Modulation of receptor-like transmembrane kinase 1 nuclear localization by DA1 peptidases in Arabidopsis.

The cleavage of intracellular domains of receptor-like kinases (RLKs) has an important functional role in the transduction of signals from the cell surface to the nucleus in many organisms. However, the peptidases that catalyze protein cleavage during signal transduction remain poorly understood despite their crucial roles in diverse signaling processes. Here, we report in the flowering plant Arabidopsis thaliana that members of the DA1 family of ubiquitin-regulated Zn metallopeptidases cleave the cytoplasmic kinase domain of transmembrane kinase 1 (TMK1), releasing it for nuclear localization where it represses auxin-responsive cell growth during apical hook formation by phosphorylation and stabilization of the transcriptional repressors IAA32 and IAA34. Mutations in DA1 family members exhibited reduced apical hook formation, and DA1 family-mediated cleavage of TMK1 was promoted by auxin treatment. Expression of the DA1 family-generated intracellular kinase domain of TMK1 by an auxin-responsive promoter fully restored apical hook formation in a tmk1 mutant, establishing the function of DA1 family peptidase activities in TMK1-mediated differential cell growth and apical hook formation. DA1 family peptidase activity therefore modulates TMK1 kinase activity between a membrane location where it stimulates acid cell growth and initiates an auxin-dependent kinase cascade controlling cell proliferation in lateral roots and a nuclear localization where it represses auxin-mediated gene expression and growth.

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis.

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.

Fast-forward breeding for a food-secure world.

Crop production systems need to expand their outputs sustainably to feed a burgeoning human population. Advances in genome sequencing technologies combined with efficient trait mapping procedures accelerate the availability of beneficial alleles for breeding and research. Enhanced interoperability between different omics and phenotyping platforms, leveraged by evolving machine learning tools, will help provide mechanistic explanations for complex plant traits. Targeted and rapid assembly of beneficial alleles using optimized breeding strategies and precise genome editing techniques could deliver ideal crops for the future. Realizing desired productivity gains in the field is imperative for securing an adequate future food supply for 10 billion people.

Genomic innovation for crop improvement.

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

Variation in Expression of the HECT E3 Ligase UPL3 Modulates LEC2 Levels, Seed Size, and Crop Yields in Brassica napus.

Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We used association analyses of oilseed rape/canola (Brassica napus) accessions to identify genetic variation that influences seed size, lipid content, and final crop yield. Variation in the promoter region of the HECT E3 ligase gene BnaUPL3 C03 made a major contribution to variation in seed weight per pod, with accessions exhibiting high seed weight per pod having lower levels of BnaUPL3 C03 expression. We defined a mechanism in which UPL3 mediated the proteasomal degradation of LEC2, a master transcriptional regulator of seed maturation. Accessions with reduced UPL3 expression had increased LEC2 protein levels, larger seeds, and prolonged expression of lipid biosynthetic genes during seed maturation. Natural variation in BnaUPL3 C03 expression appears not to have been exploited in current B napus breeding lines and could therefore be used as a new approach to maximize future yields in this important oil crop.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

The ubiquitin receptors DA1, DAR1, and DAR2 redundantly regulate endoreduplication by modulating the stability of TCP14/15 in Arabidopsis.

Organ growth involves the coordination of cell proliferation and cell growth with differentiation. Endoreduplication is correlated with the onset of cell differentiation and with cell and organ size, but little is known about the molecular mechanisms linking cell and organ growth with endoreduplication. We have previously demonstrated that the ubiquitin receptor DA1 influences organ growth by restricting cell proliferation. Here, we show that DA1 and its close family members DAR1 and DAR2 are redundantly required for endoreduplication during leaf development. DA1, DAR1, and DAR2 physically interact with the transcription factors TCP14 and TCP15, which repress endoreduplication by directly regulating the expression of cell-cycle genes. We also show that DA1, DAR1, and DAR2 modulate the stability of TCP14 and TCP15 proteins in Arabidopsis thaliana. Genetic analyses demonstrate that DA1, DAR1, and DAR2 function in a common pathway with TCP14/15 to regulate endoreduplication. Thus, our findings define an important genetic and molecular mechanism involving the ubiquitin receptors DA1, DAR1, and DAR2 and the transcription factors TCP14 and TCP15 that links endoreduplication with cell and organ growth.

Analysis of the bread wheat genome using whole-genome shotgun sequencing.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

TANG1, Encoding a Symplekin_C Domain-Contained Protein, Influences Sugar Responses in Arabidopsis.

Sugars not only serve as energy and cellular carbon skeleton but also function as signaling molecules regulating growth and development in plants. Understanding the molecular mechanisms in sugar signaling pathways will provide more information for improving plant growth and development. Here, we describe a sugar-hypersensitive recessive mutant, tang1. Light-grown tang1 mutants have short roots and increased starch and anthocyanin contents when grown on high-sugar concentration medium. Dark-grown tang1 plants exhibit sugar-hypersensitive hypocotyl elongation and enhanced dark development. The tang1 mutants also show an enhanced response to abscisic acid but reduced response to ethylene. Thus, tang1 displays a range of alterations in sugar signaling-related responses. The TANG1 gene was isolated by a map-based cloning approach and encodes a previously uncharacterized unique protein with a predicted Symplekin tight-junction protein C terminus. Expression analysis indicates that TANG1 is ubiquitously expressed at moderate levels in different organs and throughout the Arabidopsis (Arabidopsis thaliana) life cycle; however, its expression is not affected by high-sugar treatment. Genetic analysis shows that PRL1 and TANG1 have additive effects on sugar-related responses. Furthermore, the mutation of TANG1 does not affect the expression of genes involved in known sugar signaling pathways. Taken together, these results suggest that TANG1, a unique gene, plays an important role in sugar responses in Arabidopsis.

The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis.

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as auxin response factor2, apetala2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase enhancer1 OF DA1 (EOD1)/big brother to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) grain width and weight2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

The Mediator complex subunits MED25/PFT1 and MED8 are required for transcriptional responses to changes in cell wall arabinose composition and glucose treatment in Arabidopsis thaliana.

BACKGROUND: Plant cell walls are dynamic structures involved in all aspects of plant growth, environmental interactions and defense responses, and are the most abundant renewable source of carbon-containing polymers on the planet. To balance rigidity and extensibility, the composition and integrity of cell wall components need to be tightly regulated, for example during cell elongation. RESULTS: We show that mutations in the MED25/PFT1 and MED8 subunits of the Mediator transcription complex suppressed the sugar-hypersensitive hypocotyl elongation phenotype of the hsr8-1 mutant, which has cell wall defects due to arabinose deficiency that do not permit normal cell elongation. This suppression occurred independently of light and jasmonic acid (JA) signaling. Gene expression analyses revealed that the expression of genes induced in hsr8-1 that encode enzymes and proteins that are involved in cell expansion and cell wall strengthening is reduced in the pft1-2 mutant line, and the expression of genes encoding transcription factors involved in reducing hypocotyl cell elongation, genes encoding cell wall associated enzymes and proteins is up-regulated in pft1-2. PFT1 was also required for the expression of several glucose-induced genes, including those encoding cell wall components and enzymes, regulatory and enzymatic components of anthocyanin biosynthesis, and flavonoid and glucosinolate biosynthetic pathways. CONCLUSIONS: These results establish that MED25 and MED8 subunits of the Mediator transcriptional complex are required for the transcriptional regulation of genes involved in cell elongation and cell wall composition in response to defective cell walls and in sugar- responsive gene expression.

Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I.

Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far.

The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis.

Guard cell actin reorganization has been observed in stomatal responses to a wide array of stimuli. However, how the guard cell signaling machinery regulates actin dynamics is poorly understood. Here, we report the identification of an allele of the Arabidopsis thaliana ACTIN-RELATED PROTEIN C2/DISTORTED TRICHOMES2 (ARPC2) locus (encoding the ARPC2 subunit of the ARP2/3 complex) designated high sugar response3 (hsr3). The hsr3 mutant showed increased transpirational water loss that was mainly due to a lesion in stomatal regulation. Stomatal bioassay analyses revealed that guard cell sensitivity to external stimuli, such as abscisic acid (ABA), CaCl(2), and light/dark transition, was reduced or abolished in hsr3. Analysis of a nonallelic mutant of the ARP2/3 complex suggested no pleiotropic effect of ARPC2 beyond its function in the complex in regard to stomatal regulation. When treated with ABA, guard cell actin filaments underwent fast disruption in wild-type plants, whereas those in hsr3 remained largely bundled. The ABA insensitivity phenotype of hsr3 was rescued by cytochalasin D treatment, suggesting that the aberrant stomatal response was a consequence of bundled actin filaments. Our work indicates that regulation of actin reassembly through ARP2/3 complex activity is crucial for stomatal regulation.

Control of root meristem size by DA1-RELATED PROTEIN2 in Arabidopsis.

The control of organ growth by coordinating cell proliferation and differentiation is a fundamental developmental process. In plants, postembryonic root growth is sustained by the root meristem. For maintenance of root meristem size, the rate of cell differentiation must equal the rate of cell division. Cytokinin and auxin interact to affect the cell proliferation and differentiation balance and thus control root meristem size. However, the genetic and molecular mechanisms that determine root meristem size still remain largely unknown. Here, we report that da1-related protein2 (dar2) mutants produce small root meristems due to decreased cell division and early cell differentiation in the root meristem of Arabidopsis (Arabidopsis thaliana). dar2 mutants also exhibit reduced stem cell niche activity in the root meristem. DAR2 encodes a Lin-11, Isl-1, and Mec-3 domain-containing protein and shows an expression peak in the border between the transition zone and the elongation zone. Genetic analyses show that DAR2 functions downstream of cytokinin and SHORT HYPOCOTYL2 to maintain normal auxin distribution by influencing auxin transport. Further results indicate that DAR2 acts through the PLETHORA pathway to influence root stem cell niche activity and therefore control root meristem size. Collectively, our findings identify the role of DAR2 in root meristem size control and provide a novel link between several key regulators influencing root meristem size.

The peptidase DA1 cleaves and destabilizes WUSCHEL to control shoot apical meristem size.

Stem cells in plants and animals are the source of new tissues and organs. In plants, stem cells are maintained in the central zone (CZ) of multicellular meristems, and large shoot meristems with an increased stem cell population hold promise for enhancing yield. The mobile homeodomain transcription factor WUSCHEL (WUS) is a central regulator of stem cell function in plant shoot meristems. Despite its central importance, the factors that directly modulate WUS protein stability have been a long-standing question. Here, we show that the peptidase DA1 physically interacts with and cleaves the WUS protein, leading to its destabilization. Furthermore, our results reveal that cytokinin signaling represses the level of DA1 protein in the shoot apical meristem, thereby increasing the accumulation of WUS protein. Consistent with these observations, loss of DA1 function results in larger shoot apical meristems with an increased stem cell population and also influences cytokinin-induced enlargement of shoot apical meristem. Collectively, our findings uncover a previously unrecognized mechanism by which the repression of DA1 by cytokinin signaling stabilizes WUS, resulting in the enlarged shoot apical meristems with the increased stem cell number during plant growth and development.

Unlocking plant genetics with telomere-to-telomere genome assemblies.

Contiguous genome sequence assemblies will help us to realize the full potential of crop translational genomics. Recent advances in sequencing technologies, especially long-read sequencing strategies, have made it possible to construct gapless telomere-to-telomere (T2T) assemblies, thus offering novel insights into genome organization and function. Plant genomes pose unique challenges, such as a continuum of ancient to recent polyploidy and abundant highly similar and long repetitive elements. Owing to progress in sequencing approaches, for most crop plants, chromosome-scale reference genome assemblies are available, but T2T assembly construction remains challenging. Here we describe methods for haplotype-resolved, gapless T2T assembly construction in plants, including various crop species. We outline the impact of T2T assemblies in elucidating the roles of repetitive elements in gene regulation, as well as in pangenomics, functional genomics, genome-assisted breeding and targeted genome manipulation. In conjunction with sequence-enriched germplasm repositories, T2T assemblies thus hold great promise for basic and applied plant sciences.

The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica crops. The Brassica relative Arabidopsis thaliana uses the paired immune receptor complex CSA1-CHS3/DAR4 to resist Albugo infection. The CHS3/DAR4 sensor NLR, which functions together with its partner, the helper NLR CSA1, carries an integrated domain (ID) with homology to DA1 peptidases. Using domain swaps with several DA1 homologs, we show that the LIM-peptidase domain of the family member CHS3/DAR4 functions as an integrated decoy for the family member DAR3, which interacts with and inhibits the peptidase activities of the three closely related peptidases DA1, DAR1, and DAR2. Albugo infection rapidly lowers DAR3 levels and activates DA1 peptidase activity, thereby promoting endoreduplication of host tissues to support pathogen growth. We propose that the paired immune receptor CSA1-CHS3/DAR4 detects the actions of a putative Albugo effector that reduces DAR3 levels, resulting in defense activation.