Single-cell profiling reveals Müller glia coordinate retinal intercellular communication during light/dark adaptation via thyroid hormone signaling

Light adaptation enables the vertebrate visual system to operate over a wide range of ambient illumination.Regulation of phototransduction in photoreceptors is considered a major mechanism underlying light adaptation.However,various types of neurons and glial cells exist in the retina,and whether and how all retinal cells interact to adapt to light/dark conditions at the cellular and molecular levels requires systematic investigation.Therefore,we utilized single-cell RNA sequencing to dissect retinal cell-type-specific transcriptomes during light/dark adaptation in mice.The results demonstrated that,in addition to photoreceptors,other retinal cell types also showed dynamic molecular changes and specifically enriched signaling pathways under light/dark adaptation.Importantly,Müller glial cells(MGs)were identified as hub cells for intercellular interactions,displaying complex cell‒cell communication with other retinal cells.Furthermore,light increased the transcription of the deiodinase Dio2 in MGs,which converted thyroxine(T4)to active triiodothyronine(T3).Subsequently,light increased T3 levels and regulated mitochondrial respiration in retinal cells in response to light conditions.As cones specifically express the thyroid hormone receptor Thrb,they responded to the increase in T3 by adjusting light responsiveness.Loss of the expression of Dio2 specifically in MGs decreased the light responsive ability of cones.These results suggest that retinal cells display global transcriptional changes under light/dark adaptation and that MGs coordinate intercellular communication during light/dark adaptation via thyroid hormone signaling.

Optimization of the T3-induced Xenopus metamorphosis assay for detecting thyroid hormone signaling disruption of chemicals

T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone（TH）signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction.Xenopus laevis at stage 52 were exposed to series of concentrations of T3（0.31–2.5 nmol/L）for 6 days. By comparing morphological changes induced by T3, we propose head area,mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25 nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4 days as exposure duration of T3, with concentrations close to 1.25 nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.

Re-evaluation of thyroid hormone signaling antagonism of tetrabromobisphenol A for validating the T3-induced Xenopus metamorphosis assay

We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone（TH） signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500 nmol/L TBBPA in the presence of 1 nmol/L T3. After 96 hr of exposure, TBBPA in the range of 10–500 nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area（HA）, mouth width（MW）, unilateral brain width/brain length（ULBW/BL）, and hind-limb length/snout-vent length（HLL/SVL）.The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure,we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail,which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone（TH）signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3,whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.

Jasmonic acid and ethylene signaling pathways participate in the defense response of Chinese cabbage to Pectobacterium carotovorum infection

Chinese cabbage(Brassica rapa subsp.pekinensis)suffers from soft rot disease caused by Pectobacterium carotovorum(Pc).To uncover the mechanisms underlying the defense response of Chinese cabbage to Pc,we constructed a suppression subtractive hybridization(SSH)library from Pc-infected cabbage and obtained 1919 non-redundant expressed sequence tags(ESTs),which were used for cDNA microarray.We detected 800 differentially expressed genes(DEGs)in cabbage at different time points post-Pc inoculation,which were further confirmed by quantitative real-time PCR.One quarter of these DEGs were involved in the biotic stress pathways visualized by MapMan.Among them,8,8,1,3,and 2 DEGs were related to jasmonic acid(JA),ethylene(ET),JA+ET,auxin,and abscisic acid(ABA)signaling pathways,respectively,while no DEG was detected for salicylic acid(SA)signaling.Assessment of phytohormone production in the Pc-infected leaves showed that JA and ET production was increased,while SA production was decreased.Treatment with JA,methyl jasmonate(MeJA),the ET precursor 1-aminocyclopropane-1-carboxylate(ACC),or combinations thereof,reduced the disease severity,and the JA and JA+ACC treatments were superior and performed equally well.Our findings suggest that JA and ET may act synergistically against Pc infection in Chinese cabbage,and JA-mediated signaling might be the most significant.

The IDD Transcription Factors:Their Functions in Plant Development and Environmental Response

INDETERMINATE-DOMAIN proteins(IDDs)are a plant-specific transcription factor family characterized by a conserved ID domain with four zinc finger motifs.Previous studies have demonstrated that IDDs coordinate a diversity of physiological processes and functions in plant growth and development,including floral transition,plant architecture,seed and root development,and hormone signaling.In this review,we especially summarized the latest knowledge on the functions and working models of IDD members in Arabidopsis,rice,and maize,particularly focusing on their role in the regulatory network of biotic and abiotic environmental responses,such as gravity,temperature,water,and pathogens.Understanding these mechanisms underlying the function of IDD proteins in these processes is important for improving crop yields by manipulating their activity.Overall,the review offers valuable insights into the functions and mechanisms of IDD proteins in plants,providing a foundation for further research and potential applications in agriculture.

Integrative analysis of the transcriptome and metabolome reveals the response mechanism to tomato spotted wilt virus

Tomato spotted wilt virus(TSWV)is an important virus that has rapidly spread throughout the world.TSWV seriously hinders the production of tomato(Solanum lycopersicum)and other plants.In order to discover more new genes and metabolites related to TSWV resistance in tomato plants,the genes and metabolites related to the resistance of tomato plants inoculated with TSWV were identified and studied herein.The tomato TSWV-resistance line YNAU335(335)and TSWV-susceptible lines NO5 and 96172I(961)were used as the transcriptome and metabolome research materials.Transcriptomic and metabolomic techniques were used to analyze the gene and metabolite response mechanisms to TSWV inoculation.A total of 3566,2951,and 2674 differentially expressed genes(DEGs)were identified in lines 335,NO5,and961,respectively.Meanwhile,208,228,and 273 differentially accumulated metabolites(DAMs)were identified in lines 335,NO5,and 961,respectively.In line 335,the number of DEGs was the highest,but the number of DAMs was lowest.Furthermore,903 DEGs and 94 DAMs were common to the response to TSWV in the three inbred lines.The 903 DEGs and 94 DAMs were mainly enriched in the plant hormone signal transduction and flavonoid synthesis pathways.In addition,many nucleotide-binding site-leucine-rich repeat genes and transcription factors were found that might be involved in the TSWV response.These results provide new insights into TSWV resistance mechanisms.

Transcriptome Analysis of Molecular Mechanisms Underlying Phenotypic Variation in Phaseolus vulgaris Mutant‘nts’

The phenotype of a common bean plant is often closely related to its yield,and the yield of plants with reduced height or poor stem development during growth is low.Mutants serve as an essential gene resource for common bean breeding genetic research.Although model plants and crops are studied to comprehend the molecular mechanisms and genetic basis of plant phenotypes,the molecular mechanism of phenotypic variation in common beans remains underexplored.We here used the mutant‘nts’as material for transcriptome sequencing analysis.This mutant was obtained through 60Co-γirradiation from the common bean variety‘A18’.Differentially expressed genes were mainly enriched in GO functional entries such as cell wall organization,auxin response and transcription factor activity.Metabolic pathways significantly enriched in KEGG analysis included plant hormone signal transduction pathways,phenylpropanoid biosynthesis pathways,and fructose and mannose metabolic pathways.AUX1(Phvul.001G241500),the gene responsible for auxin transport,may be the key gene for auxin content inhibition.In the plant hormone signal transduction pathway,AUX1 expression was downregulated and auxin transport across the membrane was blocked,resulting in stunted growth of the mutant‘nts’.The results provide important clues for revealing the molecular mechanism of‘nts’phenotype regulation in bean mutants and offer basic materials for breeding beneficial phenotypes of bean varieties.

The GhMAX2 gene regulates plant growth and fiber development in cotton

Strigolactones(SLs)are a new type of plant endogenous hormones that have been found to regulate plant growth and architecture.At present,some genes related to the biosynthesis and signaling pathway of SLs have been isolated in plants such as Arabidopsis thaliana,Pisum sativum and Oryza sativa.However,the signaling pathway and specific mechanism of SLs in cotton remain unclear.In this study,we identified the SLs signaling gene GhMAX2 and demonstrated its function in plant growth and architecture in Gossypium hirsutum.Bioinformatics analysis showed that GhMAX2 mainly consists of anα-helix and a random coil and includes a large number of leucine-rich repeats.GhMAX2 was highly expressed in root,stem,flower,and fibers at 20 days post-anthesis(DPA).GhMAX2 promoter-drivenβ-glucuronidase expression was present exclusively in the root,main inflorescence,flower,and silique.Subcellular localization showed that GhMAX2 is targeted to the nucleus.Heterologously expressed GhMAX2 can rescue the phenotype of Arabidopsis max2-1 mutant,indicating that the function of MAX2 is highly conserved between G.hirsutum and A.thaliana species.In addition,the knockdown expression of GhMAX2 in cotton resulted in significantly reduced plant height,slow growth,short internodes,and reduced fiber length.These findings indicate that GhMAX2 probably contributes to plant growth,architecture and fiber elongation in cotton.The study reveals insights into the roles of GhMAX2-mediated SL/KAR signaling in cotton and provides a valuable foundation for the cultivation of cotton plants in the future.

Molecular Characterization and Functional Analysis of Krüppel-homolog 1(Kr-h1) in the Brown Planthopper, Nilaparvata lugens(Stl)

The brown planthopper, Nilaparvata lugens（Stl）, is the most serious insect pest of rice. It has developed high resistance to traditional insecticides because of their intensive use. Juvenile hormone（JH） analogs have been used successfully to control this species and other pest insects. However, the molecular mechanism of JH signaling is not well understood. Krüppel-homolog 1（Kr-h1） is a transcription factor involved in the JH pathway. In this study, the Kr-h1 cDNA was cloned and characterized from N. lugens by rapid amplification of cDNA ends（RACE） and reverse transcription PCR（RT-PCR）. Its spatial and temporal expression profiles were examined by real-time quantitative PCR, and its function was also studied by RNA interference（RNAi）. The open reading frame of NlKr-h1 is 1 833 bp encoding for 611 amino acids. The protein contains eight conserved zinc-finger motifs. NlKr-h1 was expressed at all life stages, with the highest mRNA level in the 4-day embryo. NlKr-h1 mRNA levels rose during each nymphal molt after the 2nd instar. In the adults, the mRNA level in males was significantly higher than that in females of either the macropterous or brachypterous type. The highest expression was observed in the female midgut. NlKr-h1 was activated by juvenile hormone III（JH III） in the 3rd-5th instar nymphs. Disruption of Nlkr-h1 expression by RNAi caused stunted wing development and malformations of both male and female external genitalia. Our findings suggest that Kr-h1 may be a useful target for pest insect management.

Transcriptome Profiling Reveals Candidate Genes Involved in Stem Swelling of Tumorous Stem Mustard

Tumorous stem mustard is well known for its swollen stem from which pickled“Fuling Mustard”is made.The molecular mechanisms governing the formation of the modified swollen stems are still poorly understood.This paper aims to identify candidate genes involved in the developmental regulation of the swollen stems.We sought to map previously published transcriptome datasets for Brassica juncea,including those derived from swollen stems at four different developmental stages and a mutant variety without swollen stems.Using pairwise comparisons of the five datasets,we identified 31368 differentially expressed genes(DEGs).A total of 55 DEGs related to plant hormone signal transduction and 259 continuously up-or downregulated transcription factors were identified during stem development using Gene Ontology(GO)analysis.Quantitative real-time PCR(qRT-PCR)results showed that the expressions of 12 important candidate DEGs were consistent with RNA-seq results.Our study provided digital gene expression profiling and a dynamic view of the swollen stem development process.Furthermore,we identified candidate genes for further studies on mechanisms of modified stem development in non-model species.

A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway ComDonents

Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid （ABA）. The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA （BRM） modulates response to ABA by preventing pre- mature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphoryla- tion/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarUy conserved. Finally, the phosphomimetic BRMs17s^D S1762D mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2- dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants.

Molecular characterization and developmental expression patterns of thyroid hormone receptors(TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone（TH） signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor（TR） agonist（T3） and antagonist（amiodarone） by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2 days induced more dramatic increases of TRβ expression in stage 27 than in stage34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax.Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.

Expanding Roles of PIFs in Signal Integration from Multiple Processes

PHYTOCHROME-INTERACTING FACTORs （PIFs） are members of the basic helix-loop-helix （bHLH） family of transcription factors in Arabidopsis. Since their discovery in phytochrome-mediated light signaling pathways, recent studies have unraveled new functions of PIFs in integrating multiple signaling pathways not only through their role as transcription factors directly targeting gene expression but also by interacting with diverse groups of factors to optimize plant growth and development. These include endogenous （e.g., hormonal） as well as abiotic （light, circadian, and elevated temperature） and biotic （defense responses） pathways. PIFs interact with key factors in each of these pathways and tailor the outcome of the signal integration among these pathways. This review discusses the roles of PIFs as pivotal signal integrators in regulating plant growth and development.

TRAF proteins as key regulators of plant development and stress responses

Tumor necrosis factor receptor-associated factor(TRAF)proteins are conserved in higher eukaryotes and play key roles in transducing cellular signals across different organelles.They are characterized by their C-terminal region(TRAF-C domain)containing seven to eight antiparallelβ-sheets,also known as the meprin and TRAF-C homology(MATH)domain.Over the past few decades,significant progress has been made toward understanding the diverse roles of TRAF proteins in mammals and plants.Compared to other eukaryotic species,the Arabidopsis thaliana and rice(Oryza sativa)genomes encode many more TRAF/MATH domaincontaining proteins;these plant proteins cluster into five classes:TRAF/MATH-only,MATH-BPM,MATH-UBP(ubiquitin protease),Seven in absentia(SINA),and MATH-Filament and MATHPEARLI-4 proteins,suggesting parallel evolution of TRAF proteins in plants.Increasing evidence now indicates that plant TRAF proteins form central signaling networks essential for multiple biological processes,such as vegetative and reproductive development,autophagosome formation,plant immunity,symbiosis,phytohormone signaling,and abiotic stress responses.Here,we summarize recent advances and highlight future prospects for understanding on the molecular mechanisms by which TRAF proteins act in plant development and stress responses.

Investigation of Mechanism of Premature Ovarian Failure Regulation by Kidney-tonifying Herbs and Liver-clearing Herbs in Dingjing Decoction

Objective To investigate the mechanisms through which kidney-tonifying herbs（KTHs） and liver-clearing herbs（LCHs） in Dingjing Decoction（DJD） regulate premature ovarian failure（POF）. Methods One hundred and fifty Sprague-Dawley rats were randomly divided into five groups such as control, model, KTHs, LCHs, and DJD groups. POF-related biological molecules were examined. Factor analysis was performed to investigate the regulatory networks and key biomolecules involved in mediating POF after treatment with KTHs and LCHs. Results The master regulatory factors in the reproductive endocrine network associated with KTHs intervention included four molecules in the pituitary-ovarian axis, cortisol（CORT） in the target gland of pituitary-adrenal axis, and some molecules in the hypothalamus. In contrast, the master regulatory factors associated with LCHs intervention included four molecules in the pituitary-ovarian axis and some molecules in the hypothalamus; No biomolecules in the pituitary-adrenal axis were involved in the LCH-mediated mechanisms.Gonadotropin-releasing hormone（Gn RH）, which was identified as a common biological molecule in the hypothalamus, was involved in regulating the reproductive endocrine network in association with KTHs intervention. Conclusion KTHs directly regulates biological molecules in the pituitary-adrenal axis and indirectly regulates those in the pituitary-adrenal axis through the hypothalamus, while the LCHs only exert its effects indirectly. Gn RH is the key biological molecule associated with KTHs intervention.