The rice NUCLEAR FACTOR-YA5 and MICRORNA169a module promotes nitrogen utilization during nitrogen deficiency.

Nitrogen (N) is essential for plant growth and development. Therefore, understanding its utilization is essential for improving crop productivity. However, much remains to be learned about plant N sensing and signaling. Here, rice (Oryza sativa) NUCLEAR FACTOR-YA5 (OsNF-YA5) expression was tightly regulated by N status and induced under N-deficient conditions. Overexpression (OE) of OsNF-YA5 in rice resulted in increased chlorophyll levels and delayed senescence compared to control plants under normal N conditions. Agronomic traits were significantly improved in OE plants and impaired in knockout mutants under N-deficient conditions. Using a dexamethasone-inducible system, we identified the putative targets of OsNF-YA5 that include amino acid, nitrate/peptide transporters, and NITRATE TRANSPORTER 1.1A (OsNRT1.1A), which functions as a key transporter in rice. OsNF-YA5 directly enhanced OsNRT1.1A expression and N uptake rate under N-deficient conditions. Besides, overexpression of OsNF-YA5 also enhanced the expression of GLUTAMINE SYNTHETASE 1/2 (GS1/2) and GLUTAMINE OXOGLUTARATE AMINOTRANSFERASE 1/2 (GOGAT1/2), increasing free amino acid contents under N-deficient conditions. Osa-miR169a expression showed an opposite pattern with OsNF-YA5 depending on N status. Further analysis revealed that osa-miR169a negatively regulates OsNF-YA5 expression and N utilization, demonstrating that an OsNF-YA5/osa-miR169a module tightly regulates rice N utilization for adaptation to N status.

Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.

Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.

Review of the technology used for structural characterization of the GMO genome using NGS data.

The molecular characterization of genetically modified organisms (GMOs) is essential for ensuring safety and gaining regulatory approval for commercialization. According to CODEX standards, this characterization involves evaluating the presence of introduced genes, insertion sites, copy number, and nucleotide sequence structure. Advances in technology have led to the increased use of next-generation sequencing (NGS) over traditional methods such as Southern blotting. While both methods provide high reproducibility and accuracy, Southern blotting is labor-intensive and time-consuming due to the need for repetitive probe design and analyses for each target, resulting in low throughput. Conversely, NGS facilitates rapid and comprehensive analysis by mapping whole-genome sequencing (WGS) data to plasmid sequences, accurately identifying T-DNA insertion sites and flanking regions. This advantage allows for efficient detection of T-DNA presence, copy number, and unintended gene insertions without additional probe work. This paper reviews the current status of GMO genome characterization using NGS and proposes more efficient strategies for this purpose.

The MYB-CC Transcription Factor PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) Functions in Phosphate Homeostasis and Affects Salt Stress Tolerance in Rice.

Inorganic phosphate (Pi) homeostasis plays an important role in plant growth and abiotic stress tolerance. Several MYB-CC transcription factors involved in Pi homeostasis have been identified in rice (Oryza sativa). PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) is a class II MYC-CC protein, in which the MYC-CC domain is located at the N terminus. In this study, we established that OsPHL7 is localized to the nucleus and that the encoding gene is induced by Pi deficiency. The Pi-responsive genes and Pi transporter genes are positively regulated by OsPHL7. The overexpression of OsPHL7 enhanced the tolerance of rice plants to Pi starvation, whereas the RNA interference-based knockdown of this gene resulted in increased sensitivity to Pi deficiency. Transgenic rice plants overexpressing OsPHL7 produced more roots than wild-type plants under both Pi-sufficient and Pi-deficient conditions and accumulated more Pi in the shoots and roots. In addition, the overexpression of OsPHL7 enhanced rice tolerance to salt stress. Together, these results demonstrate that OsPHL7 is involved in the maintenance of Pi homeostasis and enhances tolerance to Pi deficiency and salt stress in rice.

Signaling pathways underlying nitrogen transport and metabolism in plants.

Nitrogen (N) is an essential macronutrient required for plant growth and crop production. However, N in soil is usually insufficient for plant growth. Thus, chemical N fertilizer has been extensively used to increase crop production. Due to negative effects of N rich fertilizer on the environment, improving N usage has been a major issue in the field of plant science to achieve sustainable production of crops. For that reason, many efforts have been made to elucidate how plants regulate N uptake and utilization according to their surrounding habitat over the last 30 years. Here, we provide recent advances focusing on regulation of N uptake, allocation of N by N transporting system, and signaling pathway controlling N responses in plants. [BMB Reports 2023; 56(2): 56-64].

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Identification and Characterization of Key Genes Responsible for Weedy and Cultivar Growth Types in Soybean.

In cultivated plants, shoot morphology is an important factor that influences crop economic value. However, the effects of gene expression patterns on shoot morphology are not clearly understood. In this study, the molecular mechanism behind shoot morphology (including leaf, stem, and node) was analyzed using RNA sequencing to compare weedy (creeper) and cultivar (stand) growth types obtained in F7 derived from a cross of wild and cultivated soybeans. A total of 12,513 (in leaves), 14,255 (in stems), and 11,850 (in nodes) differentially expressed genes were identified among weedy and cultivar soybeans. Comparative transcriptome and expression analyses revealed 22 phytohormone-responsive genes. We found that GIBBERELLIN 2-OXIDASE 8 (GA2ox), SPINDLY (SPY), FERONIA (FER), AUXIN RESPONSE FACTOR 8 (ARF8), CYTOKININ DEHYDROGENASE-1 (CKX1), and ARABIDOPSIS HISTIDINE KINASE-3 (AHK3), which are crucial phytohormone response genes, were mainly regulated in the shoot of weedy and cultivar types. These results indicate that interactions between phytohormone signaling genes regulate shoot morphology in weedy and cultivar growth type plants. Our study provides insights that are useful for breeding and improving crops to generate high-yield soybean varieties.

Rice microRNA171f/SCL6 module enhances drought tolerance by regulation of flavonoid biosynthesis genes.

Plants have evolved sophisticated defense systems to enhance drought tolerance. These include the microRNA (miRNA) group of small noncoding RNAs that act as post-transcriptional regulators; however, details of the mechanisms by which they confer drought tolerance are not well understood. Here, we show that osa-MIR171f, a member of osa-MIR171 gene family, is mainly expressed in response to drought stress and regulates the transcript levels of SCARECROW-LIKE6-I (SCL6-I) and SCL6-II in rice (Oryza sativa). The SCL6 genes are known to be involved in shoot branching and flag leaf morphology. Osa-MIR171f-overexpressing (osa-MIR171f-OE) transgenic plants showed reduced drought symptoms compared with non-transgenic (NT) control plants under both field drought and polyethylene glycol (PEG)-mediated dehydration stress conditions. Transcriptome analysis of osa-MIR171f-OE plants and osa-mir171f-knockout (K/O) lines generated by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) revealed that osa-mature-miR171a-f (osa-miR171) regulates the expression of flavonoid biosynthesis genes, consequently leading to drought tolerance. This upregulation in the osa-MIR171f-OE plants, which did not occur in NT control plants, was observed under both normal and drought conditions. Our findings indicate that osa-miR171 plays a role in drought tolerance by regulating SCL6-I and SCL6-II transcript levels.

Whole-genome sequencing revealed different demographic histories among the Korean endemic hill pigeon (Columba rupestris), rock pigeon (Columba livia var. domestica) and oriental turtle dove (Streptopelia orientalis).

BACKGROUND: The family Columbidae is known as the pigeon family and contains approximately 351 species and 50 genera. Compared to the wealth of biological and genomic information on these Columba livia var. domesteca, information on Columba rupestris and Streptopelia orientalis has been rather limited. The C. rupestris population size is decreasing in Korea. OBJECTIVES: Whole-genome sequencing and identification of population characterization of each species based genome variation on 9 Korean pigeon and dove samples, namely, six hill pigeon (C. rupestris), one rock pigeon (C. livia var. domestica) and two oriental turtle dove (S. orientalis) samples. RESULTS: The whole genome of 9 genotypes were sequenced and mapped to the C. livia reference genome. Sequence alignment showed over 96% identity in C. rupestris and 94% identity in S. orientalis to the reference genome (GenBank assembly accession: GCA_001887795.1). Sequence variations, including single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and structural variations, revealed that intergenus (Columba vs. Streptopelia) variations were approximately four times higher than intragenus variations (C. livia vs. C. rupestris). Of the two Columba species, C. livia var. domestica is closer to S. orientalis than C. rupestris. Pairwise sequentially Markovian coalescent (PSMC) demographic history analysis revealed that the three species underwent a common population bottleneck between 105 and 120 Kya; since then, the effective population sizes of the rock pigeon and oriental turtle dove have increased. CONCLUSION: The effective population size of the hill pigeon, an Endangered Species of Grade II in Korea, has increased slowly from the second severe bottleneck that occurred approximately 0.5-1.4 × 104 years ago. Our results showed no relationship between copy number variation in the Norrie disease protein (NDP) regulatory regions and plumage color patterns. We report the first comparative analysis of three pigeon genomes.

Comparative Transcriptomics for Genes Related to Berberine and Berbamine Biosynthesis in Berberidaceae.

Berberine and berbamine are bioactive compounds of benzylisoquinoline alkaloids (BIAs) present in Berberis species. The contents of berbamine are 20 times higher than berberine in leaf tissues in three closely related species: Berberis koreana, B. thunbergii and B. amurensis. This is the first report on the quantification of berberine compared to the berbamine in the Berberis species. Comparative transcriptome analyses were carried out with mRNAs from the leaf tissues of the three-species. The comparison of the transcriptomes of B. thunbergii and B. amurensis to those of B. koreana, B. thunbergii showed a consistently higher number of differentially expressed genes than B. amurensis in KEGG and DEG analyses. All genes encoding enzymes involved in berberine synthesis were identified and their expressions were variable among the three species. There was a single copy of CYP80A/berbamunine synthase in B. koreana. Methyltransferases and cytochrome P450 mono-oxidases (CYPs) are key enzymes for BIA biosynthesis. The current report contains the copy numbers and other genomic characteristics of the methyltransferases and CYPs in Berberis species. Thus, the contents of the current research are valuable for molecular characterization for the medicinal utilization of the Berberis species.

Gene Expression and Isoform Identification of PacBio Full-Length cDNA Sequences for Berberine Biosynthesis in Berberis koreana.

Berberis koreana is a medicinal plant containing berberine, which is a bioactive compound of the benzylisoquinoline alkaloid (BIA) class. BIA is widely used in the food and drug industry for its health benefits. To investigate the berberine biosynthesis pathway, gene expression analysis was performed in leaves, flowers, and fruits at different stages of growth. This was followed by full-length cDNA sequencing analysis using the PacBio sequencer platform to determine the number of isoforms of those expressed genes. We identified 23,246 full-length unigenes, among which 8479 had more than one isoform. The number of isoforms ranged between two to thirty-one among all genes. Complete isoform analysis was carried out on the unigenes encoding BIA synthesis. Thirteen of the sixteen genes encoding enzymes for berberine synthesis were present in more than one copy. This demonstrates that gene duplication and translation into isoforms may contribute to the functional specificity of the duplicated genes and isoforms in plant alkaloid synthesis. Our study also demonstrated the streamlining of berberine biosynthesis via the absence of genes for enzymes of other BIAs, but the presence of all the genes for berberine biosynthesize in B. koreana. In addition to genes encoding enzymes for the berberine biosynthesis pathway, the genes encoding enzymes for other BIAs were not present in our dataset except for those encoding corytuberine synthase (CTS) and berbamunine synthase (BS). Therefore, this explains how B. koreana produces berberine by blocking the pathways leading to other BIAs, effectively only allowing the pathway to lead to berberine synthesis.

Identification of resurrection genes from the transcriptome of dehydrated and rehydrated Selaginella tamariscina.

Selaginella tamariscina is a lycophyta species that survives under extremely dry conditions via the mechanism of resurrection. This phenomenon involves the regulation of numerous genes that play vital roles in desiccation tolerance and subsequent rehydration. To identify resurrection-related genes, we analyzed the transcriptome between dehydration conditions and rehydration conditions of S. tamariscina. The de novo assembly generated 124,417 transcripts with an average size of 1,000 bp and 87,754 unigenes. Among these genes, 1,267 genes and 634 genes were up and down regulated by rehydration compared to dehydration. To understand gene function, we annotated Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The unigenes encoding early light-inducible protein (ELIP) were down-regulated, whereas pentatricopeptide repeat-containing protein (PPR), late embryogenesis abundant proteins (LEA), sucrose nonfermenting protein (SNF), trehalose phosphate phosphatase (TPP), trehalose phosphate synthase (TPS), and ABC transporter G family (ABCG) were significantly up-regulated in response to rehydration conditions by differentially expressed genes (DEGs) analysis. Several studies provide evidence that these genes play a role in stress environment. The ELIP and PPR genes are involved in chloroplast protection during dehydration and rehydration. LEA, SNF, and trehalose genes are known to be oxidant scavengers that protect the cell structure from the deleterious effect of drought. TPP and TPS genes were found in the starch and sucrose metabolism pathways, which are essential sugar-signaling metabolites regulating plant metabolism and other biological processes. ABC-G gene interacts with abscisic acid (ABA) phytohormone in the stomata opening during stress conditions. Our findings provide valuable information and candidate resurrection genes for future functional analysis aimed at improving the drought tolerance of crop plants.

Characterization of dwarf and narrow leaf (dnl-4) mutant in rice.

Height and leaf morphology are important agronomic traits of the major crop plant rice (Oryza sativa). In previous studies, the dwarf and narrow leaf genes (dnl1, dnl2 and dnl3) have identified in rice. Using the Ac/Ds knockout system, we found a new dwarf and narrow leaf (dnl) mutant and identified mutated gene. The dnl-4 mutant showed reduced plant height and leaf blade width compared to the wild type, and increased leaf inclination. The morphological defects of the mutant were caused by the suppressed expression of the DNL-4 gene, which encodes a pfkB carbohydrate kinase protein. These results suggest that DNL-4 expression is involved in modulating plant height and leaf growth. Furthermore, DNL-4 expression also affects productivity in rice: the dnl-4 mutant exhibited reduced panicle length and grain width compared with the wild type. To understand DNL-4 function in rice, we analyzed the expression levels of leaf growth-related genes, such as NAL1, NAL7, and CSLD4, in the dnl-4 mutant. Expression of NAL1 and NAL7 was downregulated in the dnl-4 mutant compared to the wild type. The observation that DNL-4 expression corresponded with that of NAL1 and NAL7 is consistent with the narrow leaf phenotype of the dnl-4 mutant. These results suggest that DNL-4 regulates plant height and leaf structure in rice.

Correlation of Pap smear, cervical biopsy, and clinical follow-up with an HPV typing microarray system.

A human papillomavirus (HPV) microarray system allows the determination of HPV type in clinical samples. The purpose of this study was to determine the presence of HPV in liquid-based Pap smears with the MyGene MyHPV Chip Kit HPV genotyping microarray test (MyGene Assay), and to correlate this with the cytology and biopsy diagnoses, clinical follow-up, HPV Hybrid Capture data, and HPV sequence analyses. Four hundred and two Pap smears (93 ThinPrep, 309 SurePath) were available for study. Correlation of HPV DNA detection by the MyGene Assay with the Pap smear diagnosis showed a detection rate of 19/97 (19%) for normal Pap smears, 181/242 (74%) for atypical squamous cells of undetermined significance (ASCUS), and 61/63 (97%) for squamous intraepithelial lesions (SILs). Biopsy data on 248 women were available. HPV was noted by the MyGene Assay in the Pap smear in 98/100 (98%) of the cases, for which the corresponding biopsy had been diagnosed as SIL, compared with 103/148 (69%) of the cases for which the biopsy had been negative for SIL. Clinical follow-ups were available for 200 women with ASCUS Pap smears. A significant increase was observed in the rate of biopsy-proven SILs in women with ASCUS Pap smears that were HPV-positive (63/66=95%) as compared with those that were HPV-negative (96/134=71%, P<0.05). The MyGene Assay and Hybrid Capture system gave equivalent results for all the categories studied, except for the presence of multiple infections, as determined by viral sequence analysis. Specifically, the Hybrid Capture system overestimated the presence of dual infection (low-risk and high-risk positive) by 48% and missed many cases of multiple infections, especially when 2 or more high-risk types were present. It is concluded that the MyGene Assay allows for the routine typing of HPV in liquid-based Pap smears, and that the presence of HPV DNA in ASCUS Pap smears is strongly correlated with a biopsy-proven SIL.