Plant Biology Highlights: Nature Articles 2019

I am doing this plant biology CNS (Cell - Nature - Science) paper highlights since 2016. Every winter break, it is a fun job for me to do. I enjoy as much as writing a New Year card to my favorite person. It is also a reminder for myself that lots of great science are going on and I have to do better next year and keep trying to make a difference. Here, I have previewed plant biology stories published this year in Nature. 

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on. Here they show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors — PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)—and their four homologues (DOF6, TMO6, OBP2 and HCA2), which they collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors — the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, their data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, they show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) — a complex of eight large (RbcL) and eight small (RbcS) subunits — and carbonic anhydrase. As HCO3− can diffuse through the proteinaceous carboxysome shell but CO2 cannot, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco. The shell also prevents access to reducing agents, generating an oxidizing environment. The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS. Here they have reconstituted the Rubisco–CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid–liquid phase separation in eukaryotes. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC1. Understanding carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast 

Cannabis sativa
L. has been cultivated and used around the globe for its medicinal properties for millennia. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids, and their structural complexity, which limits bulk chemical synthesis. Here they report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway. They also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency. They also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Their work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments. These biomolecular condensates are formed from processes that include liquid–liquid phase separation. The multivalent interactions necessary for liquid–liquid phase separation have been extensively studied in vitro. However, the regulation of this process in vivo is poorly understood. Here they identify an in vivo regulator of liquid–liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome. FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3′-end processing machinery. These 3′ RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3′-end processing factors to enhance polyadenylation at specific sites. Their findings show that coiled-coil proteins can promote liquid–liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies.

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation. Here they demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. They show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway substrate known as LITTLE ZIPPER 2 (ZPR2) — which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors — and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants.

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides have previously been proposed as ligands for several members of the CrRLK1L family. However, the mechanistic basis of this perception is unknown. Here they report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23–LLG1–FER and RALF23–LLG2–FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Their work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown. Here they report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together — but neither alone — assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, they isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Through the lens of evolution, climate change is an agent of natural selection that forces populations to change and adapt, or face extinction. However, current assessments of the risk of biodiversity associated with climate change do not typically take into account how natural selection influences populations differently depending on their genetic makeup. Here they make use of the extensive genome information that is available for Arabidopsis thaliana and measure how manipulation of the amount of rainfall affected the fitness of 517 natural Arabidopsis lines that were grown in Spain and Germany. This allowed us to directly infer selection along the genome. Natural selection was particularly strong in the hot-dry location in Spain, where 63% of lines were killed and where natural selection substantially changed the frequency of approximately 5% of all genome-wide variants. A significant portion of this climate-driven natural selection of variants was predictable from signatures of local adaptation (R2 = 29–52%), as genetic variants that were found in geographical areas with climates more similar to the experimental sites were positively selected. Field-validated predictions across the species range indicated that Mediterranean and western Siberian populations — at the edges of the environmental limits of this species — currently experience the strongest climate-driven selection. With more frequent droughts and rising temperatures in Europe, they forecast an increase in directional natural selection moving northwards from the southern end of Europe, putting many native A. thaliana populations at evolutionary risk.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits, and new genome editing technology is facilitating functional validation of these mutations in whole organisms. They combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase), the physiological target of cardiac glycosides. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization. We then performed CRISPR–Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides, culminating in triple mutant ‘monarch flies’ that were as insensitive to cardiac glycosides as monarch butterflies. ‘Monarch flies’ retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis. Their study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities.

The blast fungus Magnaporthe oryzae gains entry to its host plant by means of a specialized pressure-generating infection cell called an appressorium, which physically ruptures the leaf cuticle. Turgor is applied as an enormous invasive force by septin-mediated reorganization of the cytoskeleton and actin-dependent protrusion of a rigid penetration hypha. However, the molecular mechanisms that regulate the generation of turgor pressure during appressorium-mediated infection of plants remain poorly understood. Here they show that a turgor-sensing histidine–aspartate kinase, Sln1, enables the appressorium to sense when a critical turgor threshold has been reached and thereby facilitates host penetration. They found that the Sln1 sensor localizes to the appressorium pore in a pressure-dependent manner, which is consistent with the predictions of a mathematical model for plant infection. A Δsln1 mutant generates excess intracellular appressorium turgor, produces hyper-melanized non-functional appressoria and does not organize the septins and polarity determinants that are required for leaf infection. Sln1 acts in parallel with the protein kinase C cell-integrity pathway as a regulator of cAMP-dependent signalling by protein kinase A. Pkc1 phosphorylates the NADPH oxidase regulator NoxR and, collectively, these signalling pathways modulate appressorium turgor and trigger the generation of invasive force to cause blast disease.

Green plants (Viridiplantae) include around 450,000–500,000 species of great diversity and have important roles in terrestrial and aquatic ecosystems. Here, as part of the One Thousand Plant Transcriptomes Initiative, they sequenced the vegetative transcriptomes of 1,124 species that span the diversity of plants in a broad sense (Archaeplastida), including green plants (Viridiplantae), glaucophytes (Glaucophyta) and red algae (Rhodophyta). Their analysis provides a robust phylogenomic framework for examining the evolution of green plants. Most inferred species relationships are well supported across multiple species tree and supermatrix analyses, but discordance among plastid and nuclear gene trees at a few important nodes highlights the complexity of plant genome evolution, including polyploidy, periods of rapid speciation, and extinction. Incomplete sorting of ancestral variation, polyploidization and massive expansions of gene families punctuate the evolutionary history of green plants. Notably, they find that large expansions of gene families preceded the origins of green plants, land plants and vascular plants, whereas whole-genome duplications are inferred to have occurred repeatedly throughout the evolution of flowering plants and ferns. The increasing availability of high-quality plant genome sequences and advances in functional genomics are enabling research on genome evolution across the green tree of life.

The stem cell niche and the size of the root meristem in plants are maintained by intercellular interactions and signalling networks involving a peptide hormone, root meristem growth factor 1 (RGF1). Understanding how RGF1 regulates the development of the root meristem is essential for understanding stem cell function. Although five receptors for RGF1 have been identified, the downstream signalling mechanism remains unknown. Here we report a series of signalling events that follow RGF1 activity. We find that the RGF1-receptor pathway controls the distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a previously uncharacterized transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), that has a central role in mediating RGF1 signalling. Manipulating RITF1 expression leads to the redistribution of ROS along the root developmental zones. Changes in ROS distribution in turn enhance the stability of the PLETHORA2 protein, a master regulator of root stem cells. Our results thus clearly depict a signalling cascade that is initiated by RGF1, linking this peptide to mechanisms that regulate ROS.

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