Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple.

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX-LOOP-HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.

Development of Fluorenyl-Based Copolyimines with Adjustable Mechanical Properties, Recyclability and Friction Resistance.

Dynamic polyimines are a class of fascinating dynamic polymers with recyclability and reparability owing to their reversible Schiff-base reactions. However, balancing the dynamic properties and mechanical strength of dynamic polyimines presents a major challenge due to the dissociative and associative nature of the imine bonds. Herein, we introduced bulky fluorene groups and polyether amine into the skeleton of polyimine networks to achieve a tradeoff in comprehensive properties. The resulting dynamic polyimines with fluorene groups (Cardo-DPIs) were successfully synthesized by combining the rigid diamine 9,9-bis(4-aminophenyl)fluorene and the flexible polyether amine, demonstrating a high tensile strength of 64.7 MPa. Additionally, Cardo-DPIs films with more content of rigid fluorene groups exhibited higher water resistance, glass transition temperature and wear-resisting ability. Moreover, the Cardo-DPIs films not only efficiently underwent thermal reshaping, but also exhibited excellent self-healing capabilities and chemical degradation in acidic solutions. Furthermore, the resulting films can achieve fully closed-loop recovery by free amine solution for 2 h at room temperature. This study broadens the scope of dynamic polyimine materials and promotes the balanced development of their functional and mechanical properties.

The East Asian wild apples, Malus baccata (L.) Borkh and Malus hupehensis (Pamp.) Rehder., are additional contributors to the genomes of cultivated European and Chinese varieties.

The domestication process in long-lived plant perennials differs dramatically from that of annuals, with a huge amount of genetic exchange between crop and wild populations. Though apple is a major fruit crop grown worldwide, the contribution of wild apple species to the genetic makeup of the cultivated apple genome remains a topic of intense study. We used population genomics approaches to investigate the contributions of several wild apple species to European and Chinese rootstock and dessert genomes, with a focus on the extent of wild-crop gene flow. Population genetic structure inferences revealed that the East Asian wild apples, Malus baccata (L.) Borkh and M. hupehensis (Pamp.), form a single panmictic group, and that the European dessert and rootstock apples form a specific gene pool whereas the Chinese dessert and rootstock apples were a mixture of three wild gene pools, suggesting different evolutionary histories of European and Chinese apple varieties. Coalescent-based inferences and gene flow estimates indicated that M. baccata - M. hupehensis contributed to the genome of both European and Chinese cultivated apples through wild-to-crop introgressions, and not as an initial contributor as previously supposed. We also confirmed the contribution through wild-to-crop introgressions of Malus sylvestris Mill. to the cultivated apple genome. Apple tree domestication is therefore one example in woody perennials that involved gene flow from several wild species from multiple geographical areas. This study provides an example of a complex protracted process of domestication in long-lived plant perennials, and is a starting point for apple breeding programmes.

Comparative proteomic and physiological analyses reveal tribenuron-methyl phytotoxicity and nontarget-site resistance mechanisms in Brassica napus.

The application of herbicides is the most effective strategy for weed control and the development of herbicide-resistant crops will facilitate the weed management. The acetolactate synthase-inhibiting herbicide, tribenuron-methyl (TBM), is broadly used for weed control. However, its application in rapeseed field is restricted since rapeseed is sensitive to TBM. Herein, an integrated study of cytological, physiological and proteomic analysis of the TBM-resistant rapeseed mutant M342 and its wild-type (WT) plants was conducted. After TBM spraying, M342 showed improved tolerance to TBM, and proteins implicated in non-target-site resistance (NTSR) to herbicides had a significantly higher level in M342 as compared with the WT. Differentially accumulated proteins (DAPs) between these two genotypes were enriched in glutathione metabolism and oxidoreduction coenzyme metabolic process, which protected the mutant from oxidative stress triggered by TBM. Important DAPs related to stress or defence response were up-accumulated in M342 regardless of the TBM treatment, which might serve as the constitutive part of NTSR to TBM. These results provide new clues for further exploration of the NTSR mechanism in plants and establish a theoretical basis for the development of herbicide-resistant crops.

Three sesquiterpene lactones suppress lung adenocarcinoma by blocking TMEM16A-mediated Ca2+-activated Cl- channels.

Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (Emax) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

MdNup62 interactions with MdHSFs involved in flowering and heat-stress tolerance in apple.

Because of global warming, the apple flowering period is occurring significantly earlier, increasing the probability and degree of freezing injury. Moreover, extreme hot weather has also seriously affected the development of apple industry. Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport, but their roles in regulating plant development and stress responses are still unknown. Here, we analysed the components of the apple NPC and found that MdNup62 interacts with MdNup54, forming the central NPC channel. MdNup62 was localized to the nuclear pore, and its expression was significantly up-regulated in 'Nagafu No. 2' tissue-cultured seedlings subjected to heat treatments. To determine MdNup62's function, we obtained MdNup62-overexpressed (OE) Arabidopsis and tomato lines that showed significantly reduced high-temperature resistance. Additionally, OE-MdNup62 Arabidopsis lines showed significantly earlier flowering compared with wild-type. Furthermore, we identified 62 putative MdNup62-interacting proteins and confirmed MdNup62 interactions with multiple MdHSFs. The OE-MdHSFA1d and OE-MdHSFA9b Arabidopsis lines also showed significantly earlier flowering phenotypes than wild-type, but had enhanced high-temperature resistance levels. Thus, MdNUP62 interacts with multiple MdHSFs during nucleocytoplasmic transport to regulate flowering and heat resistance in apple. The data provide a new theoretical reference for managing the impact of global warming on the apple industry.

Cyclanilide Induces Lateral Bud Outgrowth by Modulating Cytokinin Biosynthesis and Signalling Pathways in Apple Identified via Transcriptome Analysis.

Cyclanilide (CYC), a plant growth regulator, is a potent shoot branching agent in apple. However, its mechanism remains unclear. The current study revealed that CYC treatment resulted in massive reprogramming of the axillary bud transcriptome, implicating several hormones in the response. We observed a marked increase (approximately 2-fold) in the level of zeatin riboside and a significant decrease (approximately 2-fold) in the level of abscisic acid (ABA). Zeatin metabolism gene cytokinin (CTK) oxidase 1 (CKX 1) was down-regulated at 168 h after CYC treatment compared with the control. Weighted gene co-expression network analysis of differentially expressed genes demonstrated the turquoise module clusters exhibited the highest positive correlation with zeatin riboside (r = 0.92) and the highest negative correlation with ABA (r = -0.8). A total of 37 genes were significantly enriched in the plant hormone signal transduction pathway in the turquoise module. Among them, the expressions of CTK receptor genes WOODEN LEG and the CTK type-A response regulators genes ARR3 and ARR9 were up-regulated. ABA signal response genes protein phosphatase 2C genes ABI2 and ABI5 were down-regulated in lateral buds after CYC treatment at 168 h. In addition, exogenous application of 6-benzylaminopurine (6-BA, a synthetic type of CTK) and CYC enhanced the inducing effect of CYC, whereas exogenous application of lovastatin (a synthetic type of inhibitor of CTK biosynthesis) or ABA and CYC weakened the promoting effect of CYC. These results collectively revealed that the stimulation of bud growth by CYC might involve CTK biosynthesis and signalling, including genes CKX1 and ARR3/9, which provided a direction for further study of the branching promoting mechanism of CYC.

Molecular mechanism of MdWUS2-MdTCP12 interaction in mediating cytokinin signaling to control axillary bud outgrowth.

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis, indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin.

Identification of apple TFL1-interacting proteins uncovers an expanded flowering network.

KEY MESSAGE: MdTFL1, a floral repressor, forms protein complexes with several proteins and could compete with MdFT1 to regulate reproductive development in apple. Floral transition is a key developmental stage in the annual growth cycle of perennial fruit trees that directly determines the fruit development in the subsequent stage. FLOWERING LOCUS T (FT)/TERMINAL FLOWER1 (TFL1) family is known to play a vital regulatory role in plant growth and flowering. In apple, the two TFL1-like genes (MdTFL1-1 and MdTFL1-2) function as floral inhibitors; however, their mechanism of action is still largely unclear. This study aimed to functionally validate MdTFL1 and probe into its mechanism of action in apple. MdTFL1-1 and MdTFL1-2 were expressed mainly in stem and apical buds of vegetative shoots, with little expression in flower buds and young fruit. Expression of MdTFL1-1 and MdTFL1-2 rapidly decreased during floral induction. On the other hand, transgenic Arabidopsis, which ectopically expressed MdTFL1-1 or MdTFL1-2, flowered later than wild-type plants; demonstrating their in planta capability to function redundantly as flower repressors. Furthermore, we identified hundreds of novel interaction proteins of the two apple MdTFL1 proteins using yeast two-hybrid screens. Independent experiments for several proteins confirmed the yeast two-hybrid interactions. Among them, the transcription factor Nuclear Factor-Y subunit C (MdNF-YC2) functions as a promoter of flowering in Arabidopsis by activating LEAFY (LFY) and APETALA1 (AP1) expression. MdFT1 showed a similar interaction pattern as MdTFL1, implying a possible antagonistic action in the regulation of flowering. These newly identified TFL1-interacting proteins (TIPs) not only expand the floral regulatory network, but may also introduce new roles for TFL1 in plant development.

Seasonal variations in water flux compositions controlled by leaf development: isotopic insights at the canopy-atmosphere interface.

Water-stable isotopes provide a valuable tool for tracing plant-water interactions, particularly evapotranspiration (ET) partitioning and leaf water dynamics at the plant-atmosphere interface. However, process-based investigations of plant/leaf development and the associated isotopic dynamics of water fluxes involving isotope enrichment at plant-atmosphere interfaces at the ecosystem scale remain challenging. In this study, in situ isotopic measurements and tracer-aided models were used to study the dynamic interactions between vegetation growth and the isotopic dynamics of water fluxes (ET, soil evaporation, and transpiration) involving isotope enrichment in canopy leaves in a multispecies grassland ecosystem. The day-to-day variations in the isotopic compositions of ET flux were mainly controlled by plant growth, which could be explained by the significant logarithmic relationship determined between the leaf area index and transpiration fraction. Leaf development promoted a significant increase in the isotopic composition of ET and led to a slight decrease in the isotopic composition of water in canopy leaves. The transpiration (evaporation) isoflux acted to increase (decrease) the δ18O of water vapor, and the total isoflux impacts depended on the seasonal tradeoffs between transpiration and evaporation. The isotopic evidence in ET fluxes demonstrates the biotic controls on day-to-day variations in water/energy flux partitioning through transpiration activity. This study emphasizes that stable isotopes of hydrogen and oxygen are effective tools for quantitative evaluations of the hydrological component partitioning of ecosystems and plant-climate interactions.

Sleep disturbance mediates the relationship between depressive symptoms and cognitive function in older adults with mild cognitive impairment.

This study examined whether and to what extent sleep disturbance mediates the effects of depressive symptoms on the cognition of individuals with mild cognitive impairment (MCI), who represent a high-risk group for developing dementia. Cross-sectional data were obtained from a sample of 204 Chinese community-dwelling older adults with MCI. MCI subjects were screened using the Montreal Cognitive Assessment, sleep quality was measured using the Pittsburgh Sleep Quality Index, and depressive symptoms were assessed using the Geriatric Depression Scale. Mediation analysis was conducted using the PROCESS macro with 10,000 bootstrap samples. The significant mediating effect of sleep quality on the association between depressive symptoms and cognition (Beta = -0.025; 95% CI, -0.054 to -0.007) explains 26% of the total effect of depressive symptoms on cognition and implies that the timely detection and management of sleep disturbance among the MCI population is highly important, especially for those with depressive symptoms.

Construction of a high-density SNP-based genetic map and identification of fruit-related QTLs and candidate genes in peach [Prunus persica (L.) Batsch].

BACKGROUND: High-density genetic mapping is a valuable tool for mapping loci that control specific traits for perennial fruit trees. Peach is an economically important fruit tree and a model Rosaceae species for genomic and genetic research. In peach, even though many molecular markers, genetic maps and QTL mappings have been reported, further research on the improvement of marker numbers, map densities, QTL accuracy and candidate gene identification is still warranted. RESULTS: A high-density single nucleotide polymorphism (SNP)-based peach linkage map was constructed using specific locus amplified fragment sequencing (SLAF-seq). This genetic map consisted of 7998 SLAF markers, spanning 1098.79 cM with an average distance of 0.17 cM between adjacent markers. A total of 40 QTLs and 885 annotated candidate genes were detected for 10 fruit-related traits, including fruit weight (FW), fruit diameter (FD), percentage of red skin colour (PSC), eating quality (EQ), fruit flavour (FV), red in flesh (RF), red around pit (RP), adherence to pit (AP), fruit development period (FDP) and fruit fibre content (FFC). Eighteen QTLs for soluble solid content (SSC) were identified along LGs 1, 4, 5, and 6 in 2015 and 2016, and 540 genes were annotated in QTL intervals. Thirty-two QTLs for fruit acidity content (FA) were detected on LG1, and 2, 4, 5, 6, and 1232 candidate genes were identified. The expression profiles of 2 candidate genes for SSC and 4 for FA were analysed in parents and their offspring. CONCLUSIONS: We constructed a high-density genetic map in peach based on SLAF-seq, which may contribute to the identification of important agronomic trait loci. Ninety QTLs for 12 fruit-related traits were identified, most of which overlapped with previous reports, and some new QTLs were obtained. A large number of candidate genes for fruit-related traits were screened and identified. These results may improve our understanding of the genetic control of fruit quality traits and provide useful information in marker-assisted selection for fruit quality in peach breeding programmes.

Genome-wide identification of SERK genes in apple and analyses of their role in stress responses and growth.

BACKGROUND: Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. RESULTS: Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein-protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of 'Nagafu 2' (CF)/CF than in dwarf trees of CF/'Malling 9' (M.9), and in CF than in the spur-type bud mutation "Yanfu 6" (YF). CONCLUSION: This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth.

Molecular role of cytokinin in bud activation and outgrowth in apple branching based on transcriptomic analysis.

KEY MESSAGE: Axillary bud activation and outgrowth were dependent on local cytokinin, and that bud activation preceded the activation of cell cycle and cell growth genes in apple branching. Cytokinin is often applied to apple trees to produce more shoot branches in apple seedlings. The molecular response of apple to the application of cytokinin, and the relationship between bud activation and cell cycle in apple branching, however, are poorly understood. In this study, RNA sequencing was used to characterize differential expression genes in axillary buds of 1-year grafted "Fuji" apple at 4 and 96 h after cytokinin application. And comparative gene expression analyses were performed in buds of decapitated shoots and buds of the treatment of biosynthetic inhibitor of cytokinin (Lovastatin) on decapitated shoots. Results indicated that decapitation and cytokinin increased ZR content in buds and internodes at 4-8 h, and induced bud elongation at 96 h after treatment, relative to buds in shoots receiving the Lovastatin treatment. RNA-seq analysis indicated that differential expression genes in auxin and cytokinin signal transduction were significantly enriched at 4 h, and DNA replication was enriched at 96 h. Cytokinin-responsive type-A response regulator, auxin polar transport, and axillary meristem-related genes were up-regulated at 4 h in the cytokinin and decapitation treatments, while qRT-PCR analysis showed that cell cycle and cell growth genes were up-regulated after 8 h. Collectively, the data indicated that bud activation and outgrowth might be dependent on local cytokinin synthesis in axillary buds or stems, and that bud activation preceded the activation of cell cycle genes during the outgrowth of ABs in apple shoots.

Exogenous application of GA3 inactively regulates axillary bud outgrowth by influencing of branching-inhibitors and bud-regulating hormones in apple (Malus domestica Borkh.).

Although gibberellin (GA) has been reported to control branching, little is known about how GA mediates signals regulating the outgrowth of axillary buds (ABs). In the current study, the effect of the exogenous application of 5.0 mM GA3 on ABs outgrowth on 1-year-old 'Nagafu No. 2'/T337/M. robusta Rehd. apple trees was investigated and compared to the bud-activating treatments, 5 mM BA or decapitation. Additionally, the expression of genes related to bud-regulating signals and sucrose levels in ABs was examined. Results indicated that GA3 did not promote ABs' outgrowth, nor down-regulate the expression of branching repressors [MdTCP40, MdTCP33, and MdTCP16 (homologs of BRANCHED1 and BRC2)], which were significantly inhibited by the BA and decapitation treatments. MdSBP12 and MdSBP18, the putative transcriptional activators of these genes, which are expressed at lower levels in BA-treated and decapitated buds, were up-regulated in the GA3 treatment in comparison to the BA treatment. Additionally, GA3 did not up-regulate the expression of CK response- and auxin transport-related genes, which were immediately induced by the BA treatment. In addition, GA3 also up-regulated the expression of several Tre6P biosynthesis genes and reduced sucrose levels in ABs. Sucrose levels, however, were still higher than what was observed in BA-treated buds, indicating that sucrose may not be limiting in GA3-controlled AB outgrowth. Although GA3 promoted cell division, it was not sufficient to induce AB outgrowth. Conclusively, some branching-inhibiting genes and bud-regulating hormones are associated with the inability of GA3 to activate AB outgrowth.

Comprehensive analysis of GASA family members in the Malus domestica genome: identification, characterization, and their expressions in response to apple flower induction.

BACKGROUND: The plant-specific gibberellic acid stimulated Arabidopsis (GASA) gene family is critical for plant development. However, little is known about these genes, particularly in fruit tree species. RESULTS: We identified 15 putative Arabidopsis thaliana GASA (AtGASA) and 26 apple GASA (MdGASA) genes. The identified genes were then characterized (e.g., chromosomal location, structure, and evolutionary relationships). All of the identified A. thaliana and apple GASA proteins included a conserved GASA domain and exhibited similar characteristics. Specifically, the MdGASA expression levels in various tissues and organs were analyzed based on an online gene expression profile and by qRT-PCR. These genes were more highly expressed in the leaves, buds, and fruits compared with the seeds, roots, and seedlings. MdGASA genes were also responsive to gibberellic acid (GA3) and abscisic acid treatments. Additionally, transcriptome sequencing results revealed seven potential flowering-related MdGASA genes. We analyzed the expression levels of these genes in response to flowering-related treatments (GA3, 6-benzylaminopurine, and sugar) and in apple varieties that differed in terms of flowering ('Nagafu No. 2' and 'Yanfu No. 6') during the flower induction period. These candidate MdGASA genes exhibited diverse expression patterns. The expression levels of six MdGASA genes were inhibited by GA3, while the expression of one gene was up-regulated. Additionally, there were expression-level differences induced by the 6-benzylaminopurine and sugar treatments during the flower induction stage, as well as in the different flowering varieties. CONCLUSION: This study represents the first comprehensive investigation of the A. thaliana and apple GASA gene families. Our data may provide useful clues for future studies and may support the hypotheses regarding the role of GASA proteins during the flower induction stage in fruit tree species.

Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus.

Although INDETERMINATE DOMAIN (IDD) genes encoding specific plant transcription factors have important roles in plant growth and development, little is known about apple IDD (MdIDD) genes and their potential functions in the flower induction. In this study, we identified 20 putative IDD genes in apple and named them according to their chromosomal locations. All identified MdIDD genes shared a conserved IDD domain. A phylogenetic analysis separated MdIDDs and other plant IDD genes into four groups. Bioinformatic analysis of chemical characteristics, gene structure, and prediction of protein-protein interactions demonstrated the functional and structural diversity of MdIDD genes. To further uncover their potential functions, we performed analysis of tandem, synteny, and gene duplications, which indicated several paired homologs of IDD genes between apple and Arabidopsis. Additionally, genome duplications also promoted the expansion and evolution of the MdIDD genes. Quantitative real-time PCR revealed that all the MdIDD genes showed distinct expression levels in five different tissues (stems, leaves, buds, flowers, and fruits). Furthermore, the expression levels of candidate MdIDD genes were also investigated in response to various circumstances, including GA treatment (decreased the flowering rate), sugar treatment (increased the flowering rate), alternate-bearing conditions, and two varieties with different-flowering intensities. Parts of them were affected by exogenous treatments and showed different expression patterns. Additionally, changes in response to alternate-bearing and different-flowering varieties of apple trees indicated that they were also responsive to flower induction. Taken together, our comprehensive analysis provided valuable information for further analysis of IDD genes aiming at flower induction.

Autocrine parathyroid hormone-like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK-ATF2-cyclinD1 signaling.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development. METHODS: The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2). RESULTS: PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists. CONCLUSIONS: Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.

Plasma levels of miR-137 and miR-124 are associated with Parkinson's disease but not with Parkinson's disease with depression.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Recently, it was reported that miR-137, miR-124, and miR-184 were widely expressed in the central nervous system and were vital to neuronal regulation. In this study, we detected the circulating levels of miR-137, miR-124, and miR-184 in PD patients, and explored the potential role of miR-124, miR-137, and miR-184 in the diagnosis of PD. We further described the relationship between these miRNAs and PD with depression (PD-Dep). The study recruited 60 controls and 60 PD patients, which were further divided into two subgroups, PD with depression (PD-Dep, n = 24) and non-depressed group (PD-NDep, n = 36) according to Hamilton Rating Scale for Depression. Plasma levels of miR-137, miR-124, and miR-184 were detected by qRT-PCR. Receiver-operating characteristic (ROC) curve was used to evaluate miR-124 and miR-137 levels as potential diagnostic biomarkers for PD. The results demonstrated that there were no significant differences in levels of miR-184 between PD patients and controls (p > 0.05). However, miR-137 levels were increased significantly for PD patients compared to controls (p < 0.05), while miR-124 levels were down-regulated (p < 0.05). The areas under the ROC curve (AUC) of miR-137 and miR-124 were 0.707 (95% CI 0.615-0.789, p < 0.05) and 0.709 (95% CI 0.618-0.633, p < 0.05), respectively. Correlation analysis revealed that there was no relationship between these two miRNAs levels and UPDRS scores or H&Y stage. There were no significant differences in miR-137 and miR-124 levels between PD-Dep and PD-NDep (p > 0.05). Thus, plasma levels of miR-137 and miR-124 are associated with Parkinson's disease and might be potential biomarkers of the diagnosis of PD. There were no associations of plasma miR-137 and miR-124 with the severity of PD motor symptoms or PD-Dep.