Seasonal pigment fluctuation in diploid and polyploid Arabidopsis revealed by machine learning-based phenotyping method PlantServation.

Long-term field monitoring of leaf pigment content is informative for understanding plant responses to environments distinct from regulated chambers but is impractical by conventional destructive measurements. We developed PlantServation, a method incorporating robust image-acquisition hardware and deep learning-based software that extracts leaf color by detecting plant individuals automatically. As a case study, we applied PlantServation to examine environmental and genotypic effects on the pigment anthocyanin content estimated from leaf color. We processed >4 million images of small individuals of four Arabidopsis species in the field, where the plant shape, color, and background vary over months. Past radiation, coldness, and precipitation significantly affected the anthocyanin content. The synthetic allopolyploid A. kamchatica recapitulated the fluctuations of natural polyploids by integrating diploid responses. The data support a long-standing hypothesis stating that allopolyploids can inherit and combine the traits of progenitors. PlantServation facilitates the study of plant responses to complex environments termed "in natura".

A low-coverage 3' RNA-seq to detect homeolog expression in polyploid wheat.

Although allopolyploid species are common among natural and crop species, it is not easy to distinguish duplicated genes, known as homeologs, during their genomic analysis. Yet, cost-efficient RNA sequencing (RNA-seq) is to be developed for large-scale transcriptomic studies such as time-series analysis and genome-wide association studies in allopolyploids. In this study, we employed a 3' RNA-seq utilizing 3' untranslated regions (UTRs) containing frequent mutations among homeologous genes, compared to coding sequence. Among the 3' RNA-seq protocols, we examined a low-cost method Lasy-Seq using an allohexaploid bread wheat, Triticum aestivum. HISAT2 showed the best performance for 3' RNA-seq with the least mapping errors and quick computational time. The number of detected homeologs was further improved by extending 1 kb of the 3' UTR annotation. Differentially expressed genes in response to mild cold treatment detected by the 3' RNA-seq were verified with high-coverage conventional RNA-seq, although the latter detected more differentially expressed genes. Finally, downsampling showed that even a 2 million sequencing depth can still detect more than half of expressed homeologs identifiable by the conventional 32 million reads. These data demonstrate that this low-cost 3' RNA-seq facilitates large-scale transcriptomic studies of allohexaploid wheat and indicate the potential application to other allopolyploid species.

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

Homeolog expression quantification methods for allopolyploids.

Genome duplication with hybridization, or allopolyploidization, occurs in animals, fungi and plants, and is especially common in crop plants. There is an increasing interest in the study of allopolyploids because of advances in polyploid genome assembly; however, the high level of sequence similarity in duplicated gene copies (homeologs) poses many challenges. Here we compared standard RNA-seq expression quantification approaches used currently for diploid species against subgenome-classification approaches which maps reads to each subgenome separately. We examined mapping error using our previous and new RNA-seq data in which a subgenome is experimentally added (synthetic allotetraploid Arabidopsis kamchatica) or reduced (allohexaploid wheat Triticum aestivum versus extracted allotetraploid) as ground truth. The error rates in the two species were very similar. The standard approaches showed higher error rates (>10% using pseudo-alignment with Kallisto) while subgenome-classification approaches showed much lower error rates (<1% using EAGLE-RC, <2% using HomeoRoq). Although downstream analysis may partly mitigate mapping errors, the difference in methods was substantial in hexaploid wheat, where Kallisto appeared to have systematic differences relative to other methods. Only approximately half of the differentially expressed homeologs detected using Kallisto overlapped with those by any other method in wheat. In general, disagreement in low-expression genes was responsible for most of the discordance between methods, which is consistent with known biases in Kallisto. We also observed that there exist uncertainties in genome sequences and annotation which can affect each method differently. Overall, subgenome-classification approaches tend to perform better than standard approaches with EAGLE-RC having the highest precision.

A Secreted Peptide and Its Receptors Shape the Auxin Response Pattern and Leaf Margin Morphogenesis.

Secreted peptides mediate intercellular communication [1, 2]. Several secreted peptides in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family regulate morphogenesis of tissues, such as stomata and inflorescences in plants [3-15]. The biological functions of other EPFL family members remain unknown. Here, we show that the EPFL2 gene is required for growth of leaf teeth. EPFL2 peptide physically interacts with ERECTA (ER) family receptor-kinases and, accordingly, the attenuation of ER family activities leads to formation of toothless leaves. During the tooth growth process, responses to the phytohormone auxin are maintained at tips of the teeth to promote their growth [16-19]. In the growing tooth tip of epfl2 and multiple er family mutants, the auxin response becomes broader. Conversely, overexpression of EPFL2 diminishes the auxin response, indicating that the EPFL2 signal restricts the auxin response to the tooth tip. Interestingly, the tip-specific auxin response in turn organizes characteristic expression patterns of ER family and EPFL2 by enhancing ER family expression at the tip while eliminating the EPFL2 expression from the tip. Our findings identify the novel ligand-receptor pairs promoting the tooth growth, and further reveal a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis.