MYB-related transcription factors control chloroplast biogenesis.

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors. However, glk mutants contain residual chlorophyll, indicating that other proteins must be involved. Here, we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana. In both species, double-mutant alleles in MYB-related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in GLK mutants. Genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins, whereas those allowing CO2 fixation, photorespiration, and photosystem assembly and repair require MYB-related proteins. Regulation between the MYB-related and GLK transcription factors appears more extensive in A. thaliana than in M. polymorpha. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

Structural insights into the chloroplast protein import in land plants.

Chloroplast proteins are imported via the translocon at the outer chloroplast membrane (TOC)-translocon at the inner chloroplast membrane (TIC) supercomplex, driven by an ATPase motor. The Ycf2-FtsHi complex has been identified as the chloroplast import motor. However, its assembly and cooperation with the TIC complex during preprotein translocation remain unclear. Here, we present the structures of the Ycf2-FtsHi and TIC complexes from Arabidopsis and an ultracomplex formed between them from Pisum. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase motor module with characteristic features. Four previously uncharacterized components of Ycf2-FtsHi were identified, which aid in complex assembly and anchoring of the motor module at a tilted angle relative to the membrane. When considering the structures of the TIC complex and the TIC-Ycf2-FtsHi ultracomplex together, it becomes evident that the tilted motor module of Ycf2-FtsHi enables its close contact with the TIC complex, thereby facilitating efficient preprotein translocation. Our study provides valuable structural insights into the chloroplast protein import process in land plants.

Reciprocal conversion between annual and polycarpic perennial flowering behavior in the Brassicaceae.

The development of perennial crops holds great promise for sustainable agriculture and food security. However, the evolution of the transition between perenniality and annuality is poorly understood. Here, using two Brassicaceae species, Crucihimalaya himalaica and Erysimum nevadense, as polycarpic perennial models, we reveal that the transition from polycarpic perennial to biennial and annual flowering behavior is a continuum determined by the dosage of three closely related MADS-box genes. Diversification of the expression patterns, functional strengths, and combinations of these genes endows species with the potential to adopt various life-history strategies. Remarkably, we find that a single gene among these three is sufficient to convert winter-annual or annual Brassicaceae plants into polycarpic perennial flowering plants. Our work delineates a genetic basis for the evolution of diverse life-history strategies in plants and lays the groundwork for the generation of diverse perennial Brassicaceae crops in the future.

The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots.

Recognition of microbe-associated molecular patterns (MAMPs) is crucial for the plant's immune response. How this sophisticated perception system can be usefully deployed in roots, continuously exposed to microbes, remains a mystery. By analyzing MAMP receptor expression and response at cellular resolution in Arabidopsis, we observed that differentiated outer cell layers show low expression of pattern-recognition receptors (PRRs) and lack MAMP responsiveness. Yet, these cells can be gated to become responsive by neighbor cell damage. Laser ablation of small cell clusters strongly upregulates PRR expression in their vicinity, and elevated receptor expression is sufficient to induce responsiveness in non-responsive cells. Finally, localized damage also leads to immune responses to otherwise non-immunogenic, beneficial bacteria. Damage-gating is overridden by receptor overexpression, which antagonizes colonization. Our findings that cellular damage can "switch on" local immune responses helps to conceptualize how MAMP perception can be used despite the presence of microbial patterns in the soil.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

Root System Depth in Arabidopsis Is Shaped by EXOCYST70A3 via the Dynamic Modulation of Auxin Transport.

Root system architecture (RSA), the distribution of roots in soil, plays a major role in plant survival. RSA is shaped by multiple developmental processes that are largely governed by the phytohormone auxin, suggesting that auxin regulates responses of roots that are important for local adaptation. However, auxin has a central role in numerous processes, and it is unclear which molecular mechanisms contribute to the variation in RSA for environmental adaptation. Using natural variation in Arabidopsis, we identify EXOCYST70A3 as a modulator of the auxin system that causes variation in RSA by acting on PIN4 protein distribution. Allelic variation and genetic perturbation of EXOCYST70A3 lead to alteration of root gravitropic responses, resulting in a different RSA depth profile and drought resistance. Overall our findings suggest that the local modulation of the pleiotropic auxin pathway can gives rise to distinct RSAs that can be adaptive in specific environments.

ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors.

During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.

RNA-Dependent Epigenetic Silencing Directs Transcriptional Downregulation Caused by Intronic Repeat Expansions.

Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.

Flavin Monooxygenase-Generated N-Hydroxypipecolic Acid Is a Critical Element of Plant Systemic Immunity.

Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection.

Plant Cell Polarity: Creating Diversity from Inside the Box.

Cell polarity in plants operates across a broad range of spatial and temporal scales to control processes from acute cell growth to systemic hormone distribution. Similar to other eukaryotes, plants generate polarity at both the subcellular and tissue levels, often through polarization of membrane-associated protein complexes. However, likely due to the constraints imposed by the cell wall and their extremely plastic development, plants possess novel polarity molecules and mechanisms highly tuned to environmental inputs. Considerable progress has been made in identifying key plant polarity regulators, but detailed molecular understanding of polarity mechanisms remains incomplete in plants. Here, we emphasize the striking similarities in the conceptual frameworks that generate polarity in both animals and plants. To this end, we highlight how novel, plant-specific proteins engage in common themes of positive feedback, dynamic intracellular trafficking, and posttranslational regulation to establish polarity axes in development. We end with a discussion of how environmental signals control intrinsic polarity to impact postembryonic organogenesis and growth.

PICKLE-mediated nucleosome condensing drives H3K27me3 spreading for the inheritance of Polycomb memory during differentiation.

Spreading of H3K27me3 is crucial for the maintenance of mitotically inheritable Polycomb-mediated chromatin silencing in animals and plants. However, how Polycomb repressive complex 2 (PRC2) accesses unmodified nucleosomes in spreading regions for spreading H3K27me3 remains unclear. Here, we show in Arabidopsis thaliana that the chromatin remodeler PICKLE (PKL) plays a specialized role in H3K27me3 spreading to safeguard cell identity during differentiation. PKL specifically localizes to H3K27me3 spreading regions but not to nucleation sites and physically associates with PRC2. Loss of PKL disrupts the occupancy of the PRC2 catalytic subunit CLF in spreading regions and leads to aberrant dedifferentiation. Nucleosome density increase endowed by the ATPase function of PKL ensures that unmodified nucleosomes are accessible to PRC2 catalytic activity for H3K27me3 spreading. Our findings demonstrate that PKL-dependent nucleosome compaction is critical for PRC2-mediated H3K27me3 read-and-write function in H3K27me3 spreading, thus revealing a mechanism by which repressive chromatin domains are established and propagated.

1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana.

Arabidopsis thaliana serves as a model organism for the study of fundamental physiological, cellular, and molecular processes. It has also greatly advanced our understanding of intraspecific genome variation. We present a detailed map of variation in 1,135 high-quality re-sequenced natural inbred lines representing the native Eurasian and North African range and recently colonized North America. We identify relict populations that continue to inhabit ancestral habitats, primarily in the Iberian Peninsula. They have mixed with a lineage that has spread to northern latitudes from an unknown glacial refugium and is now found in a much broader spectrum of habitats. Insights into the history of the species and the fine-scale distribution of genetic diversity provide the basis for full exploitation of A. thaliana natural variation through integration of genomes and epigenomes with molecular and non-molecular phenotypes.

The FLC Locus: A Platform for Discoveries in Epigenetics and Adaptation.

Our understanding of the detailed molecular mechanisms underpinning adaptation is still poor. One example for which mechanistic understanding of regulation has converged with studies of life history variation is Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC determines the need for plants to overwinter and their ability to respond to prolonged cold in a process termed vernalization. This review highlights how molecular analysis of vernalization pathways has revealed important insight into antisense-mediated chromatin silencing mechanisms that regulate FLC. In turn, such insight has enabled molecular dissection of the diversity in vernalization across natural populations of A. thaliana. Changes in both cotranscriptional regulation and epigenetic silencing of FLC are caused by noncoding polymorphisms at FLC. The FLC locus is therefore providing important concepts for how noncoding transcription and chromatin regulation influence gene expression and how these mechanisms can vary to underpin adaptation in natural populations.

Genotypes, Networks, Phenotypes: Moving Toward Plant Systems Genetics.

One of the central goals in biology is to understand how and how much of the phenotype of an organism is encoded in its genome. Although many genes that are crucial for organismal processes have been identified, much less is known about the genetic bases underlying quantitative phenotypic differences in natural populations. We discuss the fundamental gap between the large body of knowledge generated over the past decades by experimental genetics in the laboratory and what is needed to understand the genotype-to-phenotype problem on a broader scale. We argue that systems genetics, a combination of systems biology and the study of natural variation using quantitative genetics, will help to address this problem. We present major advances in these two mostly disconnected areas that have increased our understanding of the developmental processes of flowering time control and root growth. We conclude by illustrating and discussing the efforts that have been made toward systems genetics specifically in plants.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

ARGONAUTE10 controls cell fate specification and formative cell divisions in the Arabidopsis root.

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.

STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins.

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.