N6-methyladenosine-mediated feedback regulation of abscisic acid perception via phase-separated ECT8 condensates in Arabidopsis.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNAs, yet how plants recognize this chemical modification to swiftly adjust developmental plasticity under environmental stresses remains unclear. Here we show that m6A mRNA modification and its reader protein EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) act together as a key checkpoint for negative feedback regulation of abscisic acid (ABA) signalling by sequestering the m6A-modified ABA receptor gene PYRABACTIN RESISTANCE 1-LIKE 7 (PYL7) via phase-separated ECT8 condensates in stress granules in response to ABA. This partially depletes PYL7 mRNA from its translation in the cytoplasm, thus reducing PYL7 protein levels and compromising ABA perception. The loss of ECT8 results in defective sequestration of m6A-modified PYL7 in stress granules and permits more PYL7 transcripts for translation. This causes overactivation of ABA-responsive genes and the consequent ABA-hypersensitive phenotypes, including drought tolerance. Overall, our findings reveal that m6A-mediated sequestration of PYL7 by ECT8 in stress granules negatively regulates ABA perception, thereby enabling prompt feedback regulation of ABA signalling to prevent plant cell overreaction to environmental stresses.

Estimating waves via measured ship responses.

Optimisation of energy efficiency and operational performance as well as assessment of safety levels and emissions of marine operations require detailed information about the acting wave system. It is possible-with an analogy to classical wave buoys-to estimate the directional wave spectrum by processing sensor measurements of wave-induced responses (e.g., motions and structural responses) from a ship. Compared to other sources of wave data (e.g., buoys, satellites, third-generation wave models), estimation concepts using the ship itself as a buoy provide the wave spectrum at the exact spatio-temporal point, potentially increasing accuracy and with minimal associated cost. This paper gives an overview of the technology, discusses associated uncertainties, and highlights new developments made for estimating waves via measured ship responses.

N6-methyladenosine modification underlies messenger RNA metabolism and plant development.

RNA modifications constitute an essential layer of gene regulation in living organisms. As the most prevalent internal modification on eukaryotic mRNAs, N6-methyladenosine (m6A) exists in many plant species and requires the evolutionarily conserved methyltransferases, demethylases, and m6A binding proteins for writing, erasing, and reading m6A, respectively. In plants, m6A affects many aspects of mRNA metabolism, including alternative polyadenylation, secondary structure, translation, and decay, which underlies various plant developmental processes and stress responses. Here, we discuss the recent progress in understanding the roles of m6A modification in mRNA metabolism and their mechanistic link with plant development and stress responses. We also highlight some outstanding questions and provide an outlook on future prospects of m6A research in plants.

VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) family proteins are regulators of root system architecture.

Root system architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system, we identified a rice (Oryza sativa) gene, VAP-RELATED SUPPRESSOR OF TOO MANY MOUTHS1 (OsVST1), which plays a key role in controlling RSA. This gene encodes a homolog of the VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) proteins in Arabidopsis (Arabidopsis thaliana), which promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact RSA. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 did not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.

Functional Divergence of PIN1 Paralogous Genes in Rice.

Auxin is a phytohormone that plays an important role in plant growth and development by forming local concentration gradients. The regulation of auxin levels is determined by the activity of auxin efflux carrier protein PIN-formed (PIN). In Arabidopsis thaliana, PIN-formed1 (PIN1) functions in inflorescence and root development. In rice (Oryza sativa L.), there are four PIN1 homologs (OsPIN1a-1d), but their functions remain largely unexplored. Hence, in this study, we created mutant alleles of PIN1 gene-pin1a, pin1b, pin1c, pin1d, pin1a pin1b and pin1c pin1d- using CRISPR/Cas9 technology and used them to study the functions of the four OsPIN1 paralogs in rice. In wild-type rice, all four OsPIN1 genes were relatively highly expressed in the root than in other tissues. Compared with the wild type, the OsPIN1 single mutants had no dramatic phenotypes, but the pin1a pin1b double mutant had shorter shoots and primary roots, fewer crown roots, reduced root gravitropism, longer root hairs and larger panicle branch angle. Furthermore, the pin1c pin1d double mutant showed no observable phenotype at the seedling stage, but showed naked, pin-shape inflorescence at flowering. These data suggest that OsPIN1a and OsPIN1b are involved in root, shoot and inflorescence development in rice, whereas OsPIN1c and OsPIN1d mainly function in panicle formation. Our study provides basic knowledge that will facilitate the study of auxin transport and signaling in rice.

CRD1, an Xpo1 domain protein, regulates miRNA accumulation and crown root development in rice.

Crown root (CR) is the main component of the fibrous root system in cereal crops, but the molecular mechanism underlying CR development is still unclear. Here, we isolated the crown root defect 1 (crd1) mutant from ethyl methane sulfonate-mutated mutant library, which significantly inhibited CR development. The CRD1 was identified through genome resequencing and complementation analysis, which encodes an Xpo1 domain protein: the rice ortholog of Arabidopsis HASTY (HST) and human exportin-5 (XPO5). CRD1 is ubiquitously expressed, with the highest expression levels in the CR primordium at the stem base. CRD1 is a nucleocytoplasmic protein. The crd1 mutant contains significantly reduced miRNA levels in the cytoplasm and nucleus, suggesting that CRD1 is essential for maintaining normal miRNA levels in plant cells. The altered CR phenotype of crd1 was simulated by target mimicry of miR156, suggesting that this defect is due to the disruption of miR156 regulatory pathways. Our analysis of CRD1, the HST ortholog identified in monocots, expands our understanding of the molecular mechanisms underlying miRNA level and CR development.

OsSPL3, an SBP-Domain Protein, Regulates Crown Root Development in Rice.

The major root system of cereals consists of crown roots (or adventitious roots), which are important for anchoring plants in the soil and for water and nutrient uptake. However, the molecular basis of crown root formation is largely unknown. Here, we isolated a rice (Oryza sativa) mutant with fewer crown roots, named lower crown root number1 (lcrn1). Map-based cloning revealed that lcrn1 is caused by a mutation of a putative transcription factor-coding gene, O. sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (OsSPL3). We demonstrate that the point mutation in lcrn1 perturbs theO. sativa microRNA156 (OsmiR156)-directed cleavage of OsSPL3 transcripts, resulting in the mutant phenotype. Chromatin immunoprecipitation sequencing assays of OsSPL3 binding sites and RNA sequencing of differentially expressed transcripts in lcrn1 further identified potential direct targets of OsSPL3 in basal nodes, including a MADS-box transcription factor, OsMADS50. OsMADS50-overexpressing plants produced fewer crown roots, phenocopying lcrn1, while knocking out OsMADS50 in the lcrn1 background reversed this phenotype. We also show that OsSPL12, another OsmiR156 target gene, regulates OsMADS50 and crown root development. Taken together, our findings suggest a novel regulatory pathway in which the OsmiR156-OsSPL3/OsSPL12 module directly activates OsMADS50 in the node to regulate crown root development in rice.

Metajapogenins A-C, Pregnane Steroids from Shells of Metaplexis japonica.

Phytochemical investigation of the shells of Metaplexis japonica (Thunb.) Makino, belonging to the family of Apocynaceae, afforded three new pregnane steroids, metajapogenins A-C, along with three known compounds. The structures of the new compounds were elucidated as 12ß,14ß,17ß-trihydroxypregna-3,5-dien-7,20-dione, 12ß,14ß,17ß,20ß-tetrahydroxypregna-3,5-dien-7-one; 3ß,12ß,14ß,17ß-tetrahydroxypregn-5-ene-7,20-dione on the basis of extensive spectroscopic evidence derived from 1D; 2D-NMR experiments and mass spectrometry. The known compounds included pergularin; 12-O-acetylpergularin; and pergularin-3-O-ß-d-oleandropyranose; which were identified for the first time in the shells of M. japonica.

Analysis of flavonoids and hydroxycinnamic acid derivatives in rapeseeds (Brassica napus L. var. napus) by HPLC-PDA--ESI(--)-MS(n)/HRMS.

A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA-ESI(-)-MS(n)/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA-ESI(-)-MS(n)/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential.

Phenolic composition analysis and gene expression in developing seeds of yellow- and black-seeded Brassica napus.

Breeders have focused on yellow-seeded Brassica napus (rapeseed) for its better quality compared with the black-seeded variety. Moreover, flavonoids have been associated with this kind of rapeseed. In this study, we applied lipid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS(n)) to compare flavonoids in developing seeds of natural black-seeded B. napus and yellow-seeded introgression lines selected from progenies of B. napus-Sinapis alba somatic hybrids. Aside from the most abundant phenolic compounds (sinapine and sinapic acid) and 1, 2-disinapoylglucose, 16 different flavonoids were identified and quantified, including (-)-epicatechin, five monocharged oligomers of (-)-epicatechin ([DP 2](-), [DP 3](-), [DP 4] [DP 2](-) B2 and [DP 2](-) B5), quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin-sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids were lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties. Transcriptome changes of genes participating in the flavonoid pathway were discovered by quantitative reverse transcription polymerase chain reaction analysis. Consistent with flavonoid changes identified by high performance liquid chromatography analysis, the expression of most genes in the flavonoid biosynthetic pathway was also downregulated.

Use of digital gene expression to discriminate gene expression differences in early generations of resynthesized Brassica napus and its diploid progenitors.

BACKGROUND: Polyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. Brassica napus L. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. RNA-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids. RESULTS: We examined the global gene expression patterns of the first four generations of resynthesized B. napus (F1-F4), its diploid progenitors B. rapa and B. oleracea, and natural B. napus using digital gene expression analysis. Almost 42 million clean tags were generated using Illumina technology to produce the expression data for 25959 genes, which account for 63% of the annotated B. rapa genome. More than 56% of the genes were transcribed from both strands, which indicate the importance of RNA-mediated gene regulation in polyploidization. Tag mapping of the B. rapa genome generated 19023, 18547, 24383, 20659, 18881, 20692, and 19955 annotated genes for the B. rapa, B. oleracea, F1-F4 of synthesized B. napus, and natural B. napus libraries, respectively. The unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. Thousands of differentially expressed genes (DEGs) were identified and revealed the substantial changes in F1-F4. Among the 20 most DEGs are DNA binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs suggested approximately 120 biological pathways. CONCLUSIONS: The systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized B. napus. This information broadens our understanding of the mechanisms of B. napus polyploidization and contributes to molecular and genetic research by enriching the Brassica database.

FT-IR and NMR study of seed coat dissected from different colored progenies of Brassica napus-Sinapis alba hybrids.

BACKGROUND: Yellow-seeded Brassica napus is preferred by breeders for economic reasons, and seed coat is the most obvious comparable character between yellow and black rapeseed. RESULTS: Owing to an incomplete understanding of chemical components in yellow rapeseed coat, here we optimized a technique based on attenuated total reflectance-Fourier transform infrared (FT-IR) microspectroscopy to screen differences in chemical compositions of Brassica napus, Sinapis alba and four progeny lines with different yellow seed color derived from somatic hybrids between B. napus and S. alba. Nuclear magnetic resonance (NMR) analysis of seed coat from B. napus and S. alba represented two specific peaks representing components of lignin in B. napus, which were absent in the progenies. Also, the intensity of peaks related to fiber were stronger in B. napus than S. alba and yellow seed lines, indicating the differences of fiber and lignin contents in yellow-seeded lines and parent lines. Differences in the lipid, protein and carbohydrate features were identified between B. napus and S. alba, some of which were specifically inherited in progenies of the somatic hybrids. CONCLUSION: FT-IR spectrometry and NMR provide a rapid, direct bioanalytical technique for exploring the changes in molecular chemistry within the canola seed coat and for selecting rapeseed materials with high quality, which can be further used for breeding or nutrition of human and animals.