothesized that the cytoplasmic-only localization of maspin has tumor-promoting functions in LUSC. The subcellular localization of maspin and the invasive capability of LUSC cell lines were investigated using RNA sequencing (RNA-seq), Western blotting, and siRNA transfection. Maspin mRNA and protein expression were suppressed in LK-2 and RERF-LC-AI cells. Cell invasion significantly increased in response to siRNA-mediated maspin knockdown in KNS-62 cells expressing both nuclear and cytoplasmic maspin. In LK-2 cells, both nuclear and cytoplasmic maspin were re-expressed, and cell invasion and migration were significantly decreased. In contrast, re-expressed maspin in RERF-LC-AI cells was detected only in the cytoplasm (cytMaspin), and cell invasion and migration were significantly promoted. RNA-seq and downstream analyses revealed that increased cytMaspin expression downregulated the genes associated with cell adhesion and activated PYK2 and SRC, which play important roles in cancer progression. Our study demonstrates a novel biological function of cytMaspin in enhancing the invasive capabilities of LUSC cells. Understanding cytoplasm-to-nuclear maspin translocation dysregulation may develop novel therapeutic approaches to improve the prognosis of patients with LUSC. [ABSTRACT FROM AUTHOR]

This article discusses a study that used machine learning to identify and validate plant peroxisome targeting signal type 1 (PTS1) tripeptides. Peroxisomes are organelles in eukaryotic cells that are involved in metabolic pathways. The researchers trained machine learning models using a dataset of functional PTS1 sequences and non-PTS1 sequences, achieving high accuracy and recall rates in predicting peroxisome targeting. They then used the models to predict novel PTS1 tripeptides and confirmed their peroxisome targeting through experimental validation. The study expands our understanding of peroxisomes in plants and has implications for predicting peroxisomal proteins and metabolic pathways in different plant species. However, it is important to consider that the presence of a PTS1 tripeptide does not guarantee peroxisome targeting, as other factors may also influence protein localization. [Extracted from the article]

Sugars are considered primary metabolites that determine the flavor and quality of grape berries, also playing a crucial role in the plants to resist stress. Sugars Will Eventually be Exported Transporters (SWEETs) gene family has been previously reported to be involved in the growth and development of grape, while the changes in transcriptional levels under water stress remain unclear. In this study, sixteen grape SWEETs members were identified and annotated based on their homologous genes in Arabidopsis and tomato, they were classified into four clades (Clades I to IV) with VvSWEETs by phylogenetic analysis. The highly conserved motifs and gene structures of VvSWEETs indicate that they are closely evolutionary conservation. Chromosomal localization and synteny analysis found that VvSWEETs were unevenly distributed on 11 chromosomes, and the VvSWEET5a, VvSWEET5b, VvSWEET14b and VvSWEET14c existed a relatively recent evolutionary relationship. Promoter cis-acting elements showed that the clade III has more ABRE motif, especially the VvSWEET14a. The regulation of VvSWEETs is mainly influenced by the Dof and MYB families, which are associated with grape ripening, while VvSWEET14a is closely related to the bHLH, MYB, NAC, and bZIP families. RT-qPCR data and subcellular localization show that VvSWEET14a was highly induced under early water stress and is located in the vacuole membrane. The instantaneous transformation assay identified that this gene could promote to transport hexose in the vacuole to maintain normal osmotic pressure. In summary, our study provides a basis for further research on SWEET genes function and regulatory mechanism in the future, and lays the foundation for stress resistance breeding of Vitis vinifera. [ABSTRACT FROM AUTHOR]

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Abstract Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle.

Abstract Background MYB transcription factors regulate anthocyanin biosynthesis across numerous plant species. However, comprehensive genome-wide investigations regarding the R2R3-MYB gene family and its involvement in regulating anthocyanin biosynthesis in the red and white fruit color morphs of Fragaria pentaphylla remain scarce. Results A total of 101 FpR2R3-MYB genes were identified from the F. pentaphylla genome and were divided into 34 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were particularly conserved among the FpR2R3-MYB genes, especially members within the same subgroup. The FpR2R3-MYB genes were distributed over seven F. pentaphylla chromosomes. Analysis of gene duplication events revealed five pairs of tandem duplication genes and 16 pairs of segmental duplication genes, suggesting that segmental duplications are the major pattern for expansion of the FpR2R3-MYB gene family expansion in F. pentaphylla. Cis-regulatory elements of the FpR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponse. Based on the analysis of the FpR2R3-MYB gene family and transcriptome sequencing (RNA-seq) data, FpMYB9 was identified as a key transcription factor involved in the regulation of anthocyanin synthesis in F. pentaphylla fruits. The expression of FpMYB9 increases significantly during the ripening stage of red fruits, as confirmed by reverse transcription quantitative real-time PCR. In addition, subcellular localization experiments further confirmed the nuclear presence of FpMYB9, supporting its role as a transcription factor involved in anthocyanin biosynthesis. Conclusion Our results showed that the FpR2R3-MYB genes are highly conserved and play important roles in the anthocyanin biosynthesis in F. pentaphylla fruits. Our results also provide a compelling basis for further understanding of the regulatory mechanism underlying the role of FpMYB9 in anthocyanin formation in F. pentaphylla fruits.

Abstract Plant U-box genes play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance; however, their functions in coffee (Coffea canephora L.) remain largely unexplored. In this study, we identified 47 CcPUB genes in the C. canephora L. genome, clustering them into nine groups via phylogenetic tree. The CcPUB genes were unevenly distributed across the 11 chromosomes of C. canephora L., with the majority (11) on chromosome 2 and none on chromosome 8. The cis-acting elements analysis showed that CcPUB genes were involved in abiotic and biotic stresses, phytohormone responsive, and plant growth and development. RNA-seq data revealed diverse expression patterns of CcPUB genes across leaves, stems, and fruits tissues. qRT-PCR analyses under dehydration, low temperature, SA, and Colletotrichum stresses showed significant up-regulation of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 in leaves. Furthermore, subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. This study provides valuable insights into the roles of PUB genes in stress responses and phytohormone signaling in C. canephora L., and provided basis for functional characterization of PUB genes in C. canephora L.

Abstract Background The Flavonoid 3’-hydroxylase gene(F3’H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. Results In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3’H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3’H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. Conclusions The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke.

Abstract Background Sterol side-chain reductase 2 (SSR2) is a key enzyme in the synthesis of plant cholesterol pathway. Despite the importance of watermelon as a horticultural cash crop, the SSR2 gene in watermelon has not been previously studied or reported. Results In this study, 28 SSR2 genes were identified in the watermelon genome. The physicochemical properties of 28 ClaSSR proteins were predicted by bioinformatics methods, and the gene structure, conserved motif, chromosome localization, phylogenetic analysis, cis-acting elements, expression patterns, promoter activity analysis and subcellular localization of ClaSSRs were studied. The 28 ClaSSRs were unevenly distributed on 11 chromosomes, and phylogenetic analysis showed that they could be grouped into 4 groups with other related Cucurbitaceae homologous genes. Analysis of gene structure and motifs revealed similarities in exons/introns and motifs between members of the same group, further supporting phylogenetic results. The RT–qPCR results showed variations in ClaSSRs expression during watermelon fruit development. The analysis of promoter activity for ClaSSR25 showed strong activity. Subcellular localization studies confirmed that ClaSSR25 is mainly located in the cytoplasm, which aligns with the predicted outcomes. We additionally estimated the network of protein–protein interactions for ClaSSR25 and analyzed proteins that could potentially interact with ClaSSR25 in melon and Arabidopsis thaliana. Conclusions We conducted bioinformatics analysis and expression analysis of members of the watermelon SSR2 gene family in this work, and the outcomes set the stage for further investigations into the watermelon SSR2 gene. Graphical Abstract

Abstract Background The TCP (teosinte branched1/cincinnata/proliferating cell factor) family plays a prominent role in plant development and stress responses. However, TCP family genes have thus far not been identified in castor bean, and therefore an understanding of the expression and functional aspects of castor bean TCP genes is lacking. To identify the potential biological functions of castor bean (RcTCP) TCP members, the composition of RcTCP family members, their basic physicochemical properties, subcellular localizations, interacting proteins, miRNA target sites, and gene expression patterns under stress were assessed. Results The presence of 20 RcTCP genes on the nine chromosomes of castor bean was identified, all of which possess TCP domains. Phylogenetic analysis indicated a close relationship between RcTCP genes and Arabidopsis AtTCP genes, suggesting potential functional similarity. Subcellular localization experiments confirmed that RcTC01/02/03/10/16/18 are all localized in the nucleus. Protein interaction analysis revealed that the interaction quantity of RcTCP03/06/11 proteins is the highest, indicating a cascade response in the functional genes. Furthermore, it was found that the promoter region of RcTCP genes contains a large number of stress-responsive elements and hormone-induced elements, indicating a potential link between RcTCP genes and stress response functions. qRT-PCR showed that all RcTCP genes exhibit a distinct tissue-specific expression pattern and their expression is induced by abiotic stress (including low temperature, abscisic acid, drought, and high salt). Among them, RcTCP01/03/04/08/09/10/14/15/18/19 genes may be excellent stress-responsive genes. Conclusion We discovered that RcTCP genes play a crucial role in various activities, including growth and development, the stress response, and transcription. This study provides a basis for studying the function of RcTCP gene in castor.

Abstract Background The monocot chimeric jacalins (MCJ) proteins, which contain a jacalin-related lectin (JRL) domain and a dirigent domain (DIR), are specific to Poaceae. MCJ gene family is reported to play an important role in growth, development and stress response. However, their roles in maize have not been thoroughly investigated. Results In this study, eight MCJ genes in the maize genome (designated as ZmMCJs) were identified, which displayed unequal distribution across four chromosomes. Phylogenetic relationships between the ZmMCJs were evident through the identification of highly conserved motifs and gene structures. Analysis of transcriptome data revealed distinct expression patterns among the ZmMCJ genes, leading to their classification into four different modules, which were subsequently validated using RT-qPCR. Protein structures of the same module are found to be relatively similar. Subcellular localization experiments indicated that the ZmMCJs are mainly located on the cell membrane. Additionally, hemagglutination and inhibition experiments show that only part of the ZmMCJs protein has lectin activity, which is mediated by the JRL structure, and belongs to the mannose-binding type. The cis-acting elements in the promoter region of ZmMCJ genes predicted their involvement response to phytohormones, such as abscisic acid and jasmonic acid. This suggests that ZmMCJ genes may play a significant role in both biotic and abiotic stress responses. Conclusions Overall, this study adds new insights into our understanding of the gene-protein architecture, evolutionary characteristics, expression profiles, and potential functions of MCJ genes in maize.

Abstract Background Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. Results We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. Conclusion This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

Introduction: The WRKY transcription factor (TF) family is one of the largest TF families in plants and is widely involved in responses to both biotic and abiotic stresses. Methods: To clarify the function of the WRKY family in blueberries, this study identified the WRKY genes within the blueberry genome and systematically analyzed gene characteristics, phylogenetic evolution, promoter cis-elements, expression patterns, and subcellular localization of the encoded products. Results: In this study, 57 VcWRKY genes were identified, and all encoding products had a complete WRKY heptapeptide structure and zinc-finger motif. The VcWRKY genes were divided into three subgroups (I-III) by phylogenetic analysis. Group II was divided into five subgroups: IIa, IIb, IIc, IId, and IIe. 57 VcWRKY genes were distributed unevenly across 32 chromosomes. The amino acids ranged from 172 to 841, and molecular weights varied from 19.75 to 92.28 kD. Intra-group syntenic analysis identified 12 pairs of duplicate segments. Furthermore, 34 cis-element recognition sites were identified in the promoter regions of VcWRKY genes, primarily comprising phytohormone-responsive and light-responsive elements. Comparative syntenic maps were generated to investigate the evolutionary relationships of VcWRKY genes, revealing the closest homology to dicotyledonous WRKY gene families. VcWRKY genes were predominantly expressed in the fruit flesh and roots of blueberries. Gene expression analysis showed that the responses of VcWRKY genes to stress treatments were more strongly in leaves than in roots. Notably, VcWRKY13 and VcWRKY25 exhibited significant upregulation under salt stress, alkali stress, and saline-alkali stress, and VcWRKY1 and VcWRKY13 showed notable induction under drought stress. Subcellular localization analysis confirmed that VcWRKY13 and VcWRKY25 function within the nucleus. Conclusion: These findings establish a foundation for further investigation into the functions and regulatory mechanisms of VcWRKY genes and provide guidance for selecting stress-tolerant genes in the development of blueberry cultivars. [ABSTRACT FROM AUTHOR]

Introduction: Research on Glycyrrhiza uralensis, a nonhalophyte that thrives in saline-alkaline soil and a traditional Chinese medicinal component, is focused on improving its ability to tolerate salt stress to increase its productivity and preserve its "Dao-di" characteristics. Furthermore, the inoculation of bioagents such as Bacillus subtilis to increase plant responses to abiotic stressors is currently a mainstream strategy. Mitogen-activated protein kinase (MAPK), a highly conserved protein kinase, plays a significant role in plant responses to various abiotic stress pathways. Methods: This investigation involved the identification of 21 members of the GuMAPK family from the genome of G. uralensis, with an analysis of their protein conserved domains, gene structures, evolutionary relationships, and phosphorylation sites using bioinformatics tools. Results: Systematic evolutionary analysis of the 21 GuMAPKs classified them into four distinct subgroups, revealing significant differences in gene structure and exon numbers. Collinearity analysis highlighted the crucial role of segmental duplication in expanding the GuMAPK gene family, which is particularly evident in G. uralensis and shows a close phylogenetic relationship with Arabidopsis thaliana, tomato, and cucumber. Additionally, the identification of phosphorylation sites suggests a strong correlation between GuMAPK and various physiological processes, including hormonal responses, stress resistance, and growth and development. Protein interaction analysis further supported the role of GuMAPK proteins in regulating essential downstream genes. Through examination of transcriptome expression patterns, GuMAPK16-2 emerged as a prospective pivotal regulatory factor in the context of salt stress and B. subtilis inoculation, a finding supported by its subcellular localization within the nucleus. Discussion: These discoveries offer compelling evidence for the involvement of GuMAPK in the salt stress response and for the exploration of the mechanisms underlying B. subtilis' enhancement of salt tolerance in G. uralensis. [ABSTRACT FROM AUTHOR]

[Objective] WRKY, as a plant idiosyncratic transcription factor, is involved in plant growth and development. DoWRKY40 gene in yam (Dioscorea opposita Thunb.) was cloned and its expression pattern was analyzed. The protein interacting with DoWRKY40 was screened by yeast two-hybrid technique to explore the regulatory mechanism of WRKY40 in the process of yam expansion. [Method] DoWRKY40 gene was cloned from ‘Dahechangyu’ yam, and bioinformatics analysis, expression pattern analysis and subcellular localization were performed. Yeast libraries of yam tuber at different developmental stages were constructed and DoWRKY40 interacting proteins were screened. [Result] The length of open reading frame of DoWRKY40 was 927 bp; DoWRKY40 belonged to group II of the WRKY family and contained a typical WRKY transcription factor conserved domain, which was localized in the nucleus. It was closely related to Dioscorea cayenensis subsp. The expression of DoWRKY40 gene was the highest at 120 d after the growth and development of yam tuber, and it was expressed in the leaves, stems and tubers with tissue specificity. The library capacity of yam cDNA library was 1.484×108; pGBKT7-WRKY40 decoy vector had no self-activation activity. Four kinds of proteins interacting with DoWRKY40 were screened from yam library by yeast two-hybridization, which were involved in plant growth and development, cell cycle regulation and cell expansion, signal transduction and environmental stress response, respectively. [Conclusion] The DoWRKY40 gene is cloned, which responds to the growth and development process of yam. The four interacting proteins are screened, suggesting that it plays a regulatory role in the expansion and signal transduction of yam cells. [ABSTRACT FROM AUTHOR]

[Objective] SEEDSTICK (STK), a member of the MADS-box transcription factor family, plays a key role in controlling gametophyte development and seed size and morphology. This study is aimed to investigate the role of STK in quinoa gametophyte development. [Method] Transcriptomic data from two quinoa varieties with different photoperiod characteristics under different photoperiod treatments were used to screen differentially expressed genes regulated by photoperiod and related to gametophyte development. The identified gene was cloned and subjected to bioinformatics analysis, expression pattern analysis, subcellular localization analysis, and functional verification through heterologous expression in Arabidopsis. [Result] A differentially expressed gene, AUR62022366-RA, was identified. The expression pattern of this gene was consistent with phenotypic differences in plants. In short-day materials, high expression of the gene during the grain-filling stage resulted in fertile plants, while low expression led to infertile plants. Bioinformatics analysis revealed that the full-length coding sequence (CDS) of CqSTK was 672 bp, encoding 223 amino acids. Phylogenetic analysis of STK homologous genes showed that CqSTK clustered with STK genes from spinach and sugar beet, indicating a close evolutionary relationship. Furthermore, CqSTK shared a similar tertiary structure with Arabidopsis STK. Haplotype analysis indicated that the exonic sequences of CqSTK were completely conserved across ten quinoa varieties, while single nucleotide polymorphisms (SNPs) were present in the intronic regions. This gene was localized in the nucleus and cell membrane of tobacco leaf epidermal cells. Fluorescence quantitative PCR showed that CqSTK was highly expressed during flower organ formation and grain development, especially during grain development when its expression reached its peak. Overexpression and complementation experiments in Arabidopsis demonstrated that overexpressing and complementation plants had significantly delayed flowering time compared to mutants and wild-type plants, and their pod length and seed number per pod were significantly higher than those of stk mutants and complementation plants. [Conclusion] The overexpression of CqSTK delays flowering and positively regulates seed length and fruit set in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Isopentenyl diphosphate isomerase (IPI) is a key enzyme in the synthesis of isoprenoids. In this paper, the in vivo biological activity of the IPI gene from Fritillaria unibracteata (FuIPI) was investigated. Combining a color complementation experiment with High-Performance Liquid Chromatography analysis showed that the FuIPI gene could accumulate β-carotene in Escherichia coli, and Glu190 was identified as a key residue for its catalytic activity. Bioinformatics analysis together with subcellular localization indicated that the FuIPI protein was localized in chloroplasts. Compared with wild-type Arabidopsis thaliana, FuIPI transgenic plants had higher abscisic acid content and strengthening tolerance to drought and salt stress. Overall, these results indicated that the FuIPI gene had substantial biological activity in vivo, hopefully laying a foundation for its further research and application in liliaceous ornamental and medicinal plants. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals (ToGals) were identified in the Golden pompano (Trachinotus ovatus), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae, indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa, and Aeromonas hydrophila. This study lays the foundation for further research on the immune roles of Gals in teleosts. [ABSTRACT FROM AUTHOR]

Background: Sterol side-chain reductase 2 (SSR2) is a key enzyme in the synthesis of plant cholesterol pathway. Despite the importance of watermelon as a horticultural cash crop, the SSR2 gene in watermelon has not been previously studied or reported. Results: In this study, 28 SSR2 genes were identified in the watermelon genome. The physicochemical properties of 28 ClaSSR proteins were predicted by bioinformatics methods, and the gene structure, conserved motif, chromosome localization, phylogenetic analysis, cis-acting elements, expression patterns, promoter activity analysis and subcellular localization of ClaSSRs were studied. The 28 ClaSSRs were unevenly distributed on 11 chromosomes, and phylogenetic analysis showed that they could be grouped into 4 groups with other related Cucurbitaceae homologous genes. Analysis of gene structure and motifs revealed similarities in exons/introns and motifs between members of the same group, further supporting phylogenetic results. The RT–qPCR results showed variations in ClaSSRs expression during watermelon fruit development. The analysis of promoter activity for ClaSSR25 showed strong activity. Subcellular localization studies confirmed that ClaSSR25 is mainly located in the cytoplasm, which aligns with the predicted outcomes. We additionally estimated the network of protein–protein interactions for ClaSSR25 and analyzed proteins that could potentially interact with ClaSSR25 in melon and Arabidopsis thaliana. Conclusions: We conducted bioinformatics analysis and expression analysis of members of the watermelon SSR2 gene family in this work, and the outcomes set the stage for further investigations into the watermelon SSR2 gene. [ABSTRACT FROM AUTHOR]

Background: The monocot chimeric jacalins (MCJ) proteins, which contain a jacalin-related lectin (JRL) domain and a dirigent domain (DIR), are specific to Poaceae. MCJ gene family is reported to play an important role in growth, development and stress response. However, their roles in maize have not been thoroughly investigated. Results: In this study, eight MCJ genes in the maize genome (designated as ZmMCJs) were identified, which displayed unequal distribution across four chromosomes. Phylogenetic relationships between the ZmMCJs were evident through the identification of highly conserved motifs and gene structures. Analysis of transcriptome data revealed distinct expression patterns among the ZmMCJ genes, leading to their classification into four different modules, which were subsequently validated using RT-qPCR. Protein structures of the same module are found to be relatively similar. Subcellular localization experiments indicated that the ZmMCJs are mainly located on the cell membrane. Additionally, hemagglutination and inhibition experiments show that only part of the ZmMCJs protein has lectin activity, which is mediated by the JRL structure, and belongs to the mannose-binding type. The cis-acting elements in the promoter region of ZmMCJ genes predicted their involvement response to phytohormones, such as abscisic acid and jasmonic acid. This suggests that ZmMCJ genes may play a significant role in both biotic and abiotic stress responses. Conclusions: Overall, this study adds new insights into our understanding of the gene-protein architecture, evolutionary characteristics, expression profiles, and potential functions of MCJ genes in maize. [ABSTRACT FROM AUTHOR]

Background: The TCP (teosinte branched1/cincinnata/proliferating cell factor) family plays a prominent role in plant development and stress responses. However, TCP family genes have thus far not been identified in castor bean, and therefore an understanding of the expression and functional aspects of castor bean TCP genes is lacking. To identify the potential biological functions of castor bean (RcTCP) TCP members, the composition of RcTCP family members, their basic physicochemical properties, subcellular localizations, interacting proteins, miRNA target sites, and gene expression patterns under stress were assessed. Results: The presence of 20 RcTCP genes on the nine chromosomes of castor bean was identified, all of which possess TCP domains. Phylogenetic analysis indicated a close relationship between RcTCP genes and Arabidopsis AtTCP genes, suggesting potential functional similarity. Subcellular localization experiments confirmed that RcTC01/02/03/10/16/18 are all localized in the nucleus. Protein interaction analysis revealed that the interaction quantity of RcTCP03/06/11 proteins is the highest, indicating a cascade response in the functional genes. Furthermore, it was found that the promoter region of RcTCP genes contains a large number of stress-responsive elements and hormone-induced elements, indicating a potential link between RcTCP genes and stress response functions. qRT-PCR showed that all RcTCP genes exhibit a distinct tissue-specific expression pattern and their expression is induced by abiotic stress (including low temperature, abscisic acid, drought, and high salt). Among them, RcTCP01/03/04/08/09/10/14/15/18/19 genes may be excellent stress-responsive genes. Conclusion: We discovered that RcTCP genes play a crucial role in various activities, including growth and development, the stress response, and transcription. This study provides a basis for studying the function of RcTCP gene in castor. [ABSTRACT FROM AUTHOR]

Background: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. Results: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. Conclusion: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach. [ABSTRACT FROM AUTHOR]

The subcellular localization of messenger RNAs (mRNAs) is a pivotal aspect of biomolecules, tightly linked to gene regulation and protein synthesis, and offers innovative insights into disease diagnosis and drug development in the field of biomedicine. Several computational methods have been proposed to predict the subcellular localization of mRNAs within cells. However, there remains a deficiency in the accuracy of these predictions. In this study, we propose an mRCat predictor based on the gradient boosting tree algorithm specifically to predict whether mRNAs are localized in the nucleus or in the cytoplasm. This predictor firstly uses large language models to thoroughly explore hidden information within sequences and then integrates traditional sequence features to collectively characterize mRNA gene sequences. Finally, it employs CatBoost as the base classifier for predicting the subcellular localization of mRNAs. The experimental validation on an independent test set demonstrates that mRCat obtained accuracy of 0.761, F1 score of 0.710, MCC of 0.511, and AUROC of 0.751. The results indicate that our method has higher accuracy and robustness compared to other state-of-the-art methods. It is anticipated to offer deep insights for biomolecular research. [ABSTRACT FROM AUTHOR]

Key message: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at −60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) have been discovered to be extensively involved in eukaryotic epigenetic, transcriptional, and post-transcriptional regulatory processes with the advancements in sequencing technology and genomics research. Therefore, they play crucial roles in the body's normal physiology and various disease outcomes. Presently, numerous unknown lncRNA sequencing data require exploration. Establishing deep learning-based prediction models for lncRNAs provides valuable insights for researchers, substantially reducing time and costs associated with trial and error and facilitating the disease-relevant lncRNA identification for prognosis analysis and targeted drug development as the era of artificial intelligence progresses. However, most lncRNA-related researchers lack awareness of the latest advancements in deep learning models and model selection and application in functional research on lncRNAs. Thus, we elucidate the concept of deep learning models, explore several prevalent deep learning algorithms and their data preferences, conduct a comprehensive review of recent literature studies with exemplary predictive performance over the past 5 years in conjunction with diverse prediction functions, critically analyze and discuss the merits and limitations of current deep learning models and solutions, while also proposing prospects based on cutting-edge advancements in lncRNA research. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is vital for post-transcriptional gene regulation, acting as the direct template for protein synthesis. However, the methods available for predicting mRNA subcellular localization need to be improved and enhanced. Notably, few existing algorithms can annotate mRNA sequences with multiple localizations. In this work, we propose the mRNA-CLA, an innovative multi-label subcellular localization prediction framework for mRNA, leveraging a deep learning approach with a multi-head self-attention mechanism. The framework employs a multi-scale convolutional layer to extract sequence features across different regions and uses a self-attention mechanism explicitly designed for each sequence. Paired with Position Weight Matrices (PWMs) derived from the convolutional neural network layers, our model offers interpretability in the analysis. In particular, we perform a base-level analysis of mRNA sequences from diverse subcellular localizations to determine the nucleotide specificity corresponding to each site. Our evaluations demonstrate that the mRNA-CLA model substantially outperforms existing methods and tools. • Visualization of CNN and attention layers used for mRNA localization prediction. • Multiscale modeling and Bayesian optimization enhance model performance. • Integrated analyses reveal base content and sequence patterns in mRNA compartments. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are molecular chaperones that play essential roles in plant development and in response to various environmental stresses. Understanding R. delavayi HSP genes is of great importance since R. delavayi is severely affected by heat stress. In the present study, a total of 76 RdHSP genes were identified in the R. delavayi genome, which were divided into five subfamilies based on molecular weight and domain composition. Analyses of the chromosome distribution, gene structure, and conserved motif of the RdHSP family genes were conducted using bioinformatics analysis methods. Gene duplication analysis showed that 15 and 8 RdHSP genes were obtained and retained from the WGD/segmental duplication and tandem duplication, respectively. Cis-element analysis revealed the importance of RdHSP genes in plant adaptations to the environment. Moreover, the expression patterns of RdHSP family genes were investigated in R. delavayi treated with high temperature based on our RNA-seq data, which were further verified by qRT-PCR. Further analysis revealed that nine candidate genes, including six RdHSP20 subfamily genes (RdHSP20.4, RdHSP20.8, RdHSP20.6, RdHSP20.3, RdHSP20.10, and RdHSP20.15) and three RdHSP70 subfamily genes (RdHSP70.15, RdHSP70.21, and RdHSP70.16), might be involved in enhancing the heat stress tolerance. The subcellular localization of two candidate RdHSP genes (RdHSP20.8 and RdHSP20.6) showed that two candidate RdHSPs were expressed and function in the chloroplast and nucleus, respectively. These results provide a basis for the functional characterization of HSP genes and investigations on the molecular mechanisms of heat stress response in R. delavayi. [ABSTRACT FROM AUTHOR]

Main conclusion: Genome-wide identification revealed 79 BpNAC genes belonging to 16 subfamilies, and their gene structures and evolutionary relationships were characterized. Expression analysis highlighted their importance in plant selenium stress responses. Paper mulberry (Broussonetia papyrifera), a deciduous arboreal plant of the Moraceae family, is distinguished by its leaves, which are abundant in proteins, polysaccharides, and flavonoids, positioning it as a novel feedstock. NAC transcription factors, exclusive to plant species, are crucial in regulating growth, development, and response to biotic and abiotic stress. However, extensive characterization of the NAC family within paper mulberry is lacking. In this study, 79 BpNAC genes were identified from the paper mulberry genome, with an uneven distribution across 13 chromosomes. A comprehensive, genome-wide analysis of BpNACs was performed, including investigating gene structures, promoter regions, and chromosomal locations. Phylogenetic tree analysis, alongside comparisons with Arabidopsis thaliana NACs, allowed for categorizing these genes into 16 subfamilies in alignment with gene structure and motif conservation. Collinearity analysis suggested a significant homologous relationship between the NAC genes of paper mulberry and those in Morus notabilis, Ficus hispida, Antiaris toxicaria, and Cannabis sativa. Integrating transcriptome data and Se content revealed that 12 BpNAC genes were associated with selenium biosynthesis. Subsequent RT-qPCR analysis corroborated the correlation between BpNAC59, BpNAC62 with sodium selenate, and BpNAC55 with sodium selenite. Subcellular localization experiments revealed the nuclear functions of BpNAC59 and BpNAC62. This study highlights the potential BpNAC transcription factors involved in selenium metabolism, providing a foundation for strategically breeding selenium-fortified paper mulberry. [ABSTRACT FROM AUTHOR]

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The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL360 signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a Y367KQF sequence facilitates its internalization from the plasma membrane, while a Y392TSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system. [ABSTRACT FROM AUTHOR]

Molting and metamorphosis are important physiological processes in insects that are tightly controlled by ecdysone receptor (EcR) through the 20‐hydroxyecdysone (20E) signaling pathway. EcR is a steroid nuclear receptor (SR). Several FK506‐binding proteins (FKBPs) have been identified from the mammal SR complex, and are thought to be involved in the subcellular trafficking of SR. However, their roles in insects are poorly understood. To explore whether FKBPs are involved in insect molting or metamorphosis, we injected an FKBP inhibitor (FK506) into a lepidopteran insect, Spodoptera litura, and found that molting was inhibited in 61.11% of the larvae, and that the time for larvae to pupate was significantly extended. A total of 10 FKBP genes were identified from the genome of S. litura and were clustered into 2 distinct groups, according to their subcellular localization, with FKBP13 and FKBP14 belonging to the endoplasmic reticulum (ER) group and with the other members belonging to the cytoplasmic (Cy) group. All the CyFKBPs were significantly upregulated in the prepupal or pupal stages, with the opposite being observed for the ER group members. FK506 completely blocked the transfer of EcR to the nucleus under 20E induction, and significantly downregulated the transcriptional expression of many 20E signaling genes. A similar phenomenon was observed after RNA interference of 2 CyFKBPs (FKBP45 and FKBP12b), but not for FKBP13. Taken together, our data indicate that the cytoplasmic FKBPs, especially FKBP45 and FKBP12b, mediate the nuclear localization of EcR, thereby regulating the 20E signaling and ultimately affecting molting and metamorphosis in insects. [ABSTRACT FROM AUTHOR]

Key message: The utilization of transcriptome analysis, functional validation, VIGS, and DAB techniques have provided evidence that GhiPLATZ17 and GhiPLATZ22 play a pivotal role in improving the salt tolerance of upland cotton. PLATZ (Plant AT-rich sequences and zinc-binding proteins) are known to be key regulators in plant growth, development, and response to salt stress. In this study, we comprehensively analyzed the PLATZ family in ten cotton species in response to salinity stress. Gossypium herbaceum boasts 25 distinct PLATZ genes, paralleled by 24 in G. raimondii, 25 in G. arboreum, 46 in G. hirsutum, 48 in G. barbadense, 43 in G. tomentosum, 67 in G. mustelinum, 60 in G. darwinii, 46 in G. ekmanianum, and a total of 53 PLATZ genes attributed to G. stephensii. The PLATZ gene family shed light on the hybridization and allopolyploidy events that occurred during the evolutionary history of allotetraploid cotton. Ka/Ks analysis suggested that the PLATZ gene family underwent intense purifying selection during cotton evolution. Analysis of synteny and gene collinearity revealed a complex pattern of segmental and dispersed duplication events to expand PLATZ genes in cotton. Cis-acting elements and gene expressions revealed that GhiPLATZ exhibited salt stress resistance. Transcriptome analysis, functional validation, virus-induced gene silencing (VIGS), and diaminobenzidine staining (DAB) demonstrated that GhiPLATZ17 and GhiPLATZ22 enhance salt tolerance in upland cotton. The study can potentially advance our understanding of identifying salt-resistant genes in cotton. [ABSTRACT FROM AUTHOR]

Histone acetylation plays important roles in gene regulation and is catalyzed by histone acetyltransferases (HATs). The performance of histone acetylation by HATs requires the involvement of other proteins to form multi-subunit complexes. The functions of three major HAT-containing complexes, namely SAGA, NuA4, and TFIID, have been extensively studied. Recently, the MEDIATOR complex was also found to be associated with HAT in Arabidopsis. However, these complexes have rarely been investigated in other plant species. In this study, we identify the subunits of four HAT-containing complexes in six different grass species by using Arabidopsis homologs as reference sequences. We then construct the phylogenetic trees to analyze their evolutionary relationships. The results indicate that all six grass species conserve all the subunits, despite gene duplication events occurring diversely in different species. Expression analysis shows that most of the genes encoding the subunits of the four HAT-containing complexes are constitutively expressed, with only low-level expression in pollen. Consistent with their cellular function, all four HATs are found to be localized in the nucleus in sorghum. Unlike their Arabidopsis counterparts, SbGCN5 specifically acetylates H3K4, H3K9, and H3K27, while SbMYST acetylates H4K5 and H4K12 in vitro. Additionally, we discover that SbTAF1 catalyzes acetylation of H3K9 and H4K5 for the first time. These findings provide valuable insights for further investigations into the function of HAT-containing complexes in grass species. [ABSTRACT FROM AUTHOR]