Showing papers in "Plant Journal in 2022"

ROS production and signalling in chloroplasts: cornerstones and evolving concepts

Abstract: SUMMARY Reactive oxygen species (ROS) such as singlet oxygen, superoxide (O2 ●−) and hydrogen peroxide (H2O2) are the markers of living cells. Oxygenic photosynthesis produces ROS in abundance, which act as a readout of a functional electron transport system and metabolism. The concept that photosynthetic ROS production is a major driving force in chloroplast to nucleus retrograde signalling is embedded in the literature, as is the role of chloroplasts as environmental sensors. The different complexes and components of the photosynthetic electron transport chain (PETC) regulate O2 ●− production in relation to light energy availability and the redox state of the stromal Cys‐based redox systems. All of the ROS generated in chloroplasts have the potential to act as signals and there are many sulphhydryl‐containing proteins and peptides in chloroplasts that have the potential to act as H2O2 sensors and function in signal transduction. While ROS may directly move out of the chloroplasts to other cellular compartments, ROS signalling pathways can only be triggered if appropriate ROS‐sensing proteins are present at or near the site of ROS production. Chloroplast antioxidant systems serve either to propagate these signals or to remove excess ROS that cannot effectively be harnessed in signalling. The key challenge is to understand how regulated ROS delivery from the PETC to the Cys‐based redox machinery is organised to transmit redox signals from the environment to the nucleus. Redox changes associated with stromal carbohydrate metabolism also play a key role in chloroplast signalling pathways.

Bulk segregation analysis in the <scp>NGS</scp> era: a review of its teenage years

Abstract: Bulk segregation analysis (BSA) utilizes a strategy of pooling individuals with extreme phenotypes to conduct economical and rapidly linked marker screening or quantitative trait locus (QTL) mapping. With the development of next-generation sequencing (NGS) technology in the past 10 years, BSA methods and technical systems have been gradually developed and improved. At the same time, the ever-decreasing costs of sequencing accelerate NGS-based BSA application in different species, including eukaryotic yeast, grain crops, economic crops, horticultural crops, trees, aquatic animals, and insects. This paper provides a landscape of BSA methods and reviews the BSA development process in the past decade, including the sequencing method for BSA, different populations, different mapping algorithms, associated region threshold determination, and factors affecting BSA mapping. Finally, we summarize related strategies in QTL fine mapping combining BSA.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement

Abstract: SUMMARY Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole‐genome assemblies of Aegilops longissima and Aegilops speltoides. Whole‐genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome‐wide analysis of nucleotide‐binding leucine‐rich repeat (NLR) genes revealed species‐specific and lineage‐specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding.

Abstract: The assembly and scaffolding of plant crop genomes facilitate the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of Tobacco Mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We show specific examples of how HiFi and ONT datasets can complement one another to improve assembly contiguity. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all twelve chromosomes were represented as twelve contiguous sequences (N50=68.5 Mbp). This chromosome scale assembly did not require scaffolding using an orthogonal data type. The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato genome assemblies that made use of genetic maps and HiC for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1 Mbp region of the S. peruvianum genome during tomato breeding. Through marker studies and ONT-based comprehensive haplotyping we show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near complete genome sequences.

A Transcriptional Landscape Underlying Sugar Import for Grain Set in Maize.

Abstract: Developing seed depends on sugar supply for its growth and yield formation. Maize produces the largest grains among cereals. However, there is a lack of holistic understanding on the transcriptional landscape of genes controlling sucrose transport to, and utilization within, maize grains. By performing in-depth data-mining of spatial-temporal transcriptomes coupled with histological and heterologous functional analyses, we identified transporter genes specifically expressed in the maternal-filial interface, including (i) ZmSWEET11/13b in the placento-chalazal where sucrose was exported into the apoplasmic space, and (ii) ZmSTP3, ZmSWEET3a/4c (monosaccharide transporters), ZmSUT1, ZmSWEET11/13a (sucrose transporters) in the basal endosperm transfer cells for retrieval of apoplasmic sucrose or hexoses after hydrolysis by extracellular invertase. In embryo and its surrounding regions, an embryo-localized ZmSUT4 and a cohort of ZmSWEETs were specifically expressed. Interestingly, drought repressed those ZmSWEETs likely exporting sucrose but enhanced the expression of most transporter genes for uptake of apoplasmic sugars. Importantly, this drought-induced fluctuation in gene expression was largely attenuated by an increased C supply via controlled pollination, indicating that the altered gene expression is conditioned by C availability. Based on the analyses above, we proposed a holistic model on the spatial-temporal expression of genes that likely govern sugar transport and utilization across maize maternal and endosperm and embryo tissues during the critical stage of grain set. Collectively, the findings represent an advancement towards a holistic understanding of the transcriptional landscape underlying post-phloem sugar transport in maize grain and indicate that the drought-induced changes in gene expression is attributable to low C status.

The miR164a-NAM3 module confers cold tolerance by inducing ethylene production in tomato.

Abstract: Due to the high sensitivity to cold, the yield and quality of tomato (Solanum lycopersicum L.) are severely restricted by cold stress. The NAC transcription factor (TF) family has been characterized as an important player in plant growth, development, and stress response, but the role of NAC TFs in cold stress and their interaction with other post-transcriptional regulators such as microRNAs in cold tolerance remain elusive. Here, we demonstrated that SlNAM3, the predicted target of Sl-miR164a/b-5p, improved cold tolerance as evidenced by a higher maximum quantum efficiency of photosystem II (Fv/Fm), lower relative electrolyte leakage (REL) and less wilting in SlNAM3-overexpression plants than wild-type. Further genetic and molecular confirmation revealed that Sl-miR164a/b-5p functioned upstream of SlNAM3 by inhibiting the expression of the latter, thus playing a negative role in cold tolerance. Interestingly, this role is partially mediated by an ethylene-dependent pathway, as either Sl-miR164a/b-5p silencing or SlNAM3 overexpression improved cold tolerance in the transgenic lines by promoting ethylene production. Moreover, silencing the ethylene synthesis genes, SlACS1A, SlACS1B, SlACO1, and SlACO4, resulted in a significant decrease in cold tolerance. Further experiments demonstrated that NAM3 activates SlACS1A, SlACS1B, SlACO1, and SlACO4 transcription by directly binding to their promoters. Taken together, our study identified the miR164a-NAM3 module conferring cold tolerance in tomato plants via the direct regulation of SlACS1A, SlACS1B, SlACO1, and SlACO4 expression to induce ethylene synthesis.

Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis and shoot development and stress response in tea plants (Camellia sinensis).

Abstract: Tea is concocted from tea plant shoot tips that produce catechins, caffeine, theanine and terpenoids, which collectively determine the rich flavors and health benefits of the infusion. However, little is known about the integrated regulation of shoot tip development and characteristic secondary metabolite biosynthesis in tea plants. Here, we demonstrate that MYB transcription factors (TFs) play key and yet diverse roles in regulating the leaf and stem development, secondary metabolite biosynthesis, and environmental stress responses in tea plants. By integrating transcriptomic and metabolic profiling data in different tissues at a series of developmental stages or under various stress conditions, alongside biochemical and genetic analyses, we predicted the MYB TFs involved in regulating shoot development (CsMYB2, 98, 107 and 221), epidermal cell initiation (CsMYB184, 41, 139 and 219), stomatal initiation (CsMYB113, 153) and the biosynthesis of flavonoids (including catechins, anthocyanins and flavonols by CsMYB8 and 99), caffeine (CsMYB85 and 86), and theanine (CsMYB9 and 49), carotenoids (CsMYB110), mono-/sesqui-terpenoid volatiles (CsMYB68, 147, 148 and 193), lignin (CsMYB164 and 192), indolic compounds (CsMYB139, 162 and 198), as well as a likely involvement in hormone signaling-mediated environmental stresses and defense responses. We characterized the functions of some key MYBs in regulating flavonoid and carotenoid biosynthesis for tea quality and flavors. This study provides a cross family analysis of MYBs in tea alongside new insights into the coordinated regulation of tea plant shoot development and secondary metabolism, paving the way towards understanding of tea quality trait formation and genetic improvement of quality tea plants.

Fine mapping and cloning of the major seed protein quantitative trait loci on soybean chromosome 20

Abstract: Significance Statement Soybean is the most important source of protein meal worldwide and the quantitative trait loci (QTL) cqSeed protein‐003 on chromosome 20 exerts the greatest additive effect of any protein QTL mapped in the crop. Through genetic mapping and candidate gene downregulation, we identified that an insertion/deletion variant in Glyma.20G85100 is the likely gene that underlies this important QTL.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

Abstract: Section Oleifera (Theaceae) has attracted attention owing to the high levels of unsaturated fatty acids in seeds. Here, we reported the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. C. lanceoleosa underwent a whole-genome duplication event ~ 65 million years ago (Mya), prior to the C. lanceoleosa - Camellia sinensis divergence (~6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase, and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with the caffeine accumulation and distribution. Gene expression profiles, structural composition, and chromosomal location suggested that late-acting self-incompatibility of C. lanceoleosa likely favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

Role of lncRNAs in cis- and trans-regulatory responses to salt in Populus trichocarpa.

Abstract: Long non-coding RNAs (lncRNAs) are emerging as versatile regulators in diverse biological processes. However, little is known about their cis- and trans-regulatory contributions in gene expression under salt stress. Using 27 RNA-seq datasets from Populus trichocarpa leaves, stems, and roots, we identified 2,988 high-confidence lncRNAs including 1,183 salt-induced differentially expressed ones. Among them, 301 lncRNAs have a potential in positively affecting their neighboring genes predominantly by cis-regulatory manner rather than by co-transcription. Additionally, co-expression network identified six striking salt-associated modules with a total of 5,639 genes including 426 lncRNAs, and in these lncRNA sequences, DNA/RNA binding motifs are enriched. This suggests the lncRNAs might contribute to distant gene expression of the salt-associated modules by trans-regulatory manner. Moreover, we found 30 lncRNAs that have a potential in simultaneously cis- and trans-regulating salt-responsive homologous genes and Ptlinc-NAC72, especially induced under long-term salt stress, was selected for validating its regulation on homologs PtNAC72.A and PtNAC72.B (PtNAC72.A/B) expression and functional role. Transient transformation of Ptlinc-NAC72 and dual-luciferase assay of Ptlinc-NAC72 and PtNAC72.A/B promoters confirmed that Ptlinc-NAC72 can directly upregulate PtNAC72.A/B expression, and a presence/absence assay was further conducted to show that the regulation is probably mediated by Ptlinc-NAC72 recognizing the tandem elements (GAAAAA) in PtNAC72.A/B 5' UTR. Finally, overexpression of Ptlinc-NAC72 exhibits hypersensitive phenotype under salt stress. Altogether, our results shed light on lncRNAs cis- and trans-regulating gene expression in Populus and provide an example of long-term salt-induced Ptlinc-NAC72 that could be used to mitigate growth costs with plant resilience to salt stress.

Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33.

Abstract: Anthocyanin is one of the most important pigments and nutrients in fruits. Genes encoding R2R3-MYB TFs are key to anthocyanin regulation. R2R3-MYB activators have been widely studied in the anthocyanin synthesis pathway. However, the mechanism by which R2R3-MYB repressors negatively regulate anthocyanin synthesis remains poorly understood. In the present study, we characterized a subfamily 1 R2R3-MYB anthocyanin repressor gene, MYB306-like, from apple (Malus × domestica) and demonstrated its significance in anthocyanin regulation. The MdMYB306-like protein activates expression of an anthocyanin repressor gene, MdMYB17, and inhibits MdDFR (an anthocyanin structural gene) expression by directly binding to the corresponding promoters. The MdMYB306-like protein interacts with MdMYB17 and MdbHLH33 through its N-terminus. In addition, MdMYB306-like interacts with MdbHLH33 and MdMYB17 to enhance its regulatory activities on MdMYB17 and MdDFR, respectively. Our results revealed that the MdMYB306-like protein, in conjunction with MdMYB17 and MdbHLH33, forms a key regulatory module to fine-tune anthocyanin synthesis in apple.

Identification of novel regulators required for early development of vein pattern in the cotyledons by single‐cell RNA‐sequencing

Abstract: SUMMARY The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3‐day‐old cotyledons using single‐cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3‐day‐old cotyledons based on single‐cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

Insights into acylation mechanisms: co‐expression of serine carboxypeptidase‐like acyltransferases and their non‐catalytic companion paralogs

Abstract: SUMMARY Serine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Cavity surface residues of PAD4 and SAG101 contribute to EDS1 dimer signaling specificity in plant immunity.

Abstract: Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins EDS1, PAD4 and SAG101 and two sub-families of HET-S/LOB-B (HeLo)-domain "helper" NLRs, ADR1s and NRG1s. EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4 mediated pathogen resistance, but are dispensible for PAD4 mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper NLRs ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.

Mutations that alter Arabidopsis flavonoid metabolism affect the circadian clock

Abstract: SUMMARY Flavonoids are a well‐known class of specialized metabolites that play key roles in plant development, reproduction, and survival. Flavonoids are also of considerable interest from the perspective of human health, as both phytonutrients and pharmaceuticals. RNA sequencing analysis of an Arabidopsis null allele for chalcone synthase (CHS), which catalyzes the first step in flavonoid metabolism, has uncovered evidence that these compounds influence the expression of genes associated with the plant circadian clock. Analysis of promoter‐luciferase constructs further showed that the transcriptional activity of CCA1 and TOC1, two key clock genes, is altered in CHS‐deficient seedlings across the day/night cycle. Similar findings for a mutant line lacking flavonoid 3′‐hydroxylase (F3′H) activity, and thus able to synthesize mono‐ but not dihydroxylated B‐ring flavonoids, suggests that the latter are at least partially responsible; this was further supported by the ability of quercetin to enhance CCA1 promoter activity in wild‐type and CHS‐deficient seedlings. The effects of flavonoids on circadian function were also reflected in photosynthetic activity, with chlorophyll cycling abolished in CHS‐ and F3′H‐deficient plants. Remarkably, the same phenotype was exhibited by plants with artificially high flavonoid levels, indicating that neither the antioxidant potential nor the light‐screening properties of flavonoids contribute to optimal clock function, as has recently also been demonstrated in animal systems. Collectively, the current experiments point to a previously unknown connection between flavonoids and circadian cycling in plants and open the way to better understanding of the molecular basis of flavonoid action.

Direct regulation of shikimate, early phenylpropanoid and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine.

Abstract: The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in grape. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks located within -10kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 out of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates amongst which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterisation using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialised metabolism.

Root system architecture in cereals: progress, challenges and perspective

Abstract: Roots are essential multifunctional plant organs involved in water and nutrient uptake, metabolite storage, anchorage, mechanical support, and interaction with the soil environment. Understanding of this 'hidden half' provides potential for manipulation of root system architecture (RSA) traits to optimize resource use efficiency and grain yield in cereal crops. Unfortunately, root traits are highly neglected in breeding due to the challenges of phenotyping, but could have large rewards if the variability in RSA traits can be fully exploited. Until now, a plethora of genes have been characterized in detail for their potential role in improving RSA. The use of forward genetics approaches to find sequence variations in genes underpinning desirable RSA would be highly beneficial. Advances in computer vision applications have allowed image-based approaches for high-throughput phenotyping of RSA traits that can be used by any laboratory worldwide to make progress in understanding root function and dissection of the genetics. At the same time, the frontiers of root measurement include non-invasive methods like X-ray computer tomography and magnetic resonance imaging that facilitate new types of temporal studies. Root physiology and ecology are further supported by spatiotemporal root simulation modeling. The discovery of component traits providing improved resilience and yield advantage in target environments is a key necessity for mainstreaming root-based cereal breeding. The integrated use of pan-genome resources, now available in most cereals, coupled with new in-field phenotyping platforms has the potential for precise selection of superior genotypes with improved RSA.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

Abstract: The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural resistance-associated macrophage proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 to be involved in Mn utilization. Mutation of NRAMP6 in nramp1 but not wild-type (WT) background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including ER, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of yeast mutant Δsmf2 that is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening.

Abstract: Fruit ripening is a complex developmental process which is modulated by both transcriptional and post-translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production and decline in firmness. DAP-Seq combined with RNA-Seq analyses showed that MaMYB4 preferentially binds to the promoters of various ripening-associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by down-regulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, here we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post-translational levels.

Comparative analyses of American and Asian lotus genomes reveal insights into petal color, carpel thermogenesis and domestication

Abstract: SUMMARY Nelumbo lutea (American lotus), which differs from Nelumbo nucifera (Asian lotus) morphologically, is one of the two remaining species in the basal eudicot family Nelumbonaceae. Here, we assembled the 843‐Mb genome of American lotus into eight pseudochromosomes containing 31 382 protein‐coding genes. Comparative analyses revealed conserved synteny without large chromosomal rearrangements between the genomes of American and Asian lotus and identified 29 533 structural variants (SVs). Carotenoid and anthocyanin pigments determine the yellow and red petal colors of American and Asian lotus, respectively. The structural genes encoding enzymes of the carotenoid and anthocyanin biosynthesis pathways were conserved between two species but differed in expression. We detected SVs caused by repetitive sequence expansion or contraction among the anthocyanin biosynthesis regulatory MYB genes. Further transient overexpression of candidate NnMYB5 induced anthocyanin accumulation in lotus petals. Alternative oxidase (AOX), uncoupling proteins (UCPs), and sugar metabolism and transportation contributed to carpel thermogenesis. Carpels produce heat with sugars transported from leaves as the main substrates, because there was weak tonoplast sugar transporter (TST) activity, and with SWEETs were highly expressed during thermogenesis. Cell proliferation‐related activities were particularly enhanced in the warmer carpels compared with stamens during the cold night before blooming, which suggested that thermogenesis plays an important role in flower protogyny. Population genomic analyses revealed deep divergence between American and Asian lotus, and independent domestication affecting seed, rhizome, and flower traits. Our findings provide a high‐quality reference genome of American lotus for exploring the genetic divergence and variation between two species and revealed possible genomic bases for petal color, carpel thermogenesis and domestication in lotus.

Haplotype-resolved genome assembly of Bletilla striata (Thunb.) Reichb.f. to elucidate medicinal values.

Abstract: Bletilla striata, commonly known as baiji, is one of the traditional Chinese medicine species; it is highly regarded for its medicinal applications and therefore has high economic values. Here, we report a high-quality haplotype-resolved genome of B. striata, Haplotype-A (2.37 Gb with a scaffold N50 of 146.39 Mb and Contig N50 of 1.65 Mb) and Haplotype-B (2.43 Gb with a scaffold N50 of 150.22 Mb and Contig N50 of 1.66 Mb), assembled from HIFI reads and chromosome conformation capture (Hi-C) reads. We find evidence that B. striata has undergone two whole-genome duplication (WGD) events: an ancient WGD event shared by most monocots and a recent WGD event unique to all orchids. We also reconstructed for the first time the ancestral orchids karyotype (AOK) of 18 ancient chromosomes and the evolutionary trajectories of 16 modern B. striata chromosomes. Comparative genomic analysis suggested that the expanded gene families of B. striata might play important roles in secondary metabolite biosynthesis and environmental adaptation. By combining genomic and transcriptome, we first identified the core ten members from nine gene families that were probably involved in the B. striata polysaccharide (BSP) biosynthesis. Based on the virus-induced gene silencing (VIGS) and yeast two-hybrid experiments, we first exhibit a MYB transcription factor (TF), BsMYB2, which can regulate BSPs biosynthesis by directly interacting with eight key BSPs-related genes, sacA1, HK1, scrK1, scrK2, GPI1, manA1, GMPP1, and UGP2_1. Our study will enhance the understanding of orchid evolution and accelerate molecular-assisted breeding of B. striata for improving medicinal-value traits.

Advances and Applications of Single-cell Omics Technologies in Plant Research.

Abstract: Single-cell sequencing approaches ascertain the intracellular dynamics of individual cells and answer biological questions with high-dimensional catalogs of millions of cells, including genomics, transcriptomics, chromatin accessibility, and epigenomics data across species. These emerging yet thriving technologies have been fully embraced by the field of plant biology, with a constantly expanding portfolio of applications. Here, we introduce the current technical advances used for single-cell omics, especially single-cell genome and transcriptome sequencing. Firstly, we overview methods for protoplast and nucleus isolation and genome and transcriptome amplification. Subsequently, we use well-executed benchmarking studies to highlight advances made through the application of single-cell omics techniques. Looking forward, we offer a glimpse of additional hurdles and future opportunities that will introduce broad adoption of single-cell sequencing with revolutionized perspectives in plant biology.

Unsupervised and semi-supervised learning: the next frontier in machine learning for plant systems biology.

Abstract: The advancement of high-throughput omics technologies is leading plant biology research into the era of Big Data. Machine learning (ML) performs an important role in plant systems biology owing to its excellent performance and wide application in Big Data analysis. However, supervised ML algorithms require large numbers of labeled samples as training data to achieve ideal performance. In some cases, it is impossible or prohibitively expensive to obtain enough labeled training data; here, unsupervised learning (UL) and semi-supervised learning (SSL) paradigms play an indispensable role. In this review, we first introduce the basic concepts of ML techniques, as well as some representative UL and SSL algorithms, including clustering, dimensionality reduction, self-supervised learning (self-SL), positive-unlabeled (PU) learning and transfer learning. We then review recent advances and applications of UL and SSL paradigms in both plant systems biology and plant phenotyping research. Finally, we discuss the limitations and highlight the significances and challenges of UL and SSL strategies in plant systems biology.

Osa‐miR535 targets SQUAMOSA promoter binding protein‐like 4 to regulate blast disease resistance in rice

Abstract: SUMMARY Many rice microRNAs have been identified as fine‐tuning factors in the regulation of agronomic traits and immunity. Among them, Osa‐miR535 targets SQUAMOSA promoter binding protein‐like 14 (OsSPL14) to positively regulate tillers but negatively regulate yield and immunity. Here, we uncovered that Osa‐miR535 targets another SPL gene, OsSPL4, to suppress rice immunity against Magnaporthe oryzae. Overexpression of Osa‐miR535 significantly decreased the accumulation of the fusion protein SPL4TBS‐YFP that contains the target site of Osa‐miR535 in OsSPL4. Consistently, Osa‐miR535 mediated the cleavage of OsSPL4 mRNA between the 10th and 11th base pair of the predicted binding site at the 3′ untranslated region. Transgenic rice lines overexpressing OsSPL4 (OXSPL4) displayed enhanced blast disease resistance accompanied by enhanced immune responses, including increased expression of defense‐relative genes and up‐accumulated H2O2. By contrast, the knockout mutant osspl4 exhibited susceptibility. Moreover, OsSPL4 binds to the promoter of GH3.2, an indole‐3‐acetic acid‐amido synthetase, and promotes its expression. Together, these data indicate that Os‐miR535 targets OsSPL4 and OsSPL4‐GH3.2, which may parallel the OsSPL14‐WRKY45 module in rice blast disease resistance.

Comparative transcriptomic analysis unveils the deep phylogeny and secondary metabolite evolution of 116 Camellia plants.

Abstract: The Camellia includes more than 200 species of great diversity and has immense economic, ornamental, and cultural values. We here sequenced the transcriptomes of 116 Camellia plants from nearly all sections of the genus Camellia. We constructed a pan-transcriptome of Camellia plants with 89,394 gene families and then well resolved the phylogeny of genus Camellia based on 405 high-quality low-copy core genes. Most of the inferred relationships are well supported by multiple nuclear gene trees and morphological traits. We show strong evidence that Camellia plants shared a recent whole genome duplication event, followed by large expansions of transcription factor families associated with stress resistance and secondary metabolism. Secondary metabolites, particularly those associated with tea quality such as catechins and caffeine, were preferentially heavy accumulated in the Camellia plants from section Thea. We thoroughly examined the expression patterns of hundreds of genes associated with tea quality, and found some of them exhibited significantly high expression and correlations with secondary metabolite accumulations in Thea species. We also released a web-accessible database for efficient retrieval of Camellia transcriptomes. The reported transcriptome sequences and obtained novel findings will facilitate the efficient conservation and utilization of Camellia germplasm towards breeding program of cultivated tea, camellia and oil-tea plants.

B vitamin supply in plants and humans: the importance of vitamer homeostasis

Abstract: SUMMARY B vitamins are a group of water‐soluble micronutrients that are required in all life forms. With the lack of biosynthetic pathways, humans depend on dietary uptake of these compounds, either directly or indirectly, from plant sources. B vitamins are frequently given little consideration beyond their role as enzyme accessory factors and are assumed not to limit metabolism. However, it should be recognized that each individual B vitamin is a family of compounds (vitamers), the regulation of which has dedicated pathways. Moreover, it is becoming increasingly evident that individual family members have physiological relevance and should not be sidelined. Here, we elaborate on the known forms of vitamins B1, B6 and B9, their distinct functions and importance to metabolism, in both human and plant health, and highlight the relevance of vitamer homeostasis. Research on B vitamin metabolism over the past several years indicates that not only the total level of vitamins but also the oft‐neglected homeostasis of the various vitamers of each B vitamin is essential to human and plant health. We briefly discuss the potential of plant biology studies in supporting human health regarding these B vitamins as essential micronutrients. Based on the findings of the past few years we conclude that research should focus on the significance of vitamer homeostasis – at the organ, tissue and subcellular levels – which could improve the health of not only humans but also plants, benefiting from cross‐disciplinary approaches and novel technologies.

Zaxinone synthase controls arbuscular mycorrhizal colonization level in rice

Abstract: SUMMARY The Oryza sativa (rice) carotenoid cleavage dioxygenase OsZAS was described to produce zaxinone, a plant growth‐promoting apocarotenoid. A zas mutant line showed reduced arbuscular mycorrhizal (AM) colonization, but the mechanisms underlying this behavior are unknown. Here, we investigated how OsZAS and exogenous zaxinone treatment regulate mycorrhization. Micromolar exogenous supply of zaxinone rescued root growth but not the mycorrhizal defects of the zas mutant, and even reduced mycorrhization in wild‐type and zas genotypes. The zas line did not display the increase in the level of strigolactones (SLs) that was observed in wild‐type plants at 7 days post‐inoculation with AM fungus. Moreover, exogenous treatment with the synthetic SL analog GR24 rescued the zas mutant mycorrhizal phenotype, indicating that the lower AM colonization rate of zas is caused by a deficiency in SLs at the early stages of the interaction, and indicating that during this phase OsZAS activity is required to induce SL production, possibly mediated by the Dwarf14‐Like (D14L) signaling pathway. OsZAS is expressed in arbuscule‐containing cells, and OsPT11prom::OsZAS transgenic lines, where OsZAS expression is driven by the OsPT11 promoter active in arbusculated cells, exhibit increased mycorrhization compared with the wild type. Overall, our results show that the genetic manipulation of OsZAS activity in planta leads to a different effect on AM symbiosis from that of exogenous zaxinone treatment, and demonstrate that OsZAS influences the extent of AM colonization, acting as a component of a regulatory network that involves SLs.

A Solanum lycopersicoides reference genome facilitates insights into tomato specialized metabolism and immunity.

Abstract: Wild relatives of tomato are a valuable source of natural variation in tomato breeding, as many can be hybridized to the cultivated species (Solanum lycopersicum). Several, including S. lycopersicoides, have been crossed to S. lycopersicum for the development of ordered introgression lines (ILs), facilitating breeding for desirable traits. Despite the utility of these wild relatives and their associated ILs, few finished genome sequences have been produced to aid genetic and genomic studies. Here we report a chromosome-scale genome assembly for S. lycopersicoides LA2951, which contains 37,938 predicted protein-coding genes. With the aid of this genome assembly, we have precisely delimited the boundaries of the S. lycopersicoides introgressions in a set of S. lycopersicum cv. VF36 x LA2951 ILs. We demonstrate the usefulness of the LA2951 genome by identifying several quantitative trait loci (QTLs) for phenolics and carotenoids, including underlying candidate genes, and by investigating the genome organization and immunity-associated function of the clustered Pto gene family. In addition, syntenic analysis of R2R3MYB genes sheds light on the identity of the Aubergine locus underlying anthocyanin production. The genome sequence and IL map provide valuable resources for studying fruit nutrient/quality traits, pathogen resistance, and environmental stress tolerance.

Introgression from Gossypium hirsutum is a driver for population divergence and genetic diversity in Gossypium barbadense.

Abstract: During the domestication and improvement processes, interspecific introgression from Gossypium hirsutum has reorganized the genomic architecture of Gossypium barbadense; however, the introgression details and the trait-related genetic loci remain largely unknown. Here, we perform a genome-wide population analysis and genetically recategorize 365 G. barbadense accessions into four subgroups which is different with previous categorizations. A total of 315 introgression events from G. hirsutum to G. barbadense, which primarily contributed to population divergence and agronomic trait variation in G. barbadense, are identified. We find that 70% introgression from G. hirsutum have greatly increased the genetic diversity and divergence of G. barbadense. Some loci are identified with divergent haplotype selection for adaptation to the environment at high latitudes. Through genome-wide association study and genome linkage disequilibrium interval haplotyping analyses, two fiber-micronaire-related haplotype blocks are detected, one of which (FM2) is introgressed from G. hirsutum. Seven distinguished traits related to growth period, plant architecture and stronger vegetative growth habit are found to have pleiotropic effects controlled by a single gene in G. barbadense. Thus, this study provides new insights into the breeding history of G. barbadense and highlights introgression is a driver for improving cultivars in G. barbadense.

AtGH3.10 is another jasmonic acid-amido synthetase in Arabidopsis thaliana.

Abstract: Jasmonoyl-isoleucine (JA-Ile) is a key signaling molecule that activates jasmonate-regulated flower development and wound stress response. For years, the JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl-amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine and the source of persisting JA-Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate, through recombinant enzyme assays and loss-of-function mutant analyses, that AtGH3.10 functions as a JA-amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA-Ile accumulation in gh3.10-2 jar1-11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative, 12OH-JA-Ile, was undetected in the flower buds and unwounded leaves. Residual levels of JA-Ile, JA-Ala, and JA-Val were nonetheless detected in gh3.10-2 jar1-11 suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA-Ile and 12OH-JA-Ile and the expression of JA-responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM-DOMAIN1 observed in WT, gh3.10-1, and jar1-11 leaves were effectively abolished in gh3.10-2 jar1-11. Additionally, increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10-2 jar1-11. These findings conclusively show that AtGH3.10 contributes to JA-amino acid biosynthesis and functions partially redundant with AtJAR1 in sustaining the flower development and wound stress response in Arabidopsis.