Draft genome of the wheat A-genome progenitor Triticum urartu.

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Characterization of the AtSPX3 Promoter Elucidates its Complex Regulation in Response to Phosphorus Deficiency.

AtSPX3, responding to phosphate (Pi) deficiency by its expression, is an important gene involved in Pi homeostasis in Arabidopsis. To understand its transcriptional regulation, we characterized the AtSPX3 promoter by distal truncation, internal deletion and mutation of the predicted cis-elements, and identified multiple cis-elements responsive to Pi status. The P1BS (AtPHR-binding site) and AtMyb4 (putative MYB4-binding site) elements were two main cis-elements in the AtSPX3 promoter. P1BS is essential and has a dosage effect for activating expression of the gene under Pi deficiency, while the element AtMyb4 possesses a dual function: one is to enhance AtSPX3 expression in roots under Pi deficiency, and the other one is to repress AtSPX3 expression in shoots under both Pi deficiency and sufficiency. Moreover, we confirmed that AtPHR1, a key transcription factor in Pi homeostasis of plants, was required for the negative regulation function of the AtMyb4 element in shoots. Additionally, we also found that the AtSPX3 promoter had a length limitation for activating gene expression. Generally, our findings in this work are useful for understanding the molecular regulation mechanism of genes involved in Pi uptake and homeostasis.

SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana.

Histone modifications play critical roles in the perception of environmental cues by plants. Here, we report that Shk1 binding protein 1 (SKB1/AtPRMT5), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is involved in iron homeostasis in Arabidopsis. The SKB1 lesion mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency than the wild type. The expression of SKB1 was not affected by iron, but the level of H4R3sme2 mediated by SKB1 was related to iron status in plants. We showed by chromatin immunoprecipitation (ChIP) and genome-wide ChIP-seq that SKB1 associated with the chromatin of the Ib subgroup bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101), and symmetrically dimethylated histone H4R3. The quantity of SKB1 that associated with chromatin of the Ib subgroup bHLH genes and the level of H4R3sme2 corresponded to the iron status of plants (higher with increased iron supply and lower when iron was removed). We conclude that SKB1-mediated H4R3sme2 regulates iron homeostasis in Arabidopsis in the context of increasing or decreasing expression of Ib subgroup bHLH genes. Iron deficiency may cause an increase in the disassociation of SKB1 from chromatin of the bHLH genes and a decrease in the level of H4R3sme2, thereby elevating their transcription and enhancing iron uptake. Our findings provide new insight into the molecular mechanisms of iron homeostasis in strategy I plants.

Genome-wide identification and characterization of the bHLH gene family in tomato.

BACKGROUND: The basic helix-loop-helix (bHLH) proteins are a large superfamily of transcription factors, and play a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. Tomato is an important vegetable crop, and its genome sequence has been published recently. However, the bHLH gene family of tomato has not been systematically identified and characterized yet. RESULTS: In this study, we identified 159 bHLH protein-encoding genes (SlbHLH) in tomato genome and analyzed their structures. Although bHLH domains were conserved among the bHLH proteins between tomato and Arabidopsis, the intron sequences and distribution of tomato bHLH genes were extremely different compared with Arabidopsis. The gene duplication analysis showed that 58.5% and 6.3% of SlbHLH genes belonged to low-stringency and high-stringency duplication, respectively, indicating that the SlbHLH genes are mainly generated via short low-stringency region duplication in tomato. Subsequently, we classified the SlbHLH genes into 21 subfamilies by phylogenetic tree analysis, and predicted their possible functions by comparison with their homologous genes of Arabidopsis. Moreover, the expression profile analysis of SlbHLH genes from 10 different tissues showed that 21 SlbHLH genes exhibited tissue-specific expression. Further, we identified that 11 SlbHLH genes were associated with fruit development and ripening (eight of them associated with young fruit development and three with fruit ripening). The evolutionary analysis revealed that 92% SlbHLH genes might be evolved from ancestor(s) originated from early land plant, and 8% from algae. CONCLUSIONS: In this work, we systematically identified SlbHLHs by analyzing the tomato genome sequence using a set of bioinformatics approaches, and characterized their chromosomal distribution, gene structures, duplication, phylogenetic relationship and expression profiles, as well predicted their possible biological functions via comparative analysis with bHLHs of Arabidopsis. The results and information provide a good basis for further investigation of the biological functions and evolution of tomato bHLH genes.

Mediator subunit 16 functions in the regulation of iron uptake gene expression in Arabidopsis.

Iron is an essential nutrient for plant growth and development, and its absorption is tightly controlled. Under iron limitation, FIT dimerizes with the four Ib bHLH proteins and activates the expression of iron uptake genes. However, how the dimerized complex activates downstream genes remains unclear. Using forward genetics, a low-iron-sensitive mutant was screened. The corresponding gene (MED16) was isolated, and its biological functions in iron homeostasis were characterized using approaches such as gene expression, protein subcellular localization, protein-protein interaction and chromatin immunoprecipitation assay. Lesion of MED16 significantly reduced FRO2 and IRT1 expression in Arabidopsis roots. The MED16 mutants showed a low shoot iron concentration and severe leaf chlorosis under iron limitation, whereas it grew normally as wild-type under iron sufficiency. Furthermore, we showed that MED16 interacted with FIT and improved the binding of the FIT/Ib bHLH complex to FRO2 and IRT1 promoters under iron-deficient conditions. Additionally, we found that many iron-deficient response genes, which are regulated by FIT, were also controlled by MED16. In conclusion, MED16 is involved in the iron deficiency response, and modulates the iron uptake gene expression under iron limitation. Our results increase the understanding of the molecular regulation mechanisms underlying iron uptake and homeostasis in plants.

Time series analysis revealed prognostic value of continuous nasopharyngeal SARS-CoV-2 nucleic acid quantification for COVID-19: A retrospective study of >3000 COVID-19 patients from 2 centers.

BACKGROUND: Early stratification of disease progression remains one of the major challenges towards the post-coronavirus disease 2019 (COVID-19) era. The clinical relevance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid load is debated due to the heterogeneity in patients' underlying health conditions. We determined the prognostic value of nasopharyngeal viral load dynamic conversion for COVID-19. METHODS: The cycling threshold (Ct) values of 28,937 nasopharyngeal SARS-CoV-2 RT-PCRs were retrospectively collected from 3,364 COVID-19 patients during hospitalization and coordinated to the onset of disease progression. The ROC curve was utilized to determine the predictive performance of the rate of Ct value alteration between two consecutive RT-PCR runs within 48 h (ΔCt%) for disease transformation across patients with different COVID-19 severity and immune backgrounds, and further validated with 1,860 SARS-CoV-2 RT-PCR results from an independent validation cohort of 262 patients. For the 67 patients with severe COVID-19, Kaplan-Meier analysis was performed to evaluate the difference in survival between patients stratified by the magnitude of Ct value alteration between the late and early stages of hospitalization. RESULTS: The kinetics of viral nucleic acid conversion diversified across COVID-19 patients with different clinical characteristics and disease severities. The ΔCt% is a clinical characteristic- and host immune status-independent indicator for COVID-19 progression prediction (AUC = 0.79, 95 % CI = 0.76 to 0.81), which outperformed the canonical blood test markers, including c-reactive protein (AUC = 0.57, 95 % CI = 0.53 to 0.61), serum amyloid A (AUC = 0.61, 95 % CI = 0.54 to 0.68), lactate dehydrogenase (AUC = 0.61, 95 % CI = 0.56 to 0.67), d-dimer (AUC = 0.56, 95 % CI = 0.46 to 0.66), and lymphocyte count (AUC = 0.62, 95 % CI = 0.58 to 0.66). Patients with persistent high SARS-CoV-2 viral load (an increase of mean Ct value < 50 %) during the first 3 days of hospitalization demonstrated a significantly unfavorable survival (HR = 0.16, 95 % CI = 0.04 to 0.65, P = 2.41 × 10-3). CONCLUSIONS: Viral nucleic acid dynamics of SARS-CoV-2 eliminates the inter-patient variance of basic health conditions and therefore, can serve as a prognostic marker for COVID-19.

[MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis].

OBJECTIVE: To explore the expression pattern of microRNA (miRNA) in T cells of peripheral blood mononuclear cell (PBMC) from patients with primary biliary cirrhosis (PBC). METHODS: The expression profile of miRNA in T cells of PBMC was determined by microarray assay and validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: In comparison with the healthy controls, 23 miRNA were down-regulated and 2 miRNA had a higher expression (all P < 0.05). As revealed by qRT-PCR, the expressions of miR-346, miR-17-5p, miR-20a and miR-let-7b decreased obviously while miR-451 and miR-129 became up-regulated. The results were in agreement with those of microarray. CONCLUSIONS: The PBC patients and healthy controls have significantly different expression profiles of microRNA in T cells of PBMC. The differential expression of microRNA may be involved in the pathogenesis of PBC.

Tomato LeTHIC is an Fe-requiring HMP-P synthase involved in thiamine synthesis and regulated by multiple factors.

Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants.

Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana.

The Ib subgroup of the bHLH gene family in Arabidopsis contains four members (AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101). AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deficiency induced transcription factor (FIT), directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLH101 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.

The upregulation of NtAN2 expression at low temperature is required for anthocyanin accumulation in juvenile leaves of Lc-transgenic tobacco (Nicotiana tabacum L.).

Anthocyanins often accumulate in plants subjected to environmental stress, including low temperature. However, the molecular regulatory mechanism of anthocyanin biosynthesis at low temperature is largely unknown. Here, tobacco was transformed with a maize anthocyanin regulatory gene Lc driven by AtSPX3 promoter to investigate the effect of Lc upon the anthocyanin-biosynthesis pathway. We found that the anthocyanin-biosynthesis pathway could not be activated in wild type, while Lc-transgenic tobacco lines exhibited purple pigmentation in juvenile leaves at low temperature. Accordingly, the total anthocyanin contents increased specifically in juvenile leaves in Lc-transgenic lines. Transcriptional analysis showed that NtCHS and NtCHI were induced by low temperature in leaves of wild type and transgenic lines. NtDFR was uniquely expressed in Lc-transgenic lines, but its transcript was not detected in wild type, implying that NtDFR expression in tobacco leaves was dependent on Lc. Furthermore, the expression of NtAN2 (regulatory gene) and NtANS (anthocyanidin synthase gene) was coordinately upregulated in Lc-transgenic lines under low temperature, suggesting that both Lc and NtAN2 might activate the expression of NtANS. Based on our findings and previous reports, we postulated that Lc interacted with NtAN2 induced by low-temperature stress and consequently stimulated anthocyanin biosynthesis in juvenile leaves of Lc-transgenic tobacco lines.

New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat.

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding.

A snapshot of the Chinese SOL Project.

In 2003, the International Solanaceae Project (SOL) was initiated by an international consortium of ten countries including Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy and the United States. The first major effort of the SOL aimed to produce a DNA sequence map for euchromatin regions of 12 chromosomes of tomato (Solanum lycopersicum) before 2010. Here we present an update on Chinese effort for sequencing the euchromatin region of chromosome 3.

Physical mapping and identification of a candidate for the leaf rust resistance gene Lr1 of wheat.

Lr1 is a dominant leaf rust resistance gene located on chromosome 5DL of bread wheat and the wild species Aegilops tauschii. In this study, three polymorphic markers (WR001, WR002, and WR003) were developed from resistance gene analogs (RGAs) clustering around the Lr1 locus. Using these and other markers, Lr1 was mapped to a genetic interval of 0.79 cM in Ae. tauschii and 0.075 cM in wheat. The CAPS marker WR003, derived from LR1RGA1, co-segregated with Lr1 in both mapping populations of wheat and Ae. tauschii. For isolation of Lr1, two genomic BAC libraries (from Ae. tauschii and hexaploid wheat) were screened using the tightly flanking marker PSR567F and a set of nested primers derived from the conserved region of the RGA sequences. Approximately 400 kb BAC contig spanning the Lr1 locus was constructed. The LR1RGA1 encoding a CC-NBS-leucine-rich repeat (LRR) type of protein was the only one of the four RGAs at the Lr1 locus, which co-segregated with leaf rust resistance. Therefore, it represents a very good candidate for Lr1. The allelic sequences of LR1RGA1 from resistant and susceptible lines revealed a divergent DNA sequence block of approximately 605 bp encoding the LRR repeats 9-15, whereas the rest of the sequences were mostly identical. Within this sequence block, the 48 non-synonymous changes resulted in 44 amino acid differences. This indicates that LR1RGA1 likely evolved through one or more recombination or gene conversion events with unknown genes.