Showing papers in "Plant Biotechnology Journal in 2022"

Plant Public RNA‐seq Database: a comprehensive online database for expression analysis of ~45 000 plant public RNA‐Seq libraries

Abstract: Harbin Institute of Technology, Harbin, China Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China Institute of Plant and Food Science, Southern University of Science and Technology, Shenzhen, China Key Laboratory of Molecular Design for Plant Cell, Factory of Guangdong Higher Education Institutes, Southern University of Science and Technology, Shenzhen,

PhieABEs: a PAM‐less/free high‐efficiency adenine base editor toolbox with wide target scope in plants

Abstract: Summary Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site‐specific A‐to‐G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer‐adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high‐efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single‐stranded DNA‐binding domain (DBD), based on PAM‐less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n‐NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base‐editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e‐DBD‐SpRYn (hyABE8e‐SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)‐dependent off‐target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base‐editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

OsCOMT, encoding a caffeic acid O‐methyltransferase in melatonin biosynthesis, increases rice grain yield through dual regulation of leaf senescence and vascular development

Abstract: Summary Melatonin, a natural phytohormone in plants, plays multiple critical roles in plant growth and stress responses. Although melatonin biosynthesis‐related genes have been suggested to possess diverse biological functions, their roles and functional mechanisms in regulating rice grain yield remain largely unexplored. Here, we uncovered the roles of a caffeic acid O‐methyltransferase (OsCOMT) gene in mediating rice grain yield through dual regulation of leaf senescence and vascular development. In vitro and in vivo evidence revealed that OsCOMT is involved in melatonin biosynthesis. Transgenic assays suggested that OsCOMT significantly delays leaf senescence at the grain filling stage by inhibiting degradation of chlorophyll and chloroplast, which, in turn, improves photosynthesis efficiency. In addition, the number and size of vascular bundles in the culms and leaves were significantly increased in the OsCOMT‐overexpressing plants, while decreased in the knockout plants, suggesting that OsCOMT plays a positive role in vascular development of rice. Further evidence indicated that OsCOMT‐mediated vascular development might owe to the crosstalk between melatonin and cytokinin. More importantly, we found that OsCOMT is a positive regulator of grain yield, and overexpression of OsCOMT increase grain yield per plant even in a high‐yield variety background, suggesting that OsCOMT can be used as an important target for enhancing rice yield. Our findings shed novel insights into melatonin‐mediated leaf senescence and vascular development and provide a possible strategy for genetic improvement of rice grain yield.

Natural allelic variation of GmST05 controlling seed size and quality in soybean

Abstract: Summary Seed size is one of the most important agronomic traits determining the yield of crops. Cloning the key genes controlling seed size and pyramiding their elite alleles will facilitate yield improvement. To date, few genes controlling seed size have been identified in soybean, a major crop that provides half of the plant oil and one quarter of the plant protein globally. Here, through a genome‐wide association study of over 1800 soybean accessions, we determined that natural allelic variation at GmST05 (Seed Thickness 05) predominantly controlled seed thickness and size in soybean germplasm. Further analyses suggested that the two major haplotypes of GmST05 differed significantly at the transcriptional level. Transgenic experiments demonstrated that GmST05 positively regulated seed size and influenced oil and protein contents, possibly by regulating the transcription of GmSWEET10a. Population genetic diversity analysis suggested that allelic variations of GmST05 were selected during geographical differentiation but have not been fixed. In summary, natural variation in GmST05 determines transcription levels and influences seed size and quality in soybean, making it an important gene resource for soybean molecular breeding.

Leaf angle: a target of genetic improvement in cereal crops tailored for high‐density planting

Abstract: Summary High‐density planting is an effective measure for increasing crop yield per unit land area. Leaf angle (LA) is a key trait of plant architecture and a target for genetic improvement of crops. Upright leaves allow better light capture in canopy under high‐density planting, thus enhancing photosynthesis efficiency, ventilation and stress resistance, and ultimately higher grain yield. Here, we summarized the latest progress on the cellular and molecular mechanisms regulating LA formation in rice and maize. We suggest several standing out questions for future studies and then propose some promising strategies to manipulate LA for breeding of cereal crops tailored for high‐density planting.

Identification of ST1 reveals a selection involving hitchhiking of seed morphology and oil content during soybean domestication

Abstract: Summary Seed morphology and quality of cultivated soybean (Glycine max) have changed dramatically during domestication from their wild relatives, but their relationship to selection is poorly understood. Here, we describe a semi‐dominant locus, ST1 (Seed Thickness 1), affecting seed thickness and encoding a UDP‐D‐glucuronate 4‐epimerase, which catalyses UDP‐galacturonic acid production and promotes pectin biosynthesis. Interestingly, this morphological change concurrently boosted seed oil content, which, along with up‐regulation of glycolysis biosynthesis modulated by ST1, enabled soybean to become a staple oil crop. Strikingly, ST1 and an inversion controlling seed coat colour formed part of a single selective sweep. Structural variation analysis of the region surrounding ST1 shows that the critical mutation in ST1 existed in earlier wild relatives of soybean and the region containing ST1 subsequently underwent an inversion, which was followed by successive selection for both traits through hitchhiking during selection for seed coat colour. Together, these results provide direct evidence that simultaneously variation for seed morphology and quality occurred earlier than variation for seed coat colour during soybean domestication. The identification of ST1 thus sheds light on a crucial phase of human empirical selection in soybeans and provides evidence that our ancestors improved soybean based on taste.

Application of developmental regulators to improve in planta or in vitro transformation in plants

Abstract: Summary Plant genetic transformation is a crucial step for applying biotechnology such as genome editing to basic and applied plant science research. Its success primarily relies on the efficiency of gene delivery into plant cells and the ability to regenerate transgenic plants. In this study, we have examined the effect of several developmental regulators (DRs), including PLETHORA (PLT5), WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), ENHANCED SHOOT REGENERATION (ESR1), WUSHEL (WUS) and a fusion of WUS and BABY‐BOOM (WUS‐P2A‐BBM), on in planta transformation through injection of Agrobacterium tumefaciens in snapdragons (Antirrhinum majus). The results showed that PLT5, WIND1 and WUS promoted in planta transformation of snapdragons. An additional test of these three DRs on tomato (Solanum lycopersicum) further demonstrated that the highest in planta transformation efficiency was observed from PLT5. PLT5 promoted calli formation and regeneration of transformed shoots at the wound positions of aerial stems, and the transgene was stably inherited to the next generation in snapdragons. Additionally, PLT5 significantly improved the shoot regeneration and transformation in two Brassica cabbage varieties (Brassica rapa) and promoted the formation of transgenic calli and somatic embryos in sweet pepper (Capsicum annum) through in vitro tissue culture. Despite some morphological alternations, viable seeds were produced from the transgenic Bok choy and snapdragons. Our results have demonstrated that manipulation of PLT5 could be an effective approach for improving in planta and in vitro transformation efficiency, and such a transformation system could be used to facilitate the application of genome editing or other plant biotechnology application in modern agriculture.

A single nucleotide deletion in the third exon of FT‐D1 increases the spikelet number and delays heading date in wheat (Triticum aestivum L.)

Abstract: Summary The spikelet number and heading date are two crucial and correlated traits for yield in wheat. Here, a quantitative trait locus (QTL) analysis was conducted in F8 recombinant inbred lines (RILs) derived from crossing two common wheats with different spikelet numbers. A total of 15 stable QTL influencing total spikelet number (TSN) and heading date (HD) were detected. Notably, FT‐D1, a well‐known flowering time gene in wheat, was located within the finely mapped interval of a major QTL on 7DS (QTsn/Hd.cau‐7D). A causal indel of one G in the third exon of FT‐D1 was significantly associated with total spikelet number and heading date. Consistently, CRISPR/Cas9 mutant lines with homozygous mutations in FT‐D1 displayed an increase in total spikelet number and heading date when compared with wild type. Moreover, one simple and robust marker developed according to the polymorphic site of FT‐D1 revealed that this one G indel had been preferentially selected to adapt to different environments. Collectively, these data provide further insights into the genetic basis of spikelet number and heading date, and the diagnostic marker of FT‐D1 will be useful for marker‐assisted pyramiding in wheat breeding.

Engineering of rice varieties with enhanced resistances to both blast and bacterial blight diseases via CRISPR/Cas9

Abstract: Rice blast and bacterial blight represent two of major diseases having devastating impact on the yield of rice in most rice-growing countries. Developments of resistant cultivars are the most economic and effective strategy to control these diseases. Here, we used CRISPR/Cas9-mediated gene editing to rapidly install mutations in three known broad-spectrum blast-resistant genes, Bsr-d1, Pi21 and ERF922, in an indica thermosensitive genic male sterile (TGMS) rice line Longke638S (LK638S). We obtained transgene-free homozygous single or triple mutants in T1 generations. While all single and triple mutants showed increased resistance to rice blast compared with wild type, the erf922 mutants displayed the strongest blast resistance similar with triple mutants. Surprisingly, we found that Pi21 or ERF922 single mutants conferred enhanced resistance to most of tested bacterial blight. Both resistances in mutants were attribute to the up-regulation of SA- and JA-pathway associated genes. Moreover, phenotypic analysis of these single mutants in paddy fields revealed that there were no trade-offs between resistances and main agricultural traits. Together, our study provides a rapid and effective way to generate rice varieties with resistance to both rice blast and bacterial blight.

Genetic modification of flavone biosynthesis in rice enhances biofilm formation of soil diazotrophic bacteria and biological nitrogen fixation

Abstract: Summary Improving biological nitrogen fixation (BNF) in cereal crops is a long‐sought objective; however, no successful modification of cereal crops showing increased BNF has been reported. Here, we described a novel approach in which rice plants were modified to increase the production of compounds that stimulated biofilm formation in soil diazotrophic bacteria, promoted bacterial colonization of plant tissues and improved BNF with increased grain yield at limiting soil nitrogen contents. We first used a chemical screening to identify plant‐produced compounds that induced biofilm formation in nitrogen‐fixing bacteria and demonstrated that apigenin and other flavones induced BNF. We then used CRISPR‐based gene editing targeting apigenin breakdown in rice, increasing plant apigenin contents and apigenin root exudation. When grown at limiting soil nitrogen conditions, modified rice plants displayed increased grain yield. Biofilm production also modified the root microbiome structure, favouring the enrichment of diazotrophic bacteria recruitment. Our results support the manipulation of the flavone biosynthetic pathway as a feasible strategy for the induction of biological nitrogen fixation in cereals and a reduction in the use of inorganic nitrogen fertilizers.

m6A‐mediated regulation of crop development and stress responses

Abstract: Summary Dynamic chemical modifications in eukaryotic messenger RNAs (mRNAs) constitute an essential layer of gene regulation, among which N6‐methyladenosine (m6A) was unveiled to be the most abundant. m6A functionally modulates important biological processes in various mammals and plants through the regulation of mRNA metabolism, mainly mRNA degradation and translation efficiency. Physiological functions of m6A methylation are diversified and affected by intricate sequence contexts and m6A machineries. A number of studies have dissected the functional roles and the underlying mechanisms of m6A modifications in regulating plant development and stress responses. Recently, it was demonstrated that the human FTO‐mediated plant m6A removal caused dramatic yield increases in rice and potato, indicating that modulation of m6A methylation could be an efficient strategy for crop improvement. In this review, we summarize the current progress concerning the m6A‐mediated regulation of crop development and stress responses, and provide an outlook on the potential application of m6A epitranscriptome in the future improvement of crops.

<i>Brassica napus</i> genes <i>Rlm4</i> and <i>Rlm7</i> , conferring resistance to <i>Leptosphaeria maculans</i> , are alleles of the <i>Rlm9</i> wall‐associated kinase‐like resistance locus

Abstract: -

Increasing floral visitation and hybrid seed production mediated by beauty mark in Gossypium hirsutum

Abstract: Summary Hybrid crop varieties have been repeatedly demonstrated to produce significantly higher yields than their parental lines; however, the low efficiency and high cost of hybrid seed production has limited the broad exploitation of heterosis for cotton production. One option for increasing the yield of hybrid seed is to improve pollination efficiency by insect pollinators. Here, we report the molecular cloning and characterization of a semidominant gene, Beauty Mark (BM), which controls purple spot formation at the base of flower petals in the cultivated tetraploid cotton species Gossypium barbadense. BM encodes an R2R3 MYB113 transcription factor, and we demonstrate that GbBM directly targets the promoter of four flavonoid biosynthesis genes to positively regulate petal spot development. Introgression of a GbBM allele into G. hirsutum by marker‐assisted selection restored petal spot formation, which significantly increased the frequency of honeybee visits in G. hirsutum. Moreover, field tests confirmed that cotton seed yield was significantly improved in a three‐line hybrid production system that incorporated the GbBM allele. Our study thus provides a basis for the potentially broad application of this gene in improving the long‐standing problem of low seed production in elite cotton hybrid lines.

A rust fungus effector directly binds plant pre‐mRNA splice site to reprogram alternative splicing and suppress host immunity

Abstract: Summary Alternative splicing (AS) is a crucial post‐transcriptional regulatory mechanism in plant resistance. However, whether and how plant pathogens target splicing in their host remains mostly unknown. For example, although infection by Puccinia striiformis f. sp. tritici (Pst), a pathogenic fungus that severely affects the yield of wheat worldwide, has been shown to significantly influence the levels of alternatively spliced transcripts in the host, the mechanisms that govern this process, and its functional consequence have not been examined. Here, we identified Pst_A23 as a new Pst arginine‐rich effector that localizes to host nuclear speckles, nuclear regions enriched in splicing factors. We demonstrated that transient expression of Pst_A23 suppresses plant basal defence dependent on the Pst_A23 nuclear speckle localization and that this protein plays an important role in virulence, stable silencing of which improves wheat stripe rust resistance. Remarkably, RNA‐Seq data revealed that AS patterns of 588 wheat genes are altered in Pst_A23‐overexpressing lines compared to control plants. To further examine the direct relationship between Pst_A23 and AS, we confirmed direct binding between two RNA motifs predicted from these altered splicing sites and Pst_A23 in vitro. The two RNA motifs we chose occur in the cis‐element of TaXa21‐H and TaWRKY53, and we validated that Pst_A23 overexpression results in decreased functional transcripts of TaXa21‐H and TaWRKY53 while silencing of TaXa21‐H and TaWRKY53 impairs wheat resistance to Pst. Overall, this represents formal evidence that plant pathogens produce ‘splicing’ effectors, which regulate host pre‐mRNA splicing by direct engagement of the splicing sites, thereby interfering with host immunity.

Transposable element insertion: a hidden major source of domesticated phenotypic variation in Brassica rapa

Abstract: Summary Transposable element (TE) is prevalent in plant genomes. However, studies on their impact on phenotypic evolution in crop plants are relatively rare, because systematically identifying TE insertions within a species has been a challenge. Here, we present a novel approach for uncovering TE insertion polymorphisms (TIPs) using pan‐genome analysis combined with population‐scale resequencing, and we adopt this pipeline to retrieve TIPs in a Brassica rapa germplasm collection. We found that 23% of genes within the reference Chiifu‐401‐42 genome harbored TIPs. TIPs tended to have large transcriptional effects, including modifying gene expression levels and altering gene structure by introducing new introns. Among 524 diverse accessions, TIPs broadly influenced genes related to traits and acted a crucial role in the domestication of B. rapa morphotypes. As examples, four specific TIP‐containing genes were found to be candidates that potentially involved in various climatic conditions, promoting the formation of diverse vegetable crops in B. rapa. Our work reveals the hitherto hidden TIPs implicated in agronomic traits and highlights their widespread utility in studies of crop domestication.

The MdBBX22–miR858–MdMYB9/11/12 module regulates proanthocyanidin biosynthesis in apple peel

Abstract: Summary Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post‐transcriptional level are relatively rare. In this study, we investigated the function of mdm‐miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm‐miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm‐miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5′‐RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm‐miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm‐miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm‐miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm‐miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm‐miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22–miR858–MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.

Plant‐based expression and characterization of SARS‐CoV‐2 virus‐like particles presenting a native spike protein

Abstract: Summary We have investigated the use of transient expression to produce virus‐like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID‐19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full‐length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic ‘crown‐shaped’ or ‘spiky’ structure could be purified by density gradient centrifugation. Enzyme‐linked immunosorbent assays using anti‐S antibodies showed that threefold higher levels of VLPs containing the full‐length S protein were obtained by infiltration with S alone, compared to co‐infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID‐19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant‐made material is noninfectious, it does not have to be handled under conditions of high containment.

The telomere‐to‐telomere gap‐free genome of four rice parents reveals SV and PAV patterns in hybrid rice breeding

Abstract: of universally presenting key heterosis patterns of genes by PAV in F1 RNA-seq. For two-line including Y58S/9311 and LK638S/WSSM, 698 and 593 functional genes are complementary in the parental genome, respectively.

The PGS1 basic helix‐loop‐helix protein regulates Fl3 to impact seed growth and grain yield in cereals

Abstract: Summary Plant transcription factors (TFs), such as basic helix‐loop‐helix (bHLH) and AT‐rich zinc‐binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5–20 days post‐anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E‐box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand‐grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait‐associated gene for improving cereal grain yield.

SlMIR164A regulates fruit ripening and quality by controlling SlNAM2 and SlNAM3 in tomato

Abstract: Summary MiRNAs are important posttranscriptional regulators of plant development. Many miRNAs, such as the conserved miR164 species, are encoded by families of MIRNA genes, but the specific roles of individual MIRNA genes are largely undefined. Here, we characterize the functions and regulatory mechanisms of SlMIR164A, one of the primary genes of Sly‐miR164, in tomato. We show that SlMIR164A is preferentially expressed at late stages of fruit development and plays a vital role in controlling fruit ripening and quality. Loss of function of SlMIR164A by CRISPR/Cas9‐mediated mutagenesis results in accelerated fruit ripening and enhanced chloroplast development, which leads to altered sugar and organic acid contents and affects the nutritional quality of fruits. We also show that SlMIR164A modulates fruit ripening and quality through specific target genes, SlNAM2 and SlNAM3, which control key regulators of chloroplast function and fruit ripening processes. MIR164 genes have been shown to play conserved roles in regulating organ ageing, such as leaf senescence and fruit ripening, in a variety of plants, but whether and how their family members in tomato exert the same function remain to be elucidated. Our results reveal a previously undiscovered role of SlMIR164A in ripening control, which will further our understanding of the actions of MIR164 family, as well as the mechanisms of fruit ripening and quality control in tomato. Moreover, as loss of SlMIR164A exhibits minor impacts on organ morphology, our results can be leveraged in tomato breeding for specific manipulation of fruit ripening and quality to facilitate tomato improvement in agriculture.

Transposon insertions regulate genome‐wide allele‐specific expression and underpin flower colour variations in apple (Malus spp.)

Abstract: Summary Allele‐specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar ‘Royal Gala’ (RG). A high quality chromosome‐level genome was assembled using a homozygous tetra‐haploid RG plant, derived from anther cultures. Using RNA‐sequencing (RNA‐seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of ‘Golden Delicious’ (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele‐specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele‐specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome‐wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

Editing melon eIF4E associates with virus resistance and male sterility

Abstract: Summary The cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single‐nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

<i>De novo</i> chromosome‐level genome of a semi‐dwarf cultivar of <i>Prunus persica</i> identifies the aquaporin <i>PpTIP2</i> as responsible for temperature‐sensitive semi‐dwarf trait and <i>PpB3‐1</i> for flower type and size

Abstract: Peach (Prunus persica) is one of the most important fruit crops globally, but its cultivation can be hindered by large tree size. 'Zhongyoutao 14' (CN14) is a temperature-sensitive semi-dwarf (TSSD) cultivar which might be useful as breeding stock. The genome of CN14 was sequenced and assembled de novo using single-molecule real-time sequencing and chromosome conformation capture assembly. A high-quality genome was assembled and annotated, with 228.82 Mb mapped to eight chromosomes. Eighty-six re-sequenced F1 individuals and 334 previously re-sequenced accessions were used to identify candidate genes controlling TSSD and flower type and size. An aquaporin tonoplast intrinsic protein (PpTIP2) was a strong candidate gene for control of TSSD. Sequence variations in the upstream regulatory region of PpTIP2 correlated with different transcriptional activity at different temperatures. PpB3-1, a candidate gene for flower type (SH) and flower size, contributed to petal development and promoted petal enlargement. The locus of another 12 agronomic traits was identified through genome-wide association study. Most of these loci exhibited consistent and precise association signals, except for flesh texture and flesh adhesion. A 6015-bp insertion in exon 3 and a 26-bp insertion upstream of PpMYB25 were associated with fruit hairless. Along with a 70.5-Kb gap at the F-M locus in CN14, another two new alleles were identified in peach accessions. Our findings will not only promote genomic research and agronomic breeding in peach but also provide a foundation for the peach pan-genome.

Integrated multi‐omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers

Abstract: Summary Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi‐omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high‐quality chromosome‐scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the ‘Scarlet Queen’. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars.

CsMYB184 regulates caffeine biosynthesis in tea plants

Abstract: As the most well-known and globally consumed central nervous system stimulant, caffeine is a purine alkaloid natural product usually derived from tea and coffee. Caffeine has a wide range of health benefits on the human body, and plays crucial roles in pollination, resistance to herbivore attacks, and pathogen infections in plants (Zhao et al., 2020). While caffeine biosynthetic pathways have been extensively studied in tea (Camellia sinensis L) and coffee plants, the regulation of caffeine biosynthesis is not understood (Zhao et al., 2020). Tea Caffeine Synthase1 (TCS1) is the first N-methyltransferase gene reported in the tea plant, possessing the 1-N methyltransferase activity responsible for converting theobromine to caffeine (Kato et al., 2000). Studies on the structure–activity of TCS1 and genetic variations in the TCS1 gene of tea plant populations have supported that TCS1 is a determination enzyme for caffeine content (Jin et al., 2016). To explore the regulation of caffeine biosynthesis, 23 candidate transcription factors (TFs) from Weighted Gene Co-expression Network Analysis were screened in a luciferase reporter gene activation system driven by the TCS1 promoter (Figure S1). MYB184 (TEA029017) showed the highest TCS1 promoter activation with 4.7-fold (Figure 1a). Yeast one-hybrid assay showed that the 828 to 1670 bp region of TCS1 promoter, which contains an MYBCORE and a fused MYB1AT-MYBPLANT, was critically required for MYB184 recognition (Figure 1b). In planta promoter, trans-activation assays further confirmed the regions required for binding to and activating of the TCS1 promoter by MYB184 to be between 1596 and 1670 bp (Figure 1c). EMSA assay was performed to further validate the binding of MYB184 to the fused MYB1AT-MYBPLANT motif in vitro (Figure 1d–f). We then examined the function of MYB184 in regulating caffeine synthesis in tea plants. An antisense oligodeoxynucleotide (asODN) interference experiment was performed with tea plant shoot tips to knock down MYB184 expression (MYB184-KD) (Figure 1g). Accordingly, caffeine contents and the expression of TCS1 were significantly reduced in MYB184-KD shoot tips compared with the senseODN control (Figure 1h–i). However, overexpression of MYB184 (MYB184OE) in tea plant transgenic hairy root lines significantly up-regulated TCS1 transcription and thereby increased the caffeine contents as compared with wildtype root controls (Figure 1j–m). KeKecha (Camellia ptilophylla, KKC in short), belonging to the Thea section, had significantly lower caffeine but higher theobromine (Figure 1n). Although a previous study showed that TCS1 in KKC had lower NMT activity compared with TCS1 in modern tea cultivars (Jin et al., 2016), we detected a significantly lower expression level of TCS1 in KKC than in other tea cultivars (Figure 1o). To understand why TCS1 in KKC is downregulated, we cloned and compared the promoter sequences of TCS1 from KKC and from SCZ. However, the alignment of the promoter sequences did not show critical Indels or SNPs on MYB binding sites (Figure 1p). On the other hand, transcriptome analyses showed that only MYB184 expression level was significantly lower by ~14-fold in KKC than in other tea cultivars (Figure 1q,r). We thus proposed that the lower MYB184 transcript level in KKC might be the cause of the reduced TCS1 expression level. We further cloned the promoter of the MYB184 gene from KKC (proMYB184), and compared it with those from other tea cultivars. A 437-bp long terminal repeat (LTR) insertion was identified only in the proMYB184 at the site of 982 bp, but not in these promoters from other tea cultivars, as verified with both MYB184 promoter cloning and detection with PCR primers specific for the LTR insertion (Figure 1s). In tea plants, LTR insertion in a gene usually leads to suppression of the gene expression (Xia et al., 2020), which may explain the lower expression level of MYB184 in KKC than in other tea cultivars. Indeed, GUS reporter assays showed that the proMYB184 with the LTR insertion exhibited clearly lower promoter activity than four representative promoters without the LTR insertion from tea cultivars (Figure 1t). We thus concluded that the LTR insertion in the promoter of MYB184 resulted in suppressed MYB184 expression, leading to the lower TCS1 transcript level and thereby lower caffeine contents in KKC. To expand MYB184 activation of TCS1 to other Camellia species, we examined several other wild tea relatives that are known to contain significantly lower levels of caffeine compared with modern tea cultivars (Figure 1u). They also have lower TCS1 and MYB184 expression levels compared with modern tea cultivars containing higher levels of caffeine (Figures 1v,w and S2). This further supports the indispensable role of MYB184 in activation of TCS1 gene expression and caffeine biosynthesis in C. sinensis. In summary, we characterized MYB184 as the major activator of TCS1 and caffeine production in tea plants. An LTR insertion in the MYB184 promoter in wild tea C. ptilophylla explained its low TCS1 expression level and caffeine content. Our study may offer a

Genetic variation in CaTIFY4b contributes to drought adaptation in chickpea

Abstract: Summary Chickpea production is vulnerable to drought stress. Identifying the genetic components underlying drought adaptation is crucial for enhancing chickpea productivity. Here, we present the fine mapping and characterization of ‘QTL‐hotspot’, a genomic region controlling chickpea growth with positive consequences on crop production under drought. We report that a non‐synonymous substitution in the transcription factor CaTIFY4b regulates seed weight and organ size in chickpea. Ectopic expression of CaTIFY4b in Medicago truncatula enhances root growth under water deficit. Our results suggest that allelic variation in ‘QTL‐hotspot’ improves pre‐anthesis water use, transpiration efficiency, root architecture and canopy development, enabling high‐yield performance under terminal drought conditions. Gene expression analysis indicated that CaTIFY4b may regulate organ size under water deficit by modulating the expression of GRF‐INTERACTING FACTOR1 (GIF1), a transcriptional co‐activator of Growth‐Regulating Factors. Taken together, our study offers new insights into the role of CaTIFY4b and on diverse physiological and molecular mechanisms underpinning chickpea growth and production under specific drought scenarios.

Genome sequencing reveals chromosome fusion and extensive expansion of genes related to secondary metabolism in Artemisia argyi

Abstract: Summary Artemisia argyi, as famous as Artemisia annua, is a medicinal plant with huge economic value in the genus of Artemisia and has been widely used in the world for about 3000 years. However, a lack of the reference genome severely hinders the understanding of genetic basis for the active ingredient synthesis of A. argyi. Here, we firstly report a complex chromosome‐level genome assembly of A. argyi with a large size of 8.03 Gb, with features of high heterozygosity (2.36%), high repetitive sequences (73.59%) and a huge number of protein‐coding genes (279 294 in total). The assembly reveals at least three rounds of whole‐genome duplication (WGD) events, including a recent WGD event in the A. argyi genome, and a recent burst of transposable element, which may contribute to its large genome size. The genomic data and karyotype analyses confirmed that A. argyi is an allotetraploid with 34 chromosomes. Intragenome synteny analysis revealed that chromosomes fusion event occurred in the A. argyi genome, which elucidates the changes in basic chromosome numbers in Artemisia genus. Significant expansion of genes related to photosynthesis, DNA replication, stress responses and secondary metabolism were identified in A. argyi, explaining the extensive environmental adaptability and rapid growth characteristics. In addition, we analysed genes involved in the biosynthesis pathways of flavonoids and terpenoids, and found that extensive gene amplification and tandem duplication contributed to the high contents of metabolites in A. argyi. Overall, the reference genome assembly provides scientific support for evolutionary biology, functional genomics and breeding in A. argyi and other Artemisia species.

Chromosome‐level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates

Abstract: Summary Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field‐based model species for genetics and epigenetics. The availability of a high‐quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome‐level genome assembly of var. MN106‐Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32‐10) that are highly amenable to genetic transformation. We describe non‐coding RNAs, pseudogenes and transposable elements, and highlight tissue‐specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome‐wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Profiling plant histone modification at single‐cell resolution using snCUT&amp;Tag

Abstract: Chromatin immunoprecipitation with sequencing (ChIP-seq) with population cells or tissues yields ensemble epigenomic profiles that only represent the population average, which eliminates cell-to-cell epigenetic heterogeneity. Chromatin immunocleavage with sequencing (ChIC-seq) (Ku et al., 2019) that based on antibody-guided chromatin cleavage under targets is a practical alternative to ChIP-seq. Many single-cell ChIC-based methods, such as scChIC-seq (Ku et al., 2019), CUT&Tag (Kaya-Okur et al., 2019), CoBATCH (Wang et al., 2019), ACT-seq (Carter et al., 2019), and 10× scCUT&Tag (Bartosovic et al., 2021; Wu et al., 2021), have been developed and applied to study cell-type profiling, developmental trajectory, epigenetic heterogeneity, and transcriptional regulation in animals. However, due to the existence of cell walls, it is difficult to obtain single cells in plants. On the other hand, the existing scChIC methods require many single-cell barcoding procedures after tagmentation, which might lead to some DNA leakage and reduced mapping efficiency. Hence, a robust single-cell ChIC-seq method for plant epigenomic research is required emergently. In our previous study, we developed a rapid ChIC-based chromatin profiling protocol, nucleus CUT&Tag (nCUT&Tag) (Ouyang et al., 2021). Here, we combined the nCUT&Tag assay with 10× Single Cell ATAC, developing an easy-to-use single-nucleus CUT&Tag (snCUT&Tag) method in rice. We reformulated the bulk-cell nCUT&Tag with the droplet-based single-cell barcoding technology to develop snCUT&Tag (Figure 1a). Briefly, ~106 of fixed rice-seedling nuclei were collected and immuno-cleaved with protein G-Tn5 (Vazyme, Nanjing, China, cat no. TD901). Following Tn5-assisted in situ tagmentation, ~20 000 Tn5-indexed nuclei were loaded onto the 10× Genomics Single Cell ATAC microfluidics device, in which each nucleus is partitioned in a gel-bead-in-emulsion droplet and barcoded with unique oligonucleotides (Figure 1a). Finally, the chromatin features of every single nucleus could be captured by pooling and sequencing the barcoded DNA fragments. We first profiled the H3K4me3 histone mark in seedlings from the Xian group rice (Minghui 63) using snCUT&Tag. We generated over 480 million read pairs, of which 98.7% were indexed with a unique barcode (Figure 1c). After mapping the reads to Minghui 63 reference genome and removal of duplicates, we obtained the H3K4me3 profiles of 3679 single nuclei, with a median of 401 fragments per nucleus (Figure 1c). For the 3679 aggregate single nuclei, we called 28 795 peaks (Figure 1b,c). Next, we compared the aggregate snCUT&Tag data with bulk-cell nCUT&Tag (Ouyang et al., 2021) and eChIP-seq data (Zhao et al., 2020). The aggregate snCUT&Tag peaks exhibited a high degree of consistency with nCUT&Tag and eChIP-seq data (Figure 1b). Coinciding with the high consistency, global scatterplots displayed significant correlations among the snCUT&Tag, nCUT&Tag, and eChIP-seq libraries (Figure 1d). As demonstrated by few reads falling in non-peak regions (Figure 1b), the snCUT&Tag data exhibited a significantly high fraction of reads in peaks (FRiP) value (median FRiP = 0.923) (Figure 1c,e). snCUT&Tag displayed similar peak signal profiles to nCUT&Tag and eChIP-seq, with H3K4me3 peaks mainly enriching at transcription start sites (Figure 1f). These results indicated that snCUT&Tag is a robust method that can be used to profile single-nucleus chromatin features. Since the epigenomic data can be used to predict chromatin interactions (Fulco et al., 2019; Ouyang et al., 2020), we examined the H3K4me3 snCUT&Tag data and predicted cell-type specific enhancer–promoter interactions according to the activity-by-contact model (Bartosovic et al., 2021; Fulco et al., 2019). Firstly, we performed dimensionality reduction and clustering according to Bartosovic et al. (2021). The results showed that rice seedlings were partitioned into 17 cell clusters, with significantly high enrichment-signal specificity (Figure 1g). Furthermore, we annotated cell types for the 17 clusters by investigating the marker peak-associated functional genes. For instance, a marker peak in cluster 6 showed specific H3K4me3 enrichment around a malate dehydrogenase-coding gene, which was reported as a marker gene of mesophyll cells in maize (Marand et al., 2021), implying that cluster 6 should be classified as mesophyll cells. Then, we predicted enhancer–promoter interactions. Our results demonstrated that the predicted chromatin interactions were well-overlapped with H3K4me3 ChIA-PET data, and many enhancer-centred chromatin interactions showed cell-type biases that only occurred in specific cell clusters (Figure 1h). Expectedly, ~51.5% (15 881 of 30 839) of predicted enhancers overlapped with ATAC-seq peaks (Figure 1i), showing significant enrichment of ATAC-seq signals around the centre of the predicted enhancers (Figure 1j,k). Our results indicate that snCUT&Tag can be applied to dissect epigenetic heterogeneity and predict cell type-specific enhancer–promoter interactions, which might facilitate the annotation of regulatory elements and reconstruction of 3D genome structures. In conclusion, we developed a simple and practical single-nucleus ChIC-seq method (snCUT&Tag) for single-cell plant epigenomic studies. We demonstrated that snCUT&Tag could be used to dissect the cell lineage and epigenetic heterogeneity and reconstruct chromatin topology of each single nucleus. Since nCUT&Tag is applicable for studying bulk-cell chromatin features with nuclei isolated from various plant species and tissues (Ouyang et al., 2021), we believe that snCUT&Tag may have a broad spectrum of application in profiling single-nucleus histone marks for other types of plant materials as well. Expectedly, snCUT&Tag provides an avenue for plant single-cell epigenomic studies and may help understand the 3D genomic and epigenetic basis of transcription regulation in a single cell. This work was supported by the National Natural Science Foundation of China (32070612, 31771422), the funds from the Interdisciplinary Sciences Research Institute (2662021JC005), and the National Key Laboratory of Crop Genetic Improvement (ZK201906), as well as the Baichuan Project (to W.O.) from College of Life Science and Technology, Huazhong Agricultural University. No conflict of interest declared. X.L. and G.L. supervised the research. W.O. performed the experiments with assistance from X.X., M.G., and Y.Z. S.L. and W.O. performed bioinformatic analysis. W.O. and X.L. wrote the manuscript. The snCUT&Tag data have been deposited in the National Genomics Data Center (https://ngdc.cncb.ac.cn/) with accession number CRA004386.

Natural variation of HTH5 from wild rice, Oryza rufipogon Griff., is involved in conferring high‐temperature tolerance at the heading stage

Abstract: Summary Global warming is a major abiotic stress factor, which limit rice production. Exploiting the genetic basis of the natural variation in heat resistance at different reproductive stages among diverse exotic Oryza germplasms can help breeding heat‐resistant rice cultivars. Here, we identified a stable quantitative trait locus (QTL) for heat tolerance at the heading stage on chromosome 5 (qHTH5) in O. rufipogon Griff. The corresponding gene, HTH5, pertains to the pyridoxal phosphate‐binding protein PLPBP (formerly called PROSC) family, which is predicted to encode pyridoxal phosphate homeostasis protein (PLPHP) localized to the mitochondrion. Overexpression of HTH5 increased the seed‐setting rate of rice plants under heat stress at the heading stage, whereas suppression of HTH5 resulted in greater susceptibility to heat stress. Further investigation indicated that HTH5 reduces reactive oxygen species accumulation at high temperatures by increasing the heat‐induced pyridoxal 5'‐phosphate (PLP) content. Moreover, we found that two SNPs located in the HTH5 promoter region are involved with its expression level and associated with heat tolerance diversity. These findings suggest that the novel gene HTH5 might have great potential value for heightening rice tolerance to heat stress to the on‐going threat of global warming.