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Alternative cleavage and polyadenylation (APA) is increasingly recognized as an important regulatory mechanism in eukaryotic gene expression and is dynamically modulated in a developmental, tissue-specific, or environmentally responsive manner. Given the functional importance of APA and the rapid accumulation of APA sites in plants, a comprehensive and easily accessible APA site database is necessary for improved understanding of APA-mediated gene expression regulation. We present a database called PlantAPAdb that catalogs the most comprehensive APA site data derived from sequences from diverse 3′ sequencing protocols and biological samples in plants. Currently, PlantAPAdb contains APA sites in six species, Oryza sativa (japonica and indica), Arabidopsis (Arabidopsis thaliana), Medicago truncatula, Trifolium pratense, Phyllostachys edulis, and Chlamydomonas reinhardtii. APA sites in PlantAPAdb are available for bulk download and can be queried in a Google-like manner. PlantAPAdb provides rich information of the whole-genome APA sites, including genomic locations, heterogeneous cleavage sites, expression levels, and sample information. It also provides comprehensive poly(A) signals for APA sites in different genomic regions according to distinct profiles of cis-elements in plants. In addition, PlantAPAdb contains events of 3′ untranslated region shortening/lengthening resulting from APA, which helps to understand the mechanisms underlying systematic changes in 3′ untranslated region lengths. Additional information about conservation of APA sites in plants is also available, providing insights into the evolutionary polyadenylation configuration across species. As a user-friendly database, PlantAPAdb is a large and extendable resource for elucidating APA mechanisms, APA conservation, and gene expression regulation.

Low temperature is an important environmental stress that adversely affects rice (Oryza sativa) growth and productivity. Splicing of pre-mRNA is a crucial posttranscriptional regulatory step in gene expression in plants and is sensitive to temperature. DEAD-box RNA helicases belong to an RNA helicase family involved in the rearrangement of ribonucleoprotein complexes and the modification of RNA structure and are therefore involved in all aspects of RNA metabolism. In this study, we demonstrate that the rate of pre-mRNA splicing is reduced in rice at low temperatures and that the DEAD-box RNA Helicase42 (OsRH42) is necessary to support effective splicing of pre-mRNA during mRNA maturation at low temperatures. OsRH42 expression is tightly coupled to temperature fluctuation, and OsRH42 is localized in the splicing speckles and interacts directly with U2 small nuclear RNA. Retarded pre-mRNA splicing and plant growth defects were exhibited by OsRH42-knockdown transgenic lines at low temperatures, thus indicating that OsRH42 performs an essential role in ensuring accurate pre-mRNA splicing and normal plant growth under low ambient temperature. Unexpectedly, our results show that OsRH42 overexpression significantly disrupts the pre-mRNA splicing pathway, causing retarded plant growth and reducing plant cold tolerance. Combined, these results indicate that accurate control of OsRH42 homeostasis is essential for rice plants to respond to changes in ambient temperature. In addition, our study presents the molecular mechanism of DEAD-box RNA helicase function in pre-mRNA splicing, which is required for adaptation to cold stress in rice.

The second messenger calcium plays a key role in conveying specificity of signalling pathways in plant cells. Specific calcium signatures are decoded to generate correct gene expression responses and amplification of calcium signatures is vital to this process. It is not known: (1) if this amplification is an intrinsic property of all calcium-regulated gene expression responses and whether all calcium signatures have the potential to be amplified, and (2) how does a given calcium signature maintain specificity in cells containing a great number of transcription factors (TFs) and other proteins with the potential to be calcium-regulated? The work presented here uncovers the design principle by which it is possible to decode calcium signals into specific changes in gene transcription in plant cells. Regarding the first question, we found that the binding mechanism between protein components possesses an intrinsic property that will nonlinearly amplify any calcium signal. This nonlinear amplification allows plant cells to effectively distinguish the kinetics of different calcium signatures to produce specific and appropriate changes in gene expression. Regarding the second question, we found that the large number of calmodulin (CaM)-binding transcription factors (TFs) or proteins in plant cells form a buffering system such that the concentration of an active CaM-binding TF is insensitive to the concentration of any other CaM-binding protein, thus maintaining specificity. The design principle revealed by this work can be used to explain how any CaM-binding TF decodes calcium signals to generate specific gene expression responses in plant cells via transcription.

MicroRNAs (miRNAs) are small noncoding RNAs of ∼21 nt in length, which have regulatory roles in many biological processes. In animals, proper functioning of the circadian clock, which is closely linked to the fitness of almost all living organisms, is regulated by miRNAs. However, to date, there have been no reports of the roles of miRNA in regulation of the plant circadian rhythm. Here, we report a natural variant of miR397 that lengthens the circadian period and controls flowering time in Arabidopsis (Arabidopsis thaliana). Highly conserved among angiosperms, the miRNA miR397 has two members in Arabidopsis: miR397a and miR397b. However, only miR397b significantly delayed flowering. Our results suggest that miR397b controls flowering by targeting CASEIN KINASE II SUBUNIT BETA3 (CKB3), in turn modulating the circadian period of CIRCADIAN CLOCK ASSOCIATED1 (CCA1). We further demonstrated that CCA1 directly bound to the promoter of MIR397B and suppressed its expression, forming a miR397b-CKB3-CCA1 circadian regulation feedback circuit. Evolutionary analysis revealed that miR397b is a newly evolved genetic variant in Arabidopsis, and the miR397b targeting mode may have a role in enhancing plant fitness. Our results provide evidence for miRNA-mediated circadian regulation in plants and suggest the existence of a feedback loop to manipulate plant flowering through the regulation of circadian rhythm.

Members of the mitochondrial transcription terminator factor (mTERF) family, originally identified in vertebrate mitochondria, are involved in the termination of organellular transcription. In plants, mTERF proteins are mainly localized in chloroplasts and mitochondria. In Arabidopsis (Arabidopsis thaliana), mTERF8/pTAC15 was identified in the plastid-encoded RNA polymerase (PEP) complex, the major RNA polymerase of chloroplasts. In this work, we demonstrate that mTERF8 is associated with the PEP complex. An mTERF8 knockout line displayed a wild-type–like phenotype under standard growth conditions, but showed impaired efficiency of photosystem II electron flow. Transcription of most chloroplast genes was not substantially affected in the mterf8 mutant; however, the level of the psbJ transcript from the psbEFLJ polycistron was increased. RNA blot analysis showed that a larger transcript accumulates in mterf8 than in the wild type. Thus, abnormal transcription and/or RNA processing occur for the psbEFLJ polycistron. Circular reverse transcription PCR and sequence analysis showed that the psbJ transcript terminates 95 nucleotides downstream of the translation stop codon in the wild type, whereas its termination is aberrant in mterf8. Both electrophoresis mobility shift assays and chloroplast chromatin immunoprecipitation analysis showed that mTERF8 specifically binds to the 3′ terminal region of psbJ. Transcription analysis using the in vitro T7 RNA polymerase system showed that mTERF8 terminates psbJ transcription. Together, these results suggest that mTERF8 is specifically involved in the transcription termination of the chloroplast gene psbJ.

Plants perceive environmental light conditions and optimize their growth and development accordingly by regulating gene activity at multiple levels. Photoreceptors are important for light sensing and downstream gene regulation. Phytochromes, red/far-red light receptors, are believed to regulate light-responsive alternative splicing, but little is known about the underlying mechanism. Alternative splicing is primarily regulated by transacting factors, such as splicing regulators, and by cis-acting elements in precursor mRNA. In the moss Physcomitrella patens, we show that phytochrome 4 (PpPHY4) directly interacts with a splicing regulator, heterogeneous nuclear ribonucleoprotein F1 (PphnRNP-F1), in the nucleus to regulate light-responsive alternative splicing. RNA sequencing analysis revealed that PpPHY4 and PphnRNP-F1 coregulate 70% of intron retention (IR) events in response to red light. A repetitive GAA motif was identified to be an exonic splicing silencer that controls red light-responsive IR. Biochemical studies indicated that PphnRNP-F1 is recruited by the GAA motif to form RNA-protein complexes. Finally, red light elevates PphnRNP-F1 protein levels via PpPHY4, increasing levels of IR. We propose that PpPHY4 and PphnRNP-F1 regulate alternative splicing through an exonic splicing silencer to control splicing machinery activity in response to light.

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member, represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon, whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects, including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhanced miR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further, grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increased accumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover, we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax group H3K4-methyl-transferases in StMSI1-OE. Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.

Plant immunity depends on fast and specific transcriptional reprogramming triggered by the perception of biotic stresses. Numerous studies have been conducted to better understand the response of plants to the generalist herbivore two-spotted spider mite (Tetranychus urticae). However, how plants perceive mites and how this perception is translated into changes in gene expression are largely unknown. In this work, we identified a gene induced in Arabidopsis (Arabidopsis thaliana) upon spider mite attack that encodes a two-domain protein containing predicted lectin and Toll/Interleukin-1 receptor domains. The gene, previously named PP2-A5, belongs to the Phloem Protein2 family. Biotic assays showed that PP2-A5 confers tolerance to T. urticae. Overexpression or knockout of PP2-A5 leads to transcriptional reprogramming that alters the balance of hormone accumulation and corresponding signaling pathways. The nucleocytoplasmic location of this protein supports a direct interaction with regulators of gene transcription, suggesting that the combination of two putative signaling domains in a single protein may provide a novel mechanism for regulating gene expression. Together, our results suggest that PP2-A5 improves the ability to defend against T. urticae by participating in the tight regulation of hormonal cross talk upon mite feeding. Further research is needed to determine the mechanism by which this two-domain protein functions and to clarify its molecular role in signaling following a spider mite attack.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.

Carotenoid biosynthesis is regulated by plant proximity (shade) signals through specific sets of positive and negative transcriptional regulators targeting the first gene of the pathway.

Chromatin modification has gained increased attention for its role in the regulation of plant responses to environmental changes, but the specific mechanisms and molecular players remain elusive. Here, we show that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) mediates genome-wide changes in H3K36 methylation at specific genomic loci functionally relevant to nitrate treatments. Moreover, we show that the specific H3K36 methyltransferase encoded by SDG8 is required for canonical RNA processing, and that RNA isoform switching is more prominent in the sdg8-5 deletion mutant than in the wild type. To demonstrate that SDG8-mediated regulation of RNA isoform expression is functionally relevant, we examined a putative regulatory gene, CONSTANS, CO-like, and TOC1 101 (CCT101), whose nitrogen-responsive isoform-specific RNA expression is mediated by SDG8. We show by functional expression in shoot cells that the different RNA isoforms of CCT101 encode distinct regulatory proteins with different effects on genome-wide transcription. We conclude that SDG8 is involved in plant responses to environmental nitrogen supply, affecting multiple gene regulatory processes including genome-wide histone modification, transcriptional regulation, and RNA processing, and thereby mediating developmental and metabolic processes related to nitrogen use.

The life cycle of many organisms includes a quiescent stage, such as bacterial or fungal spores, insect larvae, or plant seeds. Common to these stages is their low water content and high survivability during harsh conditions. Upon rehydration, organisms need to reactivate metabolism and protein synthesis. Plant seeds contain many mRNAs that are transcribed during seed development. Translation of these mRNAs occurs during early seed germination, even before the requirement of transcription. Therefore, stored mRNAs are postulated to be important for germination. How these mRNAs are stored and protected during long-term storage is unknown. The aim of this study was to investigate how mRNAs are stored in dry seeds and whether they are indeed translated during seed germination. We investigated seed polysome profiles and the mRNAs and protein complexes that are associated with these ribosomes in seeds of the model organism Arabidopsis (Arabidopsis thaliana). We showed that most stored mRNAs are associated with monosomes in dry seeds; therefore, we focus on monosomes in this study. Seed ribosome complexes are associated with mRNA-binding proteins, stress granule (SG), and P-body proteins, which suggests regulated packing of seed mRNAs. Interestingly, approximately 17% of the mRNAs that are specifically associated with monosomes are translationally up-regulated during seed germination. These mRNAs are transcribed during seed maturation, suggesting a role for this developmental stage in determining the translational fate of mRNAs during early germination.

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and – in hypoxic conditions – nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

We present a new simulation model of the reactions in the photosynthetic electron transport chain of C3 species. We show that including recent insights about the regulation of the thylakoid proton motive force, ATP/NADPH balancing mechanisms (cyclic and non-cyclic alternative electron transport), and regulation of Rubisco activity, leads to emergent behaviors that may affect the operation and regulation of photosynthesis under different dynamic environmental conditions. The model was parameterized with experimental results in the literature, with a focus on Arabidopsis thaliana. A dataset was constructed from multiple sources, including measurements of steady-state and dynamic gas exchange, chlorophyll fluorescence and absorbance spectroscopy under different light intensities and CO2. This dataset was used to test predictions of the model under different experimental conditions. Simulations suggested that there are strong interactions between cyclic and non-cyclic alternative electron transport and that an excess capacity for alternative electron transport is required to ensure adequate redox state and lumen pH. Furthermore, the model predicted that, under specific conditions, reduction of ferredoxin by plastoquinol was possible, especially after a rapid increase in light intensity. Further analysis also revealed that the relationship between ATP synthesis and proton motive force was highly regulated by the concentrations of ATP and ADP, and this facilitated an increase in non-photochemical quenching under conditions where metabolism was limiting, such as low CO2 or high light intensity. The model may be used as an in silico platform for future research on the regulation of photosynthetic electron transport.

The development of leaf primordia is subject to light control of meristematic activity. Light regulates the expression of thousands of genes with roles in cell proliferation, organ development, and differentiation of photosynthetic cells. Previous work has highlighted roles for hormone homeostasis and the energy-dependent Target of Rapamycin (TOR) kinase in meristematic activity, yet a picture of how these two regulatory mechanisms depend on light perception and interact with each other has yet to emerge. Their relevance beyond leaf initiation also is unclear. Here, we report the discovery that the dark-arrested meristematic region of Arabidopsis (Arabidopsis thaliana) experiences a local energy deprivation state and confirm previous findings that the PIN1 auxin transporter is diffusely localized in the dark. Light triggers a rapid removal of the starvation state and the establishment of PIN1 polar membrane localization consistent with auxin export, both preceding the induction of cell cycle- and cytoplasmic growth-associated genes. We demonstrate that shoot meristematic activity can occur in the dark through the manipulation of auxin and cytokinin activity as well as through the activation of energy signaling, both targets of photomorphogenesis action, but the organ developmental outcomes differ: while TOR-dependent energy signals alone stimulate cell proliferation, the development of a normal leaf lamina requires photomorphogenesis-like hormonal responses. We further show that energy signaling adjusts the extent of cell cycle activity and growth of young leaves non-cellautonomously to available photosynthates and leads to organs constituted of a greater number of cells developing under higher irradiance. This makes energy signaling perhaps the most important biomass growth determinant under natural, unstressed conditions.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

The ambient light environment controls many aspects of plant development throughout a plant’s life cycle. Such complex control is achieved because a key repressor of light signaling, the Arabidopsis (Arabidopsis thaliana) COP1/SPA E3 ubiquitin ligase causes the degradation of multiple regulators of endogenous developmental pathways. This includes the CONSTANS (CO) transcription factor that is responsible for photoperiodic control of flowering time. There are 16 CO-like proteins whose functions are only partly understood. Here, we show that 14 CO-like (COL) proteins bind CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYTOCHROME A-105 (SPA)1 in vitro. We subsequently focused on COL12 and show that COL12 binds COP1 and SPA proteins in vivo. The COL12 protein is degraded in darkness in a COP1-dependent fashion, indicating that COL12 is a substrate of the COP1/SPA ubiquitin ligase. Overexpression of COL12 causes late flowering specifically in long day conditions by decreasing the expression of FLOWERING LOCUS T. This phenotype is genetically dependent on CO. Consistent with this finding, COL12 physically interacts with CO in vivo, suggesting that COL12 represses flowering by inhibiting CO protein function. We show that COL12 overexpression did not alter CO protein stability. It is therefore likely that COL12 represses the activity of CO rather than CO levels. Overexpression of COL12 also affects plant architecture by increasing the number of rosette branches and reducing inflorescence height. These phenotypes are CO independent. Hence, we suggest that COL12 affects plant development through CO-dependent and CO-independent mechanisms.

Microtubules at the plant cell cortex influence cell shape by patterning the deposition of cell wall materials. The elongated cells of the hypocotyl create a variety of microtubule array patterns with differing degrees of polymer coalignment and orientation to the cell's growth axis. To gain insight into the mechanisms driving array organization, we investigated the underlying microtubule array architecture in light-grown epidermal cells with explicit reference to array pattern. We discovered that all nontransverse patterns share a common underlying array architecture, having a core unimodal peak of coaligned microtubules in a split bipolarized arrangement. The growing microtubule plus ends extend toward the cell's apex and base with a region of antiparallel microtubule overlap at the cell's midzone. This core coalignment continuously shifts between ±30° from the cell's longitudinal growth axis, forming a continuum of longitudinal and oblique arrays. Transverse arrays exhibit the same unimodal core coalignment but form local domains of microtubules polymerizing in the same direction rather than a split bipolarized architecture. Quantitative imaging experiments and analysis of katanin mutants showed that the longitudinal arrays are created from microtubules originating on the outer periclinal cell face, pointing to a cell-directed, rather than self-organizing, mechanism for specifying the major array pattern classes in the hypocotyl cell.

The cytoskeletal Filamenting temperature-sensitive Z (FtsZ) ring is critical for cell division in bacteria and chloroplast division in photosynthetic eukaryotes. While bacterial FtsZ rings are composed of a single FtsZ, except in the basal glaucophytes, chloroplast division involves two heteropolymer-forming FtsZ isoforms: FtsZ1 and FtsZ2 in the green lineage and FtsZA and FtsZB in red algae. FtsZ1 and FtsZB probably arose by duplication of the more ancestral FtsZ2 and FtsZA, respectively. We expressed fluorescent fusions of FtsZ from diverse photosynthetic organisms in a heterologous system to compare their intrinsic assembly and dynamic properties. FtsZ2 and FtsZA filaments were morphologically distinct from FtsZ1 and FtsZB filaments. When coexpressed, FtsZ pairs from plants and algae colocalized, consistent with heteropolymerization. Fluorescence recovery after photobleaching experiments demonstrated that subunit exchange was greater from FtsZ1 and FtsZB filaments than from FtsZ2 and FtsZA filaments and that FtsZ1 and FtsZB increased turnover of FtsZ2 and FtsZA, respectively, from heteropolymers. GTPase activity was essential only for turnover of FtsZ2 and FtsZA filaments. Cyanobacterial and glaucophyte FtsZ properties mostly resembled those of FtsZ2 and FtsZA, though the glaucophyte protein exhibited some hybrid features. Our results demonstrate that the more ancestral FtsZ2 and FtsZA have retained functional attributes of their common FtsZ ancestor, while eukaryotic-specific FtsZ1 and FtsZB acquired new but similar dynamic properties, possibly through convergent evolution. Our findings suggest that the evolution of a second FtsZ that could copolymerize with the more ancestral form to enhance FtsZ-ring dynamics may have been essential for plastid evolution in the green and red photosynthetic lineages.

Chloroplasts develop from undifferentiated proplastids present in meristematic tissue. Thus, chloroplast biogenesis is closely connected to leaf development, which restricts our ability to study the process of chloroplast biogenesis per se. As a consequence, we know relatively little about the regulatory mechanisms behind the establishment of the photosynthetic reactions and how the activities of the two genomes involved are coordinated during chloroplast development. We developed a single cell-based experimental system from Arabidopsis (Arabidopsis thaliana) with high temporal resolution allowing for investigations of the transition from proplastids to functional chloroplasts. Using this unique cell line, we could show that the establishment of photosynthesis is dependent on a regulatory mechanism involving two distinct phases. The first phase is triggered by rapid light-induced changes in gene expression and the metabolome. The second phase is dependent on the activation of the chloroplast and generates massive changes in the nuclear gene expression required for the transition to photosynthetically functional chloroplasts. The second phase also is associated with a spatial transition of the chloroplasts from clusters around the nucleus to the final position at the cell cortex. Thus, the establishment of photosynthesis is a two-phase process with a clear checkpoint associated with the second regulatory phase allowing coordination of the activities of the nuclear and plastid genomes.

The plant hormone abscisic acid (ABA) confers drought tolerance in plants through stomatal closure and regulation of gene expression. The complex consisting of the ABA receptor PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR), type 2C protein phosphatase (PP2C), and SNF1-related protein kinase 2 (SnRK2) has a key role in ABA signaling. Basic helix-loop-helix (bHLH) transcriptional activator ABA-RESPONSIVE KINASE SUBSTRATE1 (AKS1, also known as FBH3) is released from DNA by phosphorylation-induced monomerization in response to ABA in guard cells. Here we reconstituted the release of AKS1 from DNA via the ABA signaling core complex in vitro. We first obtained evidence to confirm that AKS1 is an endogenous substrate for SnRK2s. Phosphorylation of AKS1 and activation of SnRK2 showed the same time course in response to ABA in guard cells. AKS1 was bound to SnRK2.6 in vivo. Three ABA-responsive SnRK2s (SnRK2.2/SRK2D, SnRK2.3/SRK2I, and SnRK2.6/SRK2E/OST1) phosphorylated AKS1 in vitro, and the phosphorylation was eliminated by the triple mutation of SnRK2s in plants. We reconstituted the AKS1 phosphorylation in vitro via the signaling complex containing the ABA receptor PYR1, a PP2C, HYPERSENSITIVE TO ABA1 (HAB1), and a protein kinase, SnRK2.6 in response to ABA We further reconstituted the release of AKS1 from the target gene of POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1) via the complex in response to ABA These results demonstrate that AKS1 provides a link between the signaling complex and ABA-responsive genes and furnish evidence for a minimal signaling mechanism from ABA perception to DNA.

As one of the earliest plant groups to evolve stomata, hornworts are key to understanding the origin and function of stomata. Hornwort stomata are large and scattered on sporangia that grow from their bases and release spores at their tips. We present data from development and immunocytochemistry that identify a role for hornwort stomata that is correlated with sporangial and spore maturation. We measured guard cells across the genera with stomata to assess developmental changes in size and to analyze any correlation with genome size. Stomata form at the base of the sporophyte in the green region, where they develop differential wall thickenings, form a pore, and die. Guard cells collapse inwardly, increase in surface area, and remain perched over a substomatal cavity and network of intercellular spaces that is initially fluid filled. Following pore formation, the sporophyte dries from the outside inwardly and continues to do so after guard cells die and collapse. Spore tetrads develop in spore mother cell walls within a mucilaginous matrix, both of which progressively dry before sporophyte dehiscence. A lack of correlation between guard cell size and DNA content, lack of arabinans in cell walls, and perpetually open pores are consistent with the inactivity of hornwort stomata. Stomata are expendable in hornworts, as they have been lost twice in derived taxa. Guard cells and epidermal cells of hornworts show striking similarities with the earliest plant fossils. Our findings identify an architecture and fate of stomata in hornworts that is ancient and common to plants without sporophytic leaves.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1’s impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Rice (Oryza sativa) OsNLA1 has been proposed to play a crucial role in regulating phosphate (Pi) acquisition in roots, similar to that of Arabidopsis (Arabidopsis thaliana) AtNLA. However, unlike AtNLA, OsNLA1 is not a target of miR827, a Pi starvation-induced microRNA. It is, therefore, of interest to know whether the expression of OsNLA1 depends on Pi supply and how it is regulated. In this study, we provide evidence that OsNLA1 controls Pi acquisition by directing the degradation of several OsPHT1 Pi transporters (i.e. OsPT1/2/4/7/8/12). We further show that OsNLA1 has an additional function in reproduction and uncover the mechanism of its expression regulation. Analysis of mRNA levels, promoter-GUS activity, and protoplast transient expression showed that the expression of OsNLA1.1, the most abundant transcript variant, is up-regulated in response to increasing Pi supply. The OsNLA1 promoter region was found to contain an upstream open reading frame that is required for Pi-responsive expression regulation. OsNLA1 promoter activity was observed in roots, ligules, leaves, sheaths, pollen grains, and surrounding the vascular tissues of anthers, suggesting that OsNLA1 is important throughout the development of rice. Disruption of OsNLA1 resulted in increased Pi uptake from roots as well as impaired pollen development and reduced grain production. In summary, our study reveals that Pi-induced OsNLA1 expression regulated by a unique mechanism functions in Pi acquisition, Pi translocation, and reproductive success.

Circular RNAs (circRNAs) play roles in various biological processes, but their functions in the rice (Oryza sativa) response to Magnaporthe oryzae remain elusive. Here, we demonstrate that circRNAs are involved in the rice-M. oryzae interaction using comparative circRNA-sequencing and transgenic approaches. We identified 2932 high-confidence circRNAs from young leaves of the blast-resistant accession International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake (IR25) and the blast-susceptible accession Lijiangxin Tuan Heigu (LTH) under M. oryzae-infected or uninfected conditions; 636 were detected specifically upon M. oryzae infection. The circRNAs in IR25 were significantly more diverse than those in LTH, especially under M. oryzae infection. Particularly, the number of circRNAs generated per parent gene was much higher in IR25 than in LTH and increased in IR25 but decreased in LTH upon M. oryzae infection. The higher diversity of circRNAs in IR25 was further associated with more frequent 3′ and 5′ alternative back-splicing and usage of complex splice sites. Moreover, a subset of circRNAs was differentially responsive to M. oryzae in IR25 and LTH. We further confirmed that circR5g05160 promotes rice immunity against M. oryzae. Therefore, our data indicate that circRNA diversity is associated with different responses to M. oryzae infection in rice and provide a starting point to investigate a new layer of regulation in the rice-M. oryzae interaction.

N(6)-methyladenosine (m(6)A) is the most abundant modification of eukaryotic mRNA. Although m(6)A has been demonstrated to affect almost all aspects of RNA metabolism, its global contribution to the post-transcriptional balancing of translational efficiency remains elusive in plants. In this study, we performed a parallel analysis of the transcriptome-wide mRNA m(6)A distribution and polysome profiling in two maize (Zea mays) inbred lines to assess the global correlation of m(6)A modification with translational status. m(6)A sites are widely distributed in thousands of protein-coding genes, confined to a consensus motif and primarily enriched in the 3′ untranslated regions, and highly coordinated with alternative polyadenylation usage, suggesting a role of m(6)A modification in regulating alternative polyadenylation site choice. More importantly, we identified that the m(6)A modification shows multifaceted correlations with the translational status depending on its strength and genic location. Moreover, we observed a substantial intraspecies variation in m(6)A modification, and this natural variation was shown to be partly driven by gene-specific expression and alternative splicing. Together, these findings provide an invaluable resource for ascertaining transcripts that are subject to m(6)A modification in maize and pave the way to a better understanding of natural m(6)A variation in mediating gene expression regulation.

The quiescent center (QC) of the Arabidopsis (Arabidopsis thaliana) root meristem acts as an organizer that promotes stem cell fate in adjacent cells and patterns the surrounding stem cell niche. The stem cells distal from the QC, the columella stem cells (CSCs), are maintained in an undifferentiated state by the QC-expressed transcription factor WUSCHEL RELATED HOMEOBOX5 (WOX5) and give rise to the columella cells. Differentiated columella cells provide a feedback signal via secretion of the peptide CLAVATA3/ESR-RELATED40 (CLE40), which acts through the receptor kinases ARABIDOPSIS CRINKLY4 (ACR4) and CLAVATA1 (CLV1) to control WOX5 expression. Previously, it was proposed that WOX5 protein movement from the QC into CSCs is required for CSC maintenance, and that the CLE40/CLV1/ACR4 signaling module restricts WOX5 mobility or function. Here, these assumptions were tested by exploring the function of CLE40/CLV1/ACR4 in CSC maintenance. However, no role for CLE40/CLV1/ACR4 in constricting the mobility of WOX5 or other fluorescent test proteins was identified. Furthermore, in contrast to previous observations, WOX5 mobility was not required to inhibit CSC differentiation. We propose that WOX5 acts mainly in the QC, where other short-range signals are generated that not only inhibit differentiation but also promote stem cell division in adjacent cells. Therefore, the main function of columella-derived CLE40 signal is to position the QC at a defined distance from the root tip by repressing QC-specific gene expression via the ACR4/CLV1 receptors in the distal domain and promoting WOX5 expression via the CLV2 receptor in the proximal meristem.

The turnover of abscisic acid (ABA) signaling core components modulates the plant’s response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.

Small RNAs (sRNAs) regulate gene expression, play important roles in epigenetic pathways, and have been hypothesised to contribute to hybrid vigor in plants. Prior investigations have provided valuable insights into associations between sRNAs and heterosis, often using a single hybrid genotype or tissue. However, our understanding of the role of sRNAs and their potential value to plant breeding are limited by an incomplete picture of sRNA variation between diverse genotypes and development stages. Here, we provide a deep exploration of sRNA variation and inheritance among a panel of 108 maize samples spanning five tissues from eight inbred parents and 12 hybrid genotypes, covering a spectrum of heterotic groups, genetic variation, and levels of heterosis for various traits. We document substantial developmental and genotypic influences on sRNA expression, with varying patterns for 21-nt, 22-nt and 24-nt sRNAs. We provide a detailed view of the distribution of sRNAs in the maize genome, revealing a complex make-up that also shows developmental plasticity, particularly for 22-nt sRNAs. sRNAs exhibited substantially more variation between inbreds as compared to observed variation for gene expression. In hybrids, we identify locus-specific examples of non-additive inheritance, mostly characterised as partial or complete dominance, but rarely outside the parental range. However, the global abundance of 21-nt, 22-nt and 24-nt sRNAs varies very little between inbreds and hybrids, suggesting that hybridization affects sRNA expression principally at specific loci rather than on a global scale. This study provides a valuable resource for understanding the potential role of sRNAs in hybrid vigor.One-sentence summaryCharacterizing the roles of development and genotype in driving expression variation of different small RNA populations in maize inbreds and their F1 hybrids.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis (Arabidopsis thaliana) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean (Glycine max) and cucumber (Cucumis sativus). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species.

Histone deacetylase (HDAC) proteins participate in diverse and tissue-specific developmental processes by forming various corepressor complexes with different regulatory subunits. An important HDAC machinery hub, the Histone Deacetylase Complex1 (HDC1) protein, participates in multiple protein-protein interactions and regulates organ size in plants. However, the mechanistic basis for this regulation remains unclear. Here, we identified a cucumber (Cucumis sativus) short-fruit mutant (sf2) with a phenotype that includes repressed cell proliferation. SF2 encodes an HDC1 homolog, and its expression is enriched in meristematic tissues, consistent with a role in regulating cell proliferation through the HDAC complex. A weak sf2 allele impairs HDAC targeting to chromatin, resulting in elevated levels of histone acetylation. Genome-wide mapping revealed that SF2 directly targets and promotes histone deacetylation associated with key genes involved in multiple phytohormone pathways and cell cycle regulation, by either directly repressing or activating their expression. We further show that SF2 controls fruit cell proliferation through targeting the biosynthesis and metabolism of cytokinin and polyamines. Our findings reveal a complex regulatory network of fruit cell proliferation mediated by HDC1 and elucidate patterns of HDC1-mediated regulation of gene expression.

The mechanisms by which plants activate and enhance defense responses have been well studied; however, the regulatory mechanisms that allow plants to avoid excessive defense responses are poorly understood. Here, we identified a group D mitogen-activated protein kinase (MAPK) gene from rice (Oryza sativa), OsMAPK20-5, whose expression was rapidly induced by infestation of gravid female adults of a destructive rice pest, brown planthopper (BPH, Nilaparvata lugens), but not by BPH nymphs. Expression silencing of OsMAPK20-5 (irMAPK) increased the accumulation of ethylene and nitric oxide (NO) after gravid female BPH infestation, and thereby increased rice plant resistance to BPH adults and oviposited eggs. However, when exposed to high densities of gravid BPH females, irMAPK plants wilted earlier than wild-type plants, which could be attributed to the hyperaccumulation of ethylene and NO in irMAPK plants. Interestingly, when released into the field, irMAPK plants displayed broad resistance to BPH and white-backed planthopper (Sogatella furcifera), the two most destructive pests of rice, and produced higher yield. Taken together, our study shows that although OsMAPK20-5 can reduce the resistance of rice plants to planthoppers, it also enables rice plants to control excessive defense responses and thereby prevents defense-response–related autotoxicity.

RNA N(6)-methyladenosine (m(6)A) modification is the most abundant form of RNA epigenetic modification in eukaryotes. Given that m(6)A evolution is associated with the selective constraints of nucleotide sequences in mammalian genomes, we hypothesize that m(6)A evolution can be linked, at least in part, to genomic duplication events in complex polyploid plant genomes. To test this hypothesis, we presented the maize (Zea mays) m(6)A modification landscape in a transcriptome-wide manner and identified 11,968 m(6)A peaks carried by 5,893 and 3,811 genes from two subgenomes (maize1 and maize2, respectively). Each of these subgenomes covered over 2,200 duplicate genes. Within these duplicate genes, those carrying m(6)A peaks exhibited significant differences in retention rate. This biased subgenome fractionation of m(6)A-methylated genes is associated with multiple sequence features and is influenced by asymmetric evolutionary rates. We also characterized the coevolutionary patterns of m(6)A-methylated genes and transposable elements, which can be mediated by whole genome duplication and tandem duplication. We revealed the evolutionary conservation and divergence of duplicated m(6)A functional factors and the potential role of m(6)A modification in maize responses to drought stress. This study highlights complex interplays between m(6)A modification and gene duplication, providing a reference for understanding the mechanisms underlying m(6)A evolution mediated by genome duplication events.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.

The corn leaf aphid (CLA; Rhopalosiphum maidis) is a phloem sap-sucking insect that attacks many cereal crops, including maize (Zea mays). We previously showed that the maize inbred line Mp708, which was developed by classical plant breeding, provides enhanced resistance to CLA. Here, using electrophysiological monitoring of aphid feeding behavior, we demonstrate that Mp708 provides phloem-mediated resistance to CLA. Furthermore, feeding by CLA on Mp708 plants enhanced callose deposition, a potential defense mechanism utilized by plants to limit aphid feeding and subsequent colonization. In maize, benzoxazinoids (BX) or BX-derived metabolites contribute to enhanced callose deposition by providing heightened resistance to CLA. However, BX and BX-derived metabolites were not significantly altered in CLA-infested Mp708 plants, indicating BX-independent defense against CLA. Evidence presented here suggests that the constitutively higher levels of 12-oxo-phytodienoic acid (OPDA) in Mp708 plants contributed to enhanced callose accumulation and heightened CLA resistance. OPDA enhanced the expression of ethylene biosynthesis and receptor genes, and the synergistic interactions of OPDA and CLA feeding significantly induced the expression of the transcripts encoding Maize insect resistance1-Cysteine Protease, a key defensive protein against insect pests, in Mp708 plants. Furthermore, exogenous application of OPDA on maize jasmonic acid-deficient plants caused enhanced callose accumulation and heightened resistance to CLA, suggesting that the OPDA-mediated resistance to CLA is independent of the jasmonic acid pathway. We further demonstrate that the signaling function of OPDA, rather than a direct toxic effect, contributes to enhanced CLA resistance in Mp708.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae. Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae. Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

In plants, hypoxia can be induced by submergence, and the lack of oxygen impairs mitochondrial respiration, thus affecting the plant’s energy status. Hypoxia has major effects on gene expression; these changes induce key responses that help meet the needs of the stressed plant. However, little is known about the possible role of RNA signaling in the regulation of gene expression under limited oxygen availability. Here, we report the contribution of ARGONAUTE1 (AGO1) to hypoxia-induced gene regulation in Arabidopsis (Arabidopsis thaliana). Submergence induced changes in levels of the microRNAs miR2936 and miR398, but this had no obvious effects on their putative target mRNA levels. However, we found that ago1-27 plants are intolerant to submergence and transcriptome analysis identified genes whose regulation requires functional AGO1. Analysis of mutants affected in various branches of RNA signaling highlighted the convergence of AGO1 signaling with the AGO4-dependent RNA-directed DNA methylation (RdDM) pathway. AGO4-dependent RdDM represses the expression of HOMOLOG OF RPW8 4 (HR4) and alters its response to submergence. Remarkably, methylation of the second exon of HR4 is not only reduced in ago4-1 but also in plants overexpressing a constitutively stable version of the oxygen sensor RELATED TO APETALA2 12 (RAP2.12), indicating convergence of oxygen signaling with epigenetic regulation of gene expression. Therefore, our results identify a role for AGO1 and AGO4 RNA-silencing pathways in low-oxygen signaling in Arabidopsis.

The basidiomycete Ustilago maydis causes smut disease in maize (Zea mays) by infecting all plant aerial tissues. The infection causes leaf chlorosis and stimulates the plant to produce nutrient-rich niches (i.e. tumors), where the fungus can proliferate and complete its life cycle. Previous studies have recorded high accumulation of soluble sugars and starch within these tumors. Using interdisciplinary approaches, we found that the sugar accumulation within tumors coincided with the differential expression of plant sugars will eventually be exported transporters and the proton/sucrose symporter Sucrose Transporter1. To accumulate plant sugars, the fungus deploys its own set of sugar transporters, generating a sugar gradient within the fungal cytosol, recorded by expressing a cytosolic glucose (Glc) Förster resonance energy transfer sensor. Our measurements indicated likely elevated Glc levels in hyphal tips during infection. Growing infected plants under dark conditions led to decreased plant sugar levels and loss of the fungal tip Glc gradient, supporting a tight link between fungal sugar acquisition and host supplies. Finally, the fungal infection causes a strong imbalance in plant sugar distribution, ultimately impacting seed set and yield.

Unraveling and exploiting mechanisms of disease resistance in cereal crops is currently limited by their large repeat-rich genomes and the lack of genetic recombination or cultivar (cv)-specific sequence information. We cloned the first leaf rust resistance gene Rph1 (Rph1.a) from cultivated barley (Hordeum vulgare) using “MutChromSeq,” a recently developed molecular genomics tool for the rapid cloning of genes in plants. Marker-trait association in the CI 9214/Stirling doubled haploid population mapped Rph1 to the short arm of chromosome 2H in a physical region of 1.3 megabases relative to the barley cv Morex reference assembly. A sodium azide mutant population in cv Sudan was generated and 10 mutants were confirmed by progeny-testing. Flow-sorted 2H chromosomes from Sudan (wild type) and six of the mutants were sequenced and compared to identify candidate genes for the Rph1 locus. MutChromSeq identified a single gene candidate encoding a coiled-coil nucleotide binding site Leucine-rich repeat (NLR) receptor protein that was altered in three different mutants. Further Sanger sequencing confirmed all three mutations and identified an additional two independent mutations within the same candidate gene. Phylogenetic analysis determined that Rph1 clustered separately from all previously cloned NLRs from the Triticeae and displayed highest sequence similarity (89%) with a homolog of the Arabidopsis (Arabidopsis thaliana) disease resistance protein 1 protein in Triticum urartu. In this study we determined the molecular basis for Rph1-mediated resistance in cultivated barley enabling varietal improvement through diagnostic marker design, gene editing, and gene stacking technologies.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: KX262844.1 (RsUGT72B14, R. sachalinensis); AY547304.1 (RsUGT73B6. R. sachalinensis); NP_418458.1 (malE, E. coli); NP_415214.1 (pgm, E. coli); NP_415752.1 (galU, E. coli); NM_001180688.3 (aro10, S. cerevisiae), and NP_417484.1 (yqhD, E. coli)., Edible berries are considered to be among nature's treasure chests as they contain a large number of (poly)phenols with potentially health-promoting properties. However, as berries contain complex (poly)phenol mixtures, it is challenging to associate any interesting pharmacological activity with a single compound. Thus, identification of pharmacologically interesting phenols requires systematic analyses of berry extracts. Here, raspberry (Rubus idaeus, var. Prestige) extracts were systematically analyzed to identify bioactive compounds against pathological processes of neurodegenerative diseases. Berry extracts were tested on different Saccharomyces cerevisiae strains expressing disease proteins associated with Alzheimer's, Parkinson's, or Huntington's disease or amyotrophic lateral sclerosis. After identifying bioactivity against Huntington's disease, the extract was fractionated and the obtained fractions were tested in the yeast model, which revealed that salidroside, a glycosylated phenol, displayed significant bioactivity. Subsequently, a metabolic route to salidroside was reconstructed in S. cerevisiae and Corynebacterium glutamicum. The best-performing S. cerevisiae strain was capable of producing 2.1 mM (640 mg L-1) salidroside from glucose in shake flasks, whereas an engineered C. glutamicum strain could efficiently convert the precursor tyrosol to salidroside, accumulating up to 32 mM (9,700 mg L-1) salidroside in bioreactor cultivations (yield: 0.81 mol mol-1). Targeted yeast assays verified that salidroside produced by both organisms has the same positive effects as salidroside of natural origin., We express our gratitude to Dr. Rute Neves (Chr. Hansen A/S, Denmark), Prof. Dr. Jochen Förster and Dr. Alexey Dudnik (The Nova Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark), who coordinated the BacHBerry project. We also thank Prof. Dr. Ian Macreadie (Centre of Excellence for Alzheimer’s Disease Research & Care, School of Exercise, Biomedical & Health Sciences, Edith Cowan University, WA, Australia) for providing p416_GPD-GFP_AB42; Prof. Dr. Tiago Outeiro (University Medical Center Gottingen, Department of Neurodegeneration and Restorative Research, Germany) for providing W303-1A_Syn and W303-1A TU; Prof. Dr. Flaviano Giorgini (Department of Genetics and Genome Biology, University of Leicester, UK) for providing p425GAL1_HTT103Q; and Prof. Dr. Greg Petsko (Department of Biochemistry and Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, U.S.) for providing pYES_GAL1pr-FUS-GFP and pYES_CT., info:eu-repo/semantics/publishedVersion

The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2 To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1 Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.

The plant hormone gibberellic acid (GA) controls many physiological processes, including cell differentiation, cell elongation, seed germination, and response to abiotic stress. In this study, we report that exogenous treatment of flowering Arabidopsis (Arabidopsis thaliana) plants with GA specifically affects the process of male meiotic cytokinesis leading to meiotic restitution and the production of diploid (2n) pollen grains. Similar defects in meiotic cell division and reproductive ploidy stability occur in Arabidopsis plants depleted of RGA and GAI, two members of the DELLA family that function as suppressor of GA signaling. Cytological analysis of the double rga-24 gai-t6 mutant revealed that defects in male meiotic cytokinesis are not caused by alterations in meiosis I (MI or meiosis II (MII) chromosome dynamics, but instead result from aberrations in the spatial organization of the phragmoplast-like radial microtubule arrays (RMAs) at the end of meiosis II. In line with a role for GA in the genetic regulation of the male reproductive system, we additionally show that DELLA downstream targets MYB33 and MYB65 are redundantly required for functional RMA biosynthesis and male meiotic cytokinesis. By analyzing the expression of pRGA::GFP-RGA in the wild-type Landsberg erecta background, we demonstrate that the GFP-RGA protein is specifically expressed in the anther cell layers surrounding the meiocytes and microspores, suggesting that appropriate GA signaling in the somatic anther tissue is critical for male meiotic cell wall formation and thus plays an important role in consolidating the male gametophytic ploidy consistency.

Peroxisomes are highly dynamic and metabolically active organelles that play an important role in cellular functions, including reactive oxygen species (ROS) metabolism. Peroxisomal dynamics, such as the proliferation, movement, and production of dynamic extensions called peroxules, have been associated with ROS in plant cells. However, the function and regulation of peroxules are largely unknown. Using confocal microscopy, we have shown that treatment of Arabidopsis leaves with the heavy metal cadmium produces time course-dependent changes in peroxisomal dynamics, starting with peroxule formation, followed by peroxisome proliferation, and finally returning to the normal morphology and number. These changes during Cd treatment were regulated by NADPH oxidase (C and F)-related ROS production. Peroxule formation is a general response to stimuli such as arsenic and is regulated by peroxin 11a (PEX11a), as Arabidopsis pex11a RNAi lines are unable to produce peroxules under stress conditions. The pex11a line showed higher levels of lipid peroxidation content and lower expression of genes involved in antioxidative defenses and signaling, suggesting that these extensions are involved in regulating ROS accumulation and ROS-dependent gene expression in response to stress. Our results demonstrate that PEX11a and peroxule formation play a key role in regulating stress perception and fast cell responses to environmental cues.

Hydrogen peroxide (H2O2) is a stable component of reactive oxygen species, and its production in plants represents the successful recognition of pathogen infection and pathogen-associated molecular patterns (PAMPs). This production of H2O2 is typically apoplastic but is subsequently associated with intracellular immunity pathways that regulate disease resistance, such as systemic acquired resistance and PAMP-triggered immunity. Here, we elucidate that an Arabidopsis (Arabidopsis thaliana) aquaporin (i.e. the plasma membrane intrinsic protein AtPIP1;4) acts to close the cytological distance between H2O2 production and functional performance. Expression of the AtPIP1;4 gene in plant leaves is inducible by a bacterial pathogen, and the expression accompanies H2O2 accumulation in the cytoplasm. Under de novo expression conditions, AtPIP1;4 is able to mediate the translocation of externally applied H2O2 into the cytoplasm of yeast (Saccharomyces cerevisiae) cells. In plant cells treated with H2O2, AtPIP1;4 functions as an effective facilitator of H2O2 transport across plasma membranes and mediates the translocation of externally applied H2O2 from the apoplast to the cytoplasm. The H2O2-transport role of AtPIP1;4 is essentially required for the cytoplasmic import of apoplastic H2O2 induced by the bacterial pathogen and two typical PAMPs in the absence of induced production of intracellular H2O2 As a consequence, cytoplasmic H2O2 quantities increase substantially while systemic acquired resistance and PAMP-triggered immunity are activated to repress the bacterial pathogenicity. By contrast, loss-of-function mutation at the AtPIP1;4 gene locus not only nullifies the cytoplasmic import of pathogen- and PAMP-induced apoplastic H2O2 but also cancels the subsequent immune responses, suggesting a pivotal role of AtPIP1;4 in apocytoplastic signal transduction in immunity pathways.

Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes. PYGG expression in Arabidopsis resulted in orange-colored cotyledons, which are not observed if PSY or GGPS11 are overexpressed individually. This suggests insufficient GGPP substrate availability for chlorophyll biosynthesis achieved through GGPP flux redirection to carotenogenesis. Similarly, carotenoid levels in PYGG-expressing callus exceeded that in PSY- or GGPS11-overexpression lines. The PYGG chimeric protein may assist in provitamin A biofortification of edible plant parts. Moreover, other GGPS fusions may be used to redirect metabolic flux into the synthesis of other isoprenoids of nutritional and industrial interest.

Defects in the nuclear lamina of animal cell nuclei have dramatic effects on nuclear structure and gene expression as well as diverse physiological manifestations. We report that deficiencies in CROWDED NUCLEI (CRWN), which are candidate nuclear lamina proteins in Arabidopsis (Arabidopsis thaliana), trigger widespread changes in transcript levels and whole-plant phenotypes, including dwarfing and spontaneous cell death lesions. These phenotypes are caused in part by ectopic induction of plant defense responses via the salicylic acid pathway. Loss of CRWN proteins induces the expression of the salicylic acid biosynthetic gene ISOCHORISMATE SYNTHASE1, which leads to spontaneous defense responses in crwn1 crwn2 and crwn1 crwn4 mutants, which are deficient in two of the four CRWN paralogs. The symptoms of ectopic defense response, including pathogenesis marker gene expression and cell death, increase in older crwn double mutants. These age-dependent effects are postulated to reflect an increase in nuclear dysfunction or damage over time, a phenomenon reminiscent of aging effects seen in animal nuclei and in some human laminopathy patients.

Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells and are known to contribute to fungal pathogen resistance. Syntaxin of Plants121 (SYP121), a Qa-SNARE, has been reported to function in nonhost resistance in Arabidopsis (Arabidopsis thaliana). However, the functions of SYP121 in host resistance to rice blast are largely unknown. Here, we report that the rice SYP121 protein, OsSYP121, accumulates at fungal penetration sites and mediates host resistance to rice blast. OsSYP121 is plasma membrane localized and its expression was obviously induced by the rice blast in both the blast-resistant rice landrace Heikezijing and the blast-susceptible landrace Suyunuo (Su). Overexpression of OsSYP121 in Su resulted in enhanced resistance to blast. Knockdown of OsSYP121 expression in Su resulted in a more susceptible phenotype. However, knockdown of OsSYP121 expression in the resistant landrace Heikezijing resulted in susceptibility to the blast fungus. The P(OsSYP121)::GFP-OsSYP121 accumulated at rice blast penetration sites in transgenic rice, as observed by confocal microscopy. Yeast two-hybrid results showed that OsSYP121 can interact with OsSNAP32 (Synaptosome-associated protein of 32 kD) and Vesicle-associated membrane protein714/724. The interaction between OsSYP121 and OsSNAP32 may contribute to host resistance to rice blast. Our study reveals that OsSYP121 plays an important role in rice blast resistance as it is a key component in vesicle trafficking.

Jasmonates regulate plant defense and development. In Arabidopsis (Arabidopsis thaliana), JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1/VQ22) is a repressor of jasmonate-mediated defense responses and is degraded through the ubiquitin-26S proteasome system after herbivory. We found that JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), a RING-type E3 ubiquitin ligase, interacted with JAV1. JUL1 interacted with JAV1 in the nucleus to ubiquitinate JAV1, leading to proteasomal degradation of JAV1. The transcript levels of JUL1 and JAV1 were coordinately and positively regulated by the CORONATINE INSENSITIVE1-dependent signaling pathway in the jasmonate signaling network, but in a manner that was not dependent on CORONATINE INSENSITIVE1-mediated signaling upon herbivory by Spodoptera litura. Gain or loss of function of JUL1 modulated the expression levels of the defensin gene PDF1.2 in leaves, conferring on the plants various defense properties against the generalist herbivore S. litura. Because neither the JUL1 mutant nor overexpression lines showed any obvious developmental defects, we concluded that the JAV1/JUL1 system functions as a specific coordinator of reprogramming of plant defense responses. Altogether, our findings offer insight into the mechanisms by which the JAV1/JUL1 system acts specifically to coordinate plant defense responses without interfering with plant development or growth.