A chaperonin containing T-complex polypeptide-1 facilitates the formation of the PbWoxT1-PbPTB3 ribonucleoprotein complex for long-distance RNA trafficking in Pyrus betulaefolia.

Numerous plant endogenous mRNAs move via phloem and thus affect the growth and development of long-distant organs. mRNAs are transported with RNA-binding proteins forming a ribonucleoprotein complex. However, it remains elusive how such RNP complex assembles and facilitates mRNA trafficking. Protease digestion and RNA immunoprecipitation were used to investigate the RNP assembly function of the complete Chaperonin Containing T-complex Polypeptide-1. In situ hybridization, hairy root transformation, microprojectile bombardment, and grafting experiments demonstrate the role of CCT complex in the transport of a PbWoxT1-PbPTB3 RNP complex in Pyrus betulaefolia. PbCCT5 silenced caused defective movement of GFP-PbPTB3 and GFP-PbWoxT1 from hairy roots to new leaves via the phloem. PbCCT5 is shown to interact with PbPTB3. PbCCT complex enhanced PbPTB3 stabilization and permitted assembly of PbWoxT1 and PbPTB3 into an RNP complex. Furthermore, silencing of individual CCT subunits inhibited the intercellular movement of GFP-PbPTB3 and long-distance trafficking of PbWoxT1 and PbPTB3 in grafted plants. Taken together, the CCT complex assembles PbPTB3 and PbWoxT1 into an RNP complex in the phloem in order to facilitate the long-distance trafficking of PbWoxT1 in P. betulaefolia. This study therefore provides important insights into the mechanism of RNP complex formation and transport.

Thyroid cancer incidence trend and association with obesity, physical activity in the United States.

BACKGROUND: State-level racial/ethnic and age differences and the temporal trend of thyroid cancer (TC) incidence in the USA remain unknown. Our research purposes include: Characterizing state-level temporal variation in TC incidence; examining the disparities of TC incidence by state-level race/ethnicity and age; performing an ecological correlation between TC incidence and obesity/physical activity. METHODS: TC incidence data during 2000-2017 were extracted from the United States cancer statistics. Using joinpoint regression to evaluate TC incidence trends. Annual percent change (APC), average APC (AAPC) and incidence rates were calculated. The obesity prevalence and physical activity level at the state-level were extracted from Behavioral Risk Factor Surveillance System, and the association between state-level AAPC of TC and obesity/physical activity was tested by Pearson correlation coefficient. RESULTS: We found that the TC incidence had shown an overall downward trend in recent years, but 10 states continued increasing. There were significant differences in state-level race/ethnicity (non-Hispanic Whites as a reference) and age group (45-59 age group as a reference) incidence: Incidence Rate Ratio (IRR) was 0.4-1.2 for non-Hispanic Blacks, 0.7-1.6 for non-Hispanic Asian and Pacific Islanders, 0.4-1.2 for non-Hispanic American Indians/Alaskan Natives, and 0.5-1.3 for Hispanics. High IRR in young people were distributed in northern USA, while in older people were distributed in south. The state-level obesity/physical activity level and AAPC had a weak correlation (r = 0.34, P = 0.016) and inverse weak correlation (r = -0.29, P = 0.037), respectively. The AAPC of states with a consistent increasing trend had an extremely strong correlation with obesity prevalence (r = 0.80, p = 0.006), and an inverse strong correlation with physical activity level (r = -0.65, P = 0.04). CONCLUSIONS: Thyroid cancer incidence in 10 states continued increasing. State-level variation in race/ethnicity and age group incidence were found. Lifestyle and environmental factors may interfere with the incidence trend of TC in the USA.

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry 'Lapins'.

Recent studies have shown that loss of pollen-S function in S4' pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4') in S4' pollen (pollen harboring the SFB4' gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4' did not interact with S-RNase. However, a protein in S4' pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4' pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S4-RNase as a bait to screen S4' pollen proteins. Our screen identified the protein encoded by S 4 -SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S 4 -SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S4-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4' pollen.

A Single-Nucleotide Polymorphism in the Promoter of a Hairpin RNA Contributes to Alternaria alternata Leaf Spot Resistance in Apple (Malus × domestica).

Apple leaf spot caused by the Alternaria alternata f. sp mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA) named MdhpRNA277 that produces small RNAs and is induced by ALT1 infection in 'Golden Delicious' apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in resistant apple variety 'Hanfu' and at low levels in susceptible variety 'Golden Delicious' following ALT1 infection. MdhpRNA277 was strongly induced in 'Golden Delicious' but not 'Hanfu' following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 'Golden Delicious' versus 'Hanfu'. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 'Golden Delicious' (pMdhpRNA277-GD) and 'Hanfu' (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-GD, but not to pMdhpRNA277-HF Transgenic 'GL-3' apple expressing pMdhpRNA277-GD:MdhpRNA277 was more susceptible to ALT1 infection than plants expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and reduced expression of the five target R genes. We confirmed that the SNP in pMdhpRNA277 is associated with A. alternata leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to A. alternata leaf spot.

PbWoxT1 mRNA from pear (Pyrus betulaefolia) undergoes long-distance transport assisted by a polypyrimidine tract binding protein.

Little is known about the mechanisms by which mRNAs are transported over long distances in the phloem between the rootstock and the scion in grafted woody plants. We identified an mRNA in the pear variety 'Du Li' (Pyrus betulaefolia) that was shown to be transportable in the phloem. It contains a WUSCHEL-RELATED HOMEOBOX (WOX) domain and was therefore named Wox Transport 1 (PbWoxT1). A 548-bp fragment of PbWoxT1 is critical in long-distance transport. PbWoxT1 is rich in CUCU polypyrimidine domains and its mRNAs interact with a polypyrimidine tract binding protein, PbPTB3. Furthermore, the expression of PbWoxT1 significantly increased in the stems of wild-type (WT) tobacco grafted onto the rootstocks of PbWoxT1 or PbPTB3 co-overexpressing lines, but this was not the case in WT plants grafted onto PbWoxT1 overexpressing rootstocks, suggesting that PbPTB3 mediates PbWoxT1 mRNA long-distance transport. We provide novel information that adds a new mechanism with which to explain the noncell-autonomous manner of WOX gene function, which enriches our understanding of how WOX genes work in fruit trees and other species.

Corrigendum: Research progress and trends in metabolomics of fruit trees.

[This corrects the article DOI: 10.3389/fpls.2022.881856.].

Research Progress and Trends in Metabolomics of Fruit Trees.

Metabolomics is an indispensable part of modern systems biotechnology, applied in the diseases' diagnosis, pharmacological mechanism, and quality monitoring of crops, vegetables, fruits, etc. Metabolomics of fruit trees has developed rapidly in recent years, and many important research results have been achieved in combination with transcriptomics, genomics, proteomics, quantitative trait locus (QTL), and genome-wide association study (GWAS). These research results mainly focus on the mechanism of fruit quality formation, metabolite markers of special quality or physiological period, the mechanism of fruit tree's response to biotic/abiotic stress and environment, and the genetics mechanism of fruit trait. According to different experimental purposes, different metabolomic strategies could be selected, such as targeted metabolomics, non-targeted metabolomics, pseudo-targeted metabolomics, and widely targeted metabolomics. This article presents metabolomics strategies, key techniques in metabolomics, main applications in fruit trees, and prospects for the future. With the improvement of instruments, analysis platforms, and metabolite databases and decrease in the cost of the experiment, metabolomics will prompt the fruit tree research to achieve more breakthrough results.

Functional identification of lncRNAs in sweet cherry (Prunus avium) pollen tubes via transcriptome analysis using single-molecule long-read sequencing.

Sweet cherry (Prunus avium) is a popular fruit with high nutritional value and excellent flavor. Although pollen plays an important role in the double fertilization and subsequent fruit production of this species, little is known about its pollen tube transcriptome. In this study, we identified 16,409 transcripts using single-molecule sequencing. After filtering 292 transposable elements, we conducted further analyses including mRNA classification, gene function prediction, alternative splicing (AS) analysis, and long noncoding RNA (lncRNA) identification to gain insight into the pollen transcriptome. The filtered transcripts could be matched with 3,438 coding region sequences from the sweet cherry genome. GO and KEGG analyses revealed complex biological processes during pollen tube elongation. A total of 2043 AS events were predicted, 7 of which were identified in different organs, such as the leaf, pistil and pollen tube. Using BLASTnt and the Coding-Potential Assessment Tool (CPAT), we distinguished a total of 284 lncRNAs, among which 154 qualified as natural antisense transcripts (NATs). As the NATs could be the reverse complements of coding mRNA sequences, they might bind to coding sequences. Antisense transfection assays showed that the NATs could regulate the expression levels of their complementary sequences and even affect the growth conditions of pollen tubes. In summary, this research characterizes the transcripts of P. avium pollen and lays the foundation for elucidating the physiological and biochemical mechanisms underlying sexual reproduction in the male gametes of this species.

SLFL Genes Participate in the Ubiquitination and Degradation Reaction of S-RNase in Self-compatible Peach.

It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutated PperSFB1m, PperSFB2m, PperSFB4m, and normal PperSFB2, and style determinant genes PperS1-RNase, PperS2-RNase, PperS2m-RNase, and PperS4-RNase. The mutated PperSFBs encode truncated SFB proteins due to a fragment insertion. The truncated PperSFBs and normal PperSFB2 interacted with PperS-RNases demonstrated by Y2H. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2, and HVa-HVb. The box domain of PperSFB2 did not interact with PperS-RNases, both of the box-V1 and V1-V2 had interactions with PperS-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with PperS2-RNase showed by Y2H and BiFC assay. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, named as PperSLFL1, PperSLFL2, and PperSLFL3, respectively. In phylogenetic analysis PperSLFLs clustered with Maloideae SFBB genes, and PperSFB genes were clustered into the other group with other SFB genes of Prunus. Protein interaction analysis revealed that the three PperSLFLs interacted with PperSSK1 and PperS-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules onto PperS-RNases. The above results suggest that three PperSLFLs are the appropriate candidates for the "general inhibitor," which would inactivate the S-RNases in pollen tubes, involved in the self-incompatibility of peach.

Construction of Commercial Sweet Cherry Linkage Maps and QTL Analysis for Trunk Diameter.

A cross between the sweet cherry (Prunus avium) cultivars 'Wanhongzhu' and 'Lapins' was performed to create a mapping population suitable for the construction of a linkage map. The specific-locus amplified fragment (SLAF) sequencing technique used as a single nucleotide polymorphism (SNP) discovery platform and generated 701 informative genotypic assays; these, along with 16 microsatellites (SSRs) and the incompatibility (S) gene, were used to build a map which comprised 8 linkage groups (LGs) and covered a genetic distance of 849.0 cM. The mean inter-marker distance was 1.18 cM and there were few gaps > 5 cM in length. Marker collinearity was maintained with the established peach genomic sequence. The map was used to show that trunk diameter (TD) is under the control of 4 loci, mapping to 3 different LGs. Different locus influenced TD at a varying stage of the tree's development. The high density 'W×L' genetic linkage map has the potential to enable high-resolution identification of QTLs of agronomically relevant traits, and accelerate sweet cherry breeding.

Prognostic value of pretreatment serum alkaline phosphatase in nasopharyngeal carcinoma.

BACKGROUND: The prognostic value of serum alkaline phosphatase (S-ALP) has not been fully validated for nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: S-ALP levels were measured in 601 patients newly diagnosed with NPC before radical treatment, and possible associations of these levels with 5-year overall survival (OS) and tumor-free survival (TFS) were explored using univariate and multivariate analyses. RESULTS: Elevated pretreatment S-ALP (>85 U/L) was significantly less frequent among patients classified as T1+2 or stage I+II than among those classified as T3+4 or stage III+IV. Multivariate analysis showed that elevated pretreatment S-ALP (>85 U/L), age, T classification and N stage were independent predictors of poor OS and TFS. CONCLUSIONS: Pretreatment S-ALP may be a reliable biomarker to evaluate the long-term prognosis of patients with NPC.

Prognostic value of pretreatment serum levels of lactate dehydrogenase in nonmetastatic nasopharyngeal carcinoma: single-site analysis of 601 patients in a highly endemic area.

BACKGROUND: Numerous studies have generated promising but incomplete evidence for the prognostic value of pretreatment serum levels of lactate dehydrogenase (S-LDH) in nasopharyngeal carcinoma (NPC). METHODS: Pretreatment serum levels of S-LDH in 601 patients with NPC were measured before treatment, and their associations with overall survival and tumor-free survival were studied. Univariate and multivariate analysis of subgroups was used to evaluate the prognostic value of S-LDH in early-stage and late-stage NPC separately. RESULTS: Pretreatment S-LDH levels were significantly lower in T1+2 patients than in T3+4 patients, lower in N0+1 patients than in N2+3 ones, and lower in stage I + II patients than in III + IV ones. Multivariate analysis showed that among patients with late-stage NPC, high pretreatment S-LDH levels >225 U/L were an independent predictor of poor overall survival and tumor-free survival. Among patients with early-stage NPC, pretreatment S-LDH levels >171 U/L, which overlap with the normal range, were an independent predictor of shorter overall survival and tumor-free survival. CONCLUSION: Pretreatment S-LDH levels may be a reliable biomarker for predicting the long-term prognosis of patients with early-stage or late-stage NPC.

Prognostic value of the pretreatment serum level of cytokeratin fraction 21-1 in undifferentiated nasopharyngeal carcinoma: a study of 332 cases.

BACKGROUND: Prognostic value of serum cytokeratin fraction 21-1 (CYFRA 21-1) has not been fully validated for nasopharyngeal carcinoma (NPC). METHODS: Serum CYFRA 21-1 levels of 332 patients with NPC were measured before treatment, and their association with overall survival (OS), tumor-free survival (TFS), time to local recurrence (TLR), and time to distant recurrence (TDR) was studied. RESULTS: Pretreatment serum CYFRA 21-1 level of patients with classification of T1+2 , N0 , stage I+II was significantly lower than that of those with T3+4 , N1+2+3 and III+IV, respectively. Multivariate analysis showed that a high pretreatment serum level of CYFRA 21-1 and T classification were independent predictors of poor OS and TDR. A high pretreatment level of CYFRA 21-1 was an independent predictor of shorter TFS and TLR. CONCLUSION: The pretreatment serum level of CYFRA 21-1 would be a reliable biomarker to evaluate the long-term prognosis of patients with undifferentiated NPC.

Cloning and characterization of miRNAs and their targets, including a novel miRNA-targeted NBS-LRR protein class gene in apple (Golden Delicious).

MicroRNA (miRNA) has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple (Malus domestica cv. Golden Delicious) by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 (Malus domestica microRNA Ln11), targeted an apple nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class protein coding gene (Md-NBS). 5' RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety 'JiGuan' were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety 'FuJi' infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.

Characterization of sck1, a novel Castanea mollissima mutant with the extreme short catkins and decreased gibberellin.

A novel Chinese chestnut (Castanea mollissima Bl.) mutant with extreme short catkins, here was named sck1 and has been characterized in the present study. This sck1 caused 6-fold shorter than wild-type catkins. Endogenous gibberellic acids markedly decreased in the mutant, and application of exogenous GA(3) could partially restore the sck1 phenotype to the wild-type phenotype. Paclobutrazol (PP(333)), an antagonist of GAs biosynthesis, could significantly inhibit the wild-type catkins growth, and lead to a short catkins phenotype similar to the sck1. In addition, compared to the wild-type catkins, the mRNA expression level of ent-kaurenoic acid oxidase (KAO), a gibberellin biosynthesis key gene, was significantly down-regulated (P<0.01) in the sck1. Importantly, transient over-expression of a normal CmKAO gene in short catkins also could partially restore the wild-type phenotype. Real-time PCR and semi-quantitative analysis showed that the mRNA expression level of KAO was significantly up-regulated. In addition, transient RNA interference of CmKAO in wild-type catkins led the mRNA expression level of KAO decrease significantly and inhibited the wild-type catkins elongation strongly. Taken together, our results suggest that the lower gibberellic acids content that is due to decreased CmKAO expression level may contribute to the generation of the extreme short male catkins, sck1.