Plant Biology Highlights: Nature Articles 2020

Every winter I go back and look at some major plant biology stories appeared in Cell, Nature, and Science. I have been doing this since 2016. Yes - 5 years in a row! Here is plant biology stories published this year in Nature

Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis

Hydrogen peroxide (H2O2) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues. H2O2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H2O2 also exist on the cell surface remains unknown. In plant cells, H2O2 triggers an influx of Ca2+ ions, which is thought to be involved in H2O2 sensing and signalling. Here, by using forward genetic screens based on Ca2+ imaging, they isolated hydrogen-peroxide-induced Ca2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H2O2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H2O2-induced activation of Ca2+ channels in guard cells and is required for stomatal closure. Their findings help to identify how the perception of extracellular H2O2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

Mass-spectrometry-based draft of the Arabidopsis proteome

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities. Here they present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Their analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). They present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions. 

FERONIA controls pectin- and nitric oxide-mediated male–female interaction

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release. Here they show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. They demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Their results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

A plant genetic network for preventing dysbiosis in the phyllosphere 

The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here they show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec), simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment. 

Plant 22-nt siRNAs mediate translational repression and stress adaptation 

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here they report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases—NIA1 and NIA2—produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, their research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses. 

Early Holocene crop cultivation and landscape modification in Amazonia 

The onset of plant cultivation is one of the most important cultural transitions in human history. Southwestern Amazonia has previously been proposed as an early centre of plant domestication, on the basis of molecular markers that show genetic similarities between domesticated plants and wild relatives. However, the nature of the early human occupation of southwestern Amazonia, and the history of plant cultivation in this region, are poorly understood. Here we document the cultivation of squash (Cucurbita sp.) at about 10,250 calibrated years before present (cal. yr BP), manioc (Manihot sp.) at about 10,350 cal. yr BP and maize (Zea mays) at about 6,850 cal. yr BP, in the Llanos de Moxos (Bolivia). We show that, starting at around 10,850 cal. yr BP, inhabitants of this region began to create a landscape that ultimately comprised approximately 4,700 artificial forest islands within a treeless, seasonally flooded savannah. Their results confirm that the Llanos de Moxos is a hotspot for early plant cultivation and demonstrate that—ever since their arrival in Amazonia—humans have markedly altered the landscape, with lasting repercussions for habitat heterogeneity and species conservation. 

Ligand-induced monoubiquitination of BIK1 regulates plant immunity 

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)–BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2–BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Their study reveals the intertwined regulation of PRR–RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling. 

Extensive signal integration by the phytohormone protein network 

Plant hormones coordinate responses to environmental cues with developmental programs, and are fundamental for stress resilience and agronomic yield. The core signalling pathways underlying the effects of phytohormones have been elucidated by genetic screens and hypothesis-driven approaches, and extended by interactome studies of select pathways. However, fundamental questions remain about how information from different pathways is integrated. Genetically, most phenotypes seem to be regulated by several hormones, but transcriptional profiling suggests that hormones trigger largely exclusive transcriptional programs. They hypothesized that protein–protein interactions have an important role in phytohormone signal integration. Here, they experimentally generated a systems-level map of the Arabidopsis phytohormone signalling network, consisting of more than 2,000 binary protein–protein interactions. In the highly interconnected network, they identify pathway communities and hundreds of previously unknown pathway contacts that represent potential points of crosstalk. Functional validation of candidates in seven hormone pathways reveals new functions for 74% of tested proteins in 84% of candidate interactions, and indicates that a large majority of signalling proteins function pleiotropically in several pathways. Moreover, they identify several hundred largely small-molecule-dependent interactions of hormone receptors. Comparison with previous reports suggests that noncanonical and nontranscription-mediated receptor signalling is more common than hitherto appreciated. 

Transcriptional regulation of strigolactone signalling in Arabidopsis 

Plant hormones known as strigolactones control plant development and interactions between host plants and symbiotic fungi or parasitic weeds. In Arabidopsis thaliana and rice, the proteins DWARF14 (D14), MORE AXILLARY GROWTH 2 (MAX2), SUPPRESSOR OF MAX2-LIKE 6, 7 and 8 (SMXL6, SMXL7 and SMXL8) and their orthologues form a complex upon strigolactone perception and play a central part in strigolactone signalling. However, whether and how strigolactones activate downstream transcription remains largely unknown. Here they use a synthetic strigolactone to identify 401 strigolactone-responsive genes in Arabidopsis, and show that these plant hormones regulate shoot branching, leaf shape and anthocyanin accumulation mainly through transcriptional activation of the BRANCHED 1, TCP DOMAIN PROTEIN 1 and PRODUCTION OF ANTHOCYANIN PIGMENT 1 genes. They find that SMXL6 targets 729 genes in the Arabidopsis genome and represses the transcription of SMXL6, SMXL7 and SMXL8 by binding directly to their promoters, showing that SMXL6 serves as an autoregulated transcription factor to maintain the homeostasis of strigolactone signalling. These findings reveal an unanticipated mechanism through which a transcriptional repressor of hormone signalling can directly recognize DNA and regulate transcription in higher plants. 

Temperature-dependent growth contributes to long-term cold sensing 

Temperature is a key factor in the growth and development of all organisms. Plants have to interpret temperature fluctuations, over hourly to monthly timescales, to align their growth and development with the seasons. Much is known about how plants respond to acute thermal stresses, but the mechanisms that integrate long-term temperature exposure remain unknown. The slow, winter-long upregulation of VERNALIZATION INSENSITIVE 3 (VIN3), a PHD protein that functions with Polycomb repressive complex 2 to epigenetically silence FLOWERING LOCUS C (FLC) during vernalization, is central to plants interpreting winter progression. Here, by a forward genetic screen, they identify two dominant mutations of the transcription factor NTL8 that constitutively activate VIN3 expression and alter the slow VIN3 cold induction profile. In the wild type, the NTL8 protein accumulates slowly in the cold, and directly upregulates VIN3 transcription. Through combining computational simulation and experimental validation, they show that a major contributor to this slow accumulation is reduced NTL8 dilution due to slow growth at low temperatures. Temperature-dependent growth is thus exploited through protein dilution to provide the long-term thermosensory information for VIN3 upregulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures. 

Massive haplotypes underlie ecotypic differentiation in sunflowers 

Species often include multiple ecotypes that are adapted to different environments. However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are maintained despite hybridization with non-adapted populations. Here, by resequencing 1,506 wild sunflowers from 3 species (Helianthus annuus, Helianthus petiolaris and Helianthus argophyllus), they identify 37 large (1–100 Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes of the silverleaf sunflower H. argophyllus (probably through deletion of a homologue of FLOWERING LOCUS T (FT)), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other—possibly now-extinct—congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation. 

A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis 

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms. The evening complex consists of EARLY FLOWERING 3 (ELF3), a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat, embedded within a predicted prion domain (PrD). Here they find that the length of the polyQ repeat correlates with thermal responsiveness. They show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism. 

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity 

Perception of biotic and abiotic stresses often leads to stomatal closure in plants. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive. Here they report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid—a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses. 

Structural basis of salicylic acid perception by Arabidopsis NPR proteins 

Salicylic acid (SA) is a plant hormone that is critical for resistance to pathogens. The NPR proteins have previously been identified as SA receptors, although how they perceive SA and coordinate hormonal signalling remain unknown. Here they report the mapping of the SA-binding core of Arabidopsis thaliana NPR4 and its ligand-bound crystal structure. The SA-binding core domain of NPR4 refolded with SA adopts an α-helical fold that completely buries SA in its hydrophobic core. The lack of a ligand-entry pathway suggests that SA binding involves a major conformational remodelling of the SA-binding core of NPR4, which they validated using hydrogen–deuterium-exchange mass spectrometry analysis of the full-length protein and through SA-induced disruption of interactions between NPR1 and NPR4. They show that, despite the two proteins sharing nearly identical hormone-binding residues, NPR1 displays minimal SA-binding activity compared to NPR4. They further identify two surface residues of the SA-binding core, the mutation of which can alter the SA-binding ability of NPR4 and its interaction with NPR1. They also demonstrate that expressing a variant of NPR4 that is hypersensitive to SA could enhance SA-mediated basal immunity without compromising effector-triggered immunity, because the ability of this variant to re-associate with NPR1 at high levels of SA remains intact. By revealing the structural mechanisms of SA perception by NPR proteins, our work paves the way for future investigation of the specific roles of these proteins in SA signalling and their potential for engineering plant immunity. 

Structures of fungal and plant acetohydroxyacid synthases 

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids. It is the target for more than 50 commercial herbicides. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here they describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. They found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. They also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms. 

Quinone perception in plants via leucine-rich-repeat receptor-like kinases 

Quinones are produced and sensed in all kingdoms of life. Plants are primary producers of quinone, but the role of quinone as a signalling agent in plants remains largely unknown. One well-documented role of quinone is in the induction of haustoria (specialized feeding structures) in plants that parasitize roots, which occurs in the presence of the host-derived quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ). However, how parasitic plants sense DMBQ remains unclear, as is whether nonparasitic plants are capable of sensing quinones. Here they use Arabidopsis thaliana and DMBQ as a model plant and quinone to show that DMBQ signalling occurs in Arabidopsis via elevation of cytosolic Ca2+ concentration. They performed a forward genetic screen in Arabidopsis that isolated DMBQ-unresponsive mutants, which we named cannot respond to DMBQ 1 (card1). The CANNOT RESPOND TO DMBQ 1 (CARD1; At5g49760, also known as HPCA1) gene encodes a leucine-rich-repeat receptor-like kinase that is highly conserved in land plants. In Arabidopsis, DMBQ triggers defence-related gene expression, and card1 mutants show impaired immunity against bacterial pathogens. In Phtheirospermum japonicum (a plant that parasitizes roots), DMBQ initiates Ca2+ signalling in the root and is important for the development of the haustorium. Furthermore, CARD1 homologues from this parasitic plant complement DMBQ-induced elevation of cytosolic Ca2+ concentration in the card1 mutant. Their results demonstrate that plants—unlike animals and bacteria—use leucine-rich-repeat receptor-like kinases for quinone signalling. This work provides insights into the role of quinone signalling and CARD1 functions in plants that help us to better understand the signalling pathways used during the formation of the haustorium in parasitic plants and in plant immunity in nonparasitic plants. 

Multiple wheat genomes reveal global variation in modern breeding 

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome, and the lack of genome-assembly data for multiple wheat lines. Here they generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses. They provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars. 

The barley pan-genome reveals the hidden legacy of mutation breeding 

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the ‘pan-genome’). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions. Here they report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley—comprising landraces, cultivars and a wild barley—that were selected as representatives of global barley diversity. They catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. They discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding. 

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