Although photosynthesis is essential to sustain life on Earth, not all plants use sunlight to synthesize nutrients from carbon dioxide and water. Holoparasitic plants, which are important in agricultural and natural ecosystems, are dependent on other plants for nutrients. Phytohormones are crucial in holoparasitic plant-host interactions, from seed germination to senescence, not only because they act as growth and developmental regulators, but also because of their central role in the regulation of host photosynthesis and source-sink relations between the host and the holoparasitic plant. Here, we compile and discuss current knowledge on the impact and ecophysiology of holoparasitic plants (such as the broomrapes Orobanche sp. and Phelipanche sp.) that infest economically important dicotyledonous crops in Mediterranean agroecosystems (legumes [Fabaceae], sunflowers [Helianthus sp.], or tomato [Solanum lycopersicum] plants). We also highlight the role of holoparasitic plant-host interactions (such as those between Cytinus hypocistis and various shrubs of the genus Cistus) in shaping natural Mediterranean ecosystems. The roles of phytohormones in controlling plant-host interactions, abiotic factors in parasitism, and the biological significance of natural seed banks and how dormancy and germination are regulated, will all be discussed. Holoparasitic plants are unique organisms; improving our understanding of their interaction with hosts as study models will help us to better manage parasitic plants, both in agricultural and natural ecosystems.

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Comment in Plant Physiol. 2021 Apr 23;185(4):1479-1480.

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

The lifestyle of parasitic plants is associated with peculiar morphological, genetic, and physiological adaptations that existing online plant-specific resources fail to adequately represent. Here, we introduce the Web Application for the Research of Parasitic Plants (WARPP) as an online resource dedicated to advancing research and development of parasitic plant biology. WARPP is a framework to facilitate international efforts by providing a central hub of curated evolutionary, ecological, and genetic data. The first version of WARPP provides a community hub for researchers to test this web application, for which curated data revolving around the economically important Broomrape family (Orobanchaceae) is readily accessible. The initial set of WARPP online tools includes a genome browser that centralizes genomic information for sequenced parasitic plant genomes, an orthogroup summary detailing the presence and absence of orthologous genes in parasites compared with nonparasitic plants, and an ancestral trait explorer showing the evolution of life-history preferences along phylogenies. WARPP represents a project under active development and relies on the scientific community to populate the web app's database and further the development of new analysis tools. The first version of WARPP can be securely accessed at https://parasiticplants.app. The source code is licensed under GNU GPLv2 and is available at https://github.com/wickeLab/WARPP.

Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

Aquaporins such as the plasma membrane intrinsic proteins (PIPs) allow water to move through cell membranes and are vital for stomatal movement in plants. Despite their importance, the dynamic changes in aquaporins during water efflux and influx have not been directly observed in real time in vivo. Here, to determine which factors regulate these changes during the bidirectional translocation of water, we examined aquaporin dynamics during the stomatal immune response to the bacterial flagellin-derived peptide flg22. The Arabidopsis (Arabidopsis thaliana) aquaporin mutant pip2;1 showed defects in the flg22-induced stomatal response. Variable-angle total internal reflection fluorescence microscopy revealed that the movement dynamics and dwell times of AQ6]GFP-AtPIP2;1 in guard cells and subsidiary cells exhibited cell type-specific dependencies on flg22. The cytoskeleton, rather than the cell wall, was the major factor regulating AtPIP2;1 dynamics, although both the cytoskeleton and cell wall might form bounded domains that restrict the diffusion of AtPIP2;1 in guard cells and subsidiary cells. Finally, our analysis revealed the different roles of cortical actin and microtubules in regulating AtPIP2;1 dynamics in guard cells, as well as subsidiary cells, under various conditions. Our observations shed light on the heterogeneous mechanisms that regulate membrane protein dynamics in plants in response to pathogens.

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth, and non-photochemical quenching (NPQ) of excess light energy prevents the generation of reactive oxygen species and maintains efficient photosynthesis under high light. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and ultra-violet (UV) light photoreceptors, but the biological significance of photoprotection activation by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared with that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that C. reinhardtii senses relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis (Arabidopsis thaliana) preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that C. reinhardtii developed a UV response distinct from that of land plants.

Meiotic recombination (MR) drives novel combinations of alleles and contributes to genomic diversity in eukaryotes. In this study, we showed that heat stress (36°C-38°C) over the fertile threshold fully abolished crossover formation in Arabidopsis (Arabidopsis thaliana). Cytological and genetic studies in wild-type plants and syn1 and rad51 mutants suggested that heat stress reduces generation of SPO11-dependent double-strand breaks (DSBs). In support, the abundance of recombinase DMC1, which is required for MR-specific DSB repair, was significantly reduced under heat stress. In addition, high temperatures induced disassembly and/or instability of the ASY4- but not the SYN1-mediated chromosome axis. At the same time, the ASY1-associated lateral element of the synaptonemal complex (SC) was partially affected, while the ZYP1-dependent central element of SC was disrupted, indicating that heat stress impairs SC formation. Moreover, expression of genes involved in DSB formation; e.g. SPO11-1, PRD1, 2, and 3 was not impacted; however, recombinase RAD51 and chromosome axis factors ASY3 and ASY4 were significantly downregulated under heat stress. Taken together, these findings revealed that heat stress inhibits MR via compromised DSB formation and homolog synapsis, which are possible downstream effects of the impacted chromosome axis. Our study thus provides evidence shedding light on how increasing environmental temperature influences MR in Arabidopsis.

Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine (HIS). Here, we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases (LOXs), the fatty acid hydroperoxides. LOX activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a LOX inhibitor, but not by HIS, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the LOX-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of LOX substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of LOXs showed downregulation of LOX protein and activity. The lines with downregulated LOX displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared with wild type. The results show that LOXs can serve as an enzymatic source of "dark" 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.

Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.

Comment in Plant Physiol. 2021 Apr 23;185(4):1473-1475.

The R2R3 transcription factor MdMYB88 has previously been reported to function in biotic and abiotic stress responses. Here, we identify BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (MdBES1), a vital component of brassinosteroid (BR) signaling in apple (Malus × domestica) that directly binds to the MdMYB88 promoter, regulating the expression of MdMYB88 in a dynamic and multifaceted mode. MdBES1 positively regulated expression of MdMYB88 under cold stress and pathogen attack, but negatively regulated its expression under control and drought conditions. Consistently, MdBES1 was a positive regulator for cold tolerance and disease resistance in apple, but a negative regulator for drought tolerance. In addition, MdMYB88 participated in BR biosynthesis by directly regulating the BR biosynthetic genes DE ETIOLATED 2 (MdDET2), DWARF 4 (MdDWF4), and BRASSINOSTEROID 6 OXIDASE 2 (MdBR6OX2). Applying exogenous BR partially rescued the erect leaf and dwarf phenotypes, as well as defects in stress tolerance in MdMYB88/124 RNAi plants. Moreover, knockdown of MdMYB88 in MdBES1 overexpression (OE) plants decreased resistance to a pathogen and C-REPEAT BINDING FACTOR1 expression, whereas overexpressing MdMYB88 in MdBES1 OE plants increased expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 （MdSPL3） and BR biosynthetic genes, suggesting that MdMYB88 contributes to MdBES1 function during BR biosynthesis and the stress response. Taken together, our results reveal multifaceted regulation of MdBES1 on MdMYB88 in BR biosynthesis and stress tolerance.

Comment in Plant Physiol. 2021 Apr 23;185(4):1476-1478.

Photosynthesis is not only essential for plants, but it also sustains life on Earth. Phytohormones play crucial roles in developmental processes, from organ initiation to senescence, due to their role as growth and developmental regulators, as well as their central role in the regulation of photosynthesis. Furthermore, phytohormones play a major role in photoprotection of the photosynthetic apparatus under stress conditions. Here, in addition to discussing our current knowledge on the role of the phytohormones auxin, cytokinins, gibberellins, and strigolactones in promoting photosynthesis, we will also highlight the role of abscisic acid beyond stomatal closure in modulating photosynthesis and photoprotection under various stress conditions through crosstalk with ethylene, salicylates, jasmonates, and brassinosteroids. Furthermore, the role of phytohormones in controlling the production and scavenging of photosynthesis-derived reactive oxygen species, the duration and extent of photo-oxidative stress and redox signaling under stress conditions will be discussed in detail. Hormones have a significant impact on the regulation of photosynthetic processes in plants under both optimal and stress conditions, with hormonal interactions, complementation, and crosstalk being important in the spatiotemporal and integrative regulation of photosynthetic processes during organ development at the whole-plant level.

Invasive holoparasitic plants of the genus Cuscuta (dodder) threaten African ecosystems due to their rapid spread and attack on various host plant species. Most Cuscuta species cannot photosynthesize and hence rely on host plants for nourishment. After attachment through a peg-like organ called a haustorium, the parasites deprive hosts of water and nutrients, which negatively affects host growth and development. Despite their rapid spread in Africa, dodders have attracted limited research attention, although data on their taxonomy, host range, and epidemiology are critical for their management. Here, we combine taxonomy and phylogenetics to reveal the presence of field dodder (Cuscuta campestris) and C. kilimanjari (both either naturalized or endemic to East Africa), in addition to the introduction of the giant dodder (C. reflexa), a south Asian species, in continental Africa. These parasites have a wide host range, parasitizing species across 13 angiosperm orders. We evaluated the possibility of C. reflexa to expand this host range to tea (Camelia sinensis), coffee (Coffea arabica), and mango (Mangifera indica), crops of economic importance to Africa, for which haustorial formation and vascular-bundle connections in all three crops revealed successful parasitism. However, only mango mounted a successful postattachment resistance response. Furthermore, species distribution models predicted high habitat suitability for Cuscuta spp. across major tea- and coffee-growing regions of Eastern Africa, suggesting an imminent risk to these crops. Our findings provide relevant insights into a poorly understood threat to biodiversity and economic wellbeing in Eastern Africa, and provide critical information to guide development of management strategies to avert Cuscuta spp. spread.

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic "currency" of the membrane. The dynamics of membrane voltage-so-called action, systemic, and variation potentials-have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport-an electrical "substrate"-and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory.

The high-affinity K+ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K+ acquisition and plant growth at low micromolar K+ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K+, whereas residues G67, Y70, and G71 may shape the selective filter for K+, which resembles that of K+shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K+ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. Four distinct trichomatic nectaries develop in cotton (Gossypium hirsutum), one floral and three extrafloral, and the nectars they secrete serve different purposes. Floral nectar attracts bees for promoting pollination, while extrafloral nectar attracts predatory insects as a means of indirect protection from herbivores. Cotton therefore provides an ideal system for contrasting mechanisms of nectar production and nectar composition between different nectary types. Here, we report the transcriptome and ultrastructure of the four cotton nectary types throughout development and compare these with the metabolomes of secreted nectars. Integration of these datasets supports specialization among nectary types to fulfill their ecological niche, while conserving parallel coordination of the merocrine-based and eccrine-based models of nectar biosynthesis. Nectary ultrastructures indicate an abundance of rough endoplasmic reticulum positioned parallel to the cell walls and a profusion of vesicles fusing to the plasma membranes, supporting the merocrine model of nectar biosynthesis. The eccrine-based model of nectar biosynthesis is supported by global transcriptomics data, which indicate a progression from starch biosynthesis to starch degradation and sucrose biosynthesis and secretion. Moreover, our nectary global transcriptomics data provide evidence for novel metabolic processes supporting de novo biosynthesis of amino acids secreted in trace quantities in nectars. Collectively, these data demonstrate the conservation of nectar-producing models among trichomatic and extrafloral nectaries.

The Polycomb Repressive Complex 2 (PRC2) is well-known for its role in controlling developmental transitions by suppressing the premature expression of key developmental regulators. Previous work revealed that PRC2 also controls the onset of senescence, a form of developmental programmed cell death (PCD) in plants. Whether the induction of PCD in response to stress is similarly suppressed by the PRC2 remained largely unknown. In this study, we explored whether PCD triggered in response to immunity- and disease-promoting pathogen effectors is associated with changes in the distribution of the PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) modification in Arabidopsis thaliana. We furthermore tested the distribution of the heterochromatic histone mark H3K9me2, which is established, to a large extent, by the H3K9 methyltransferase KRYPTONITE, and occupies chromatin regions generally not targeted by PRC2. We report that effector-induced PCD caused major changes in the distribution of both repressive epigenetic modifications and that both modifications have a regulatory role and impact on the onset of PCD during pathogen infection. Our work highlights that the transition to pathogen-induced PCD is epigenetically controlled, revealing striking similarities to developmental PCD.

Increasing sea levels associated with climate change threaten the survival of coastal forests, yet the mechanisms by which seawater exposure causes tree death remain poorly understood. Despite the potentially crucial role of nonstructural carbohydrate (NSC) reserves in tree survival, their dynamics in the process of death under seawater exposure are unknown. Here we monitored progressive tree mortality and associated NSC storage in Sitka-spruce (Picea sitchensis) trees dying under ecosystem-scale increases in seawater exposure in western Washington, USA. All trees exposed to seawater, because of monthly tidal intrusion, experienced declining crown foliage during the sampling period, and individuals with a lower percentage of live foliated crown (PLFC) died faster. Tree PLFC was strongly correlated with subsurface salinity and needle ion contents. Total NSC concentrations in trees declined remarkably with crown decline, and reached extremely low levels at tree death (2.4% and 1.6% in leaves and branches, respectively, and 0.4% in stems and roots). Starch in all tissues was almost completely consumed, while sugars remained at a homeostatic level in foliage. The decreasing NSC with closer proximity to death and near zero starch at death are evidences that carbon starvation occurred during Sitka-spruce mortality during seawater exposure. Our results highlight the importance of carbon storage as an indicator of tree mortality risks under seawater exposure.

Chemical signals known as strigolactones (SLs) were discovered more than 50 years ago as host-derived germination stimulants of parasitic plants in the Orobanchaceae. Strigolactone-responsive germination is an essential adaptation of obligate parasites in this family, which depend upon a host for survival. Several species of obligate parasites, including witchweeds (Striga, Alectra spp.) and broomrapes (Orobanche, Phelipanche spp.), are highly destructive agricultural weeds that pose a significant threat to global food security. Understanding how parasites sense SLs and other host-derived stimulants will catalyze the development of innovative chemical and biological control methods. This review synthesizes the recent discoveries of strigolactone receptors in parasitic Orobanchaceae, their signaling mechanism, and key steps in their evolution.

The presence of mixed-linkage (1,3;1,4)-β-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

Jumonji C (JmjC) domain proteins are histone lysine demethylases that require ferrous iron and alpha-ketoglutarate (or α-KG) as cofactors in the oxidative demethylation reaction. In plants, α-KG is produced by isocitrate dehydrogenases (ICDHs) in different metabolic pathways. It remains unclear whether fluctuation of α-KG levels affects JmjC demethylase activity and epigenetic regulation of plant gene expression. In this work, we studied the impact of loss of function of the cytosolic ICDH (cICDH) gene on the function of histone demethylases in Arabidopsis thaliana. Loss of cICDH resulted in increases of overall histone H3 lysine 4 trimethylation (H3K4me3) and enhanced mutation defects of the H3K4me3 demethylase gene JMJ14. Genetic analysis suggested that the cICDH mutation may affect the activity of other demethylases, including JMJ15 and JMJ18 that function redundantly with JMJ14 in the plant thermosensory response. Furthermore, we show that mutation of JMJ14 affected both the gene activation and repression programs of the plant thermosensory response and that JMJ14 and JMJ15 repressed a set of genes that are likely to play negative roles in the process. The results provide evidence that histone H3K4 demethylases are involved in the plant response to elevated ambient temperature.

Seeds of the root parasitic plant Striga hermonthica can sense very low concentrations of strigolactones (SLs) exuded from host roots. The S. hermonthica hyposensitive to light (ShHTL) proteins are putative SL receptors, among which ShHTL7 reportedly confers sensitivity to picomolar levels of SL when expressed in Arabidopsis thaliana. However, the molecular mechanism underlying ShHTL7 sensitivity is unknown. Here we determined the ShHTL7 crystal structure and quantified its interactions with various SLs and key interacting proteins. We established that ShHTL7 has an active-site pocket with broad-spectrum response to different SLs and moderate affinity. However, in contrast to other ShHTLs, we observed particularly high affinity of ShHTL7 for F-box protein AtMAX2. Furthermore, ShHTL7 interacted with AtMAX2 and with transcriptional regulator AtSMAX1 in response to nanomolar SL concentration. ShHTL7 mutagenesis analyses identified surface residues that contribute to its high-affinity binding to AtMAX2 and residues in the ligand binding pocket that confer broad-spectrum response to SLs with various structures. Crucially, yeast-three hybrid experiments showed that AtMAX2 confers responsiveness of the ShHTL7-AtSMAX1 interaction to picomolar levels of SL in line with the previously reported physiological sensitivity. These findings highlight the key role of SL-induced MAX2-ShHTL7-SMAX1 complex formation in determining the sensitivity to SL. Moreover, these data suggest a strategy to screen for compounds that could promote suicidal seed germination at physiologically relevant levels.

Less than 40% of the nitrogen (N) fertilizer applied to soil is absorbed by crops. Thus, improving the N use efficiency of crops is critical for agricultural development. However, the underlying regulation of these processes remains largely unknown, particularly in woody plants. By conducting yeast two-hybrid assays, we identified one interacting protein of MdMYB88 and MdMYB124 in apple (Malus × domestica), namely BTB and TAZ domain protein 2 (MdBT2). Ubiquitination and protein stabilization analysis revealed that MdBT2 ubiquitinates and degrades MdMYB88 and MdMYB124 via the 26S proteasome pathway. MdBT2 negatively regulates nitrogen usage as revealed by the reduced fresh weight, dry weight, N concentration, and N usage index of MdBT2 overexpression calli under low-N conditions. In contrast, MdMYB88 and MdMYB124 increase nitrate absorption, allocation, and remobilization by regulating expression of MdNRT2.4, MdNRT1.8, MdNRT1.7, and MdNRT1.5 under N limitation, thereby regulating N usage. The results obtained illustrate the mechanism of a regulatory module comprising MdBT2-MdMYB88/MdMYB124-MdNRTs, through which plants modulate N usage. These data contribute to a molecular approach to improve the N usage of fruit crops under limited N acquisition.

In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3'-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants.

Dodder (Cuscuta spp., Convolvulaceae) is a genus of parasitic plants with worldwide distribution. Dodders are able to simultaneously parasitize two or more adjacent hosts, forming dodder-connected plant clusters. Nitrogen (N) deficiency is a common challenge to plants. To date, it has been unclear whether dodder transfers N-systemic signals between hosts grown in N-heterogeneous soil. Transcriptome and methylome analyses were carried out to investigate whether dodder (Cuscuta campestris) transfers N-systemic signals between N-replete and N-depleted cucumber (Cucumis sativus) hosts, and it was found that N-systemic signals from the N-deficient cucumber plants were rapidly translocated through C. campestris to the N-replete cucumber plants. Unexpectedly, certain systemic signals were also transferred from the N-replete to N-depleted cucumber hosts. We demonstrate that these systemic signals are able to regulate large transcriptome and DNA methylome changes in the recipient hosts. Importantly, N stress also induced many long-distance mobile mRNA transfers between C. campestris and hosts, and the bilateral N-systemic signaling between N-replete and N-depleted hosts had a strong impact on the inter-plant mobile mRNAs. Our 15N labeling experiment indicated that under N-heterogeneous conditions, N-systemic signals from the N-deficient cucumber hosts did not obviously change the N-uptake activity of the N-replete cucumber hosts; however, in plant clusters comprising C. campestris-connected cucumber and soybean (Glycine max) plants, if the soybean plants were N-starved, the cucumber plants exhibited increased N-uptake activity. This study reveals that C. campestris facilitates plant-plant communications under N-stress conditions by enabling extensive bilateral N-systemic signaling between different hosts.

Parasitic plants are mostly viewed as pests. This is caused by several species causing serious damage to agriculture and forestry. There is however much more to parasitic plants than presumed weeds. Many parasitic plans exert even positive effects on natural ecosystems and human society, which we review in this paper. Plant parasitism generally reduces the growth and fitness of the hosts. The network created by a parasitic plant attached to multiple host plant individuals may however trigger transferring systemic signals among these. Parasitic plants have repeatedly been documented to play the role of keystone species in the ecosystems. Harmful effects on community dominants, including invasive species, may facilitate species coexistence and thus increase biodiversity. Many parasitic plants enhance nutrient cycling and provide resources to other organisms like herbivores or pollinators, which contributes to facilitation cascades in the ecosystems. There is also a long tradition of human use of parasitic plants for medicinal and cultural purposes worldwide. Few species provide edible fruits. Several parasitic plants are even cultivated by agriculture/forestry for efficient harvesting of their products. Horticultural use of some parasitic plant species has also been considered. While providing multiple benefits, parasitic plants should always be used with care. In particular, parasitic plant species should not be cultivated outside their native geographical range to avoid the risk of their uncontrolled spread and the resulting damage to ecosystems.

The plant hormone ethylene is important for the ripening of climacteric fruit, such as pear (Pyrus ussuriensis), and the brassinosteroid (BR) class of phytohormones affects ethylene biosynthesis during ripening via an unknown molecular mechanism. Here, we observed that exogenous BR treatment suppressed ethylene production and delayed fruit ripening, whereas treatment with a BR biosynthesis inhibitor promoted ethylene production and accelerated fruit ripening in pear, suggesting BR is a ripening suppressor. The expression of the transcription factor BRASSINAZOLE-RESISTANT 1PuBZR1 was enhanced by BR treatment during pear fruit ripening. PuBZR1 interacted with PuACO1, which converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, and suppressed its activity. BR-activated PuBZR1 bound to the promoters of PuACO1 and of PuACS1a, which encodes ACC synthase, and directly suppressed their transcription. Moreover, PuBZR1 suppressed the expression of transcription factor PuERF2 by binding its promoter, and PuERF2 bound to the promoters of PuACO1 and PuACS1a. We concluded that PuBZR1 indirectly suppresses the transcription of PuACO1 and PuACS1a through its regulation of PuERF2. Ethylene production and expression profiles of corresponding apple (Malus domestica) homologs showed similar changes following epibrassinolide treatment. Together, these results suggest that BR-activated BZR1 suppresses ACO1 activity and the expression of ACO1 and ACS1, thereby reducing ethylene production and suppressing fruit ripening. This likely represents a conserved mechanism by which BR suppresses ethylene biosynthesis during climacteric fruit ripening.

Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

Erratum in Plant Physiol. 2021 Dec 4;187(4):2880.

Nonphotosynthetic holoparasites exploit flexible targeting of phylloquinone biosynthesis to facilitate plasma membrane redox signaling. Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane (PM) preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here, we provide evidence of PM phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by PM-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of P. aegyptiaca phylloquinone genes toward coexpression with oxidoreductases involved in PM electron transport. Genes encoding the PM phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

The Striga, particularly S. he rmonthica, problem has become a major threat to food security, exacerbating hunger and poverty in many African countries. A number of Striga control strategies have been proposed and tested during the past decade, however, further research efforts are still needed to provide sustainable and effective solutions to the Striga problem. In this paper, we provide an update on the recent progress and the approaches used in Striga management, and highlight emerging opportunities for developing new technologies to control this enigmatic parasite.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Comment in Plant Physiol. 2021 Apr 23;185(4):1471-1472.

Parasitic plants are plants that connect with a haustorium to the vasculature of another, host, plant from which they absorb water, assimilates, and nutrients. Because of this parasitic lifestyle, parasitic plants need to coordinate their lifecycle with that of their host. Parasitic plants have evolved a number of host detection/host response mechanisms of which the germination in response to chemical host signals in one of the major families of parasitic plants, the Orobanchaceae, is a striking example. In this update review, we discuss these germination stimulants. We review the different compound classes that function as germination stimulants, how they are produced, and in which host plants. We discuss why they are reliable signals, how parasitic plants have evolved mechanisms that detect and respond to them, and whether they play a role in host specificity. The advances in the knowledge underlying this signaling relationship between host and parasitic plant have greatly improved our understanding of the evolution of plant parasitism and are facilitating the development of more effective control measures in cases where these parasitic plants have developed into weeds.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Parasitic plants pose a major biotic threat to plant growth and development and lead to losses in crop productivity of billions of USD annually. By comparison with "normal" autotrophic plants, parasitic plants live a heterotrophic lifestyle and rely on water, solutes and to a greater (holoparasitic plants) or lesser extent (hemiparasitic plants) on sugars from other host plants. Most hosts are unable to detect an infestation by plant parasites or unable to fend off these parasitic invaders. However, a few hosts have evolved defense strategies to avoid infestation or protect themselves actively post-attack often leading to full or partial resistance. Here, we review the current state of our understanding of the defense strategies to plant parasitism used by host plants with emphasis on the active molecular resistance mechanisms. Furthermore, we outline the perspectives and the potential of future studies that will be indispensable to develop and breed resistant crops.

Abscission of plant organs is induced by developmental signals and diverse environmental stimuli and involves multiple regulatory networks, including biotic or abiotic stress-impaired auxin flux in the abscission zone (AZ). Depletion of auxin activates AZ ethylene (ETH) production and triggers acceleration of abscission, a process that requires hydrogen peroxide (H2O2). However, the interaction between these networks and the underlying mechanisms that control abscission are poorly understood. Here, we found that expression of tonoplast intrinsic proteins, which belong to the aquaporin (AQP) family in the AZ was important for tomato (Solanum lycopersicum) pedicel abscission. Liquid chromatography-tandem mass spectrometry and in situ hybridization revealed that SlTIP1;1 was most abundant and specifically present in the tomato pedicel AZ. SlTIP1;1 localized in the plasma membrane and tonoplast. Knockout of SlTIP1;1 resulted in delayed abscission, whereas overexpression of SlTIP1;1 accelerated abscission. Further analysis indicated that SlTIP1;1 mediated abscission via gating of cytoplasmic H2O2 concentrations and osmotic water permeability (Pf). Elevated cytoplasmic levels of H2O2 caused a suppressed auxin signal in the early abscission stage and enhanced ETH production during abscission. Furthermore, we found that increasing Pf was required to enhance the turgor pressure to supply the break force for AZ cell separation. Moreover, we observed that SlERF52 bound directly to the SlTIP1;1 promoter to regulate its expression, demonstrating a positive loop in which cytoplasmic H2O2 activates ETH production, which activates SlERF52. This, in turn, induces SlTIP1;1, which leads to elevated cytoplasmic H2O2 and water influx.

Comment in Plant Physiol. 2021 Apr 23;185(4):1481-1482.

Members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors play key roles in the promotion of plant growth and development. Many GRFs are post-transcriptionally repressed by microRNA (miRNA) miR396, an evolutionarily conserved small RNA, which restricts their expression to proliferative tissue. We performed a comprehensive analysis of the GRF family in eudicot plants and found that in many species all the GRFs have a miR396-binding site. Yet, we also identified GRFs with mutations in the sequence recognized by miR396, suggesting a partial or complete release of their post-transcriptional repression. Interestingly, Brassicaceae species share a group of GRFs that lack miR396 regulation, including Arabidopsis GRF5 and GRF6. We show that instead of miR396-mediated post-transcriptional regulation, the spatiotemporal control of GRF5 is achieved through evolutionarily conserved promoter sequences, and that AUXIN RESPONSE FACTOR 2 (ARF2) binds to such conserved sequences to repress GRF5 expression. Furthermore, we demonstrate that the unchecked expression of GRF5 in arf2 mutants is responsible for the increased cell number of arf2 leaves. The results describe a switch in the repression mechanisms that control the expression of GRFs and mechanistically link the control of leaf growth by miR396, GRFs, and ARF2 transcription factors.

Jasmonates (JAs) are phytohormones with crucial roles in plant defense. Plants accumulate JAs in response to wounding or herbivore attack, but how JA biosynthesis is triggered remains poorly understood. Here we show that herbivory by cotton bollworm (Helicoverpa armigera) induced both ethylene (ET) and JA production in tomato (Solanum lycopersicum) leaves. Using RNA-seq, ET mutants, and inhibitors of ET signaling, we identified ET-induced ETHYLENE RESPONSE FACTOR 15 (ERF15) and ERF16 as critical regulators of JA biosynthesis in tomato plants. Transcripts of ERF15 and ERF16 were markedly upregulated and peaked at 60 and 15 min, respectively, after simulated herbivore attack. While mutation in ERF16 resulted in the attenuated expression of JA biosynthetic genes and decreased JA accumulation 15 min after the simulated herbivory treatment, these changes were not observed in erf15 mutants until 60 min after treatment. Electrophoretic mobility shift assays and dual-luciferase assays demonstrated that both ERFs15 and 16 are transcriptional activators of LIPOXYGENASE D, ALLENE OXIDE CYCLASE, and 12-OXO-PHYTODIENOIC ACID REDUCTASE 3, key genes in JA biosynthesis. Furthermore, JA-activated MYC2 and ERF16 also function as the transcriptional activators of ERF16, contributing to dramatic increases in ERF16 expression. Taken together, our results demonstrated that ET signaling is involved in the rapid induction of the JA burst. ET-induced ERF15 and ERF16 function as powerful transcriptional activators that trigger the JA burst in response to herbivore attack.

Interactions between plant hormones and environmental signals are important for the maintenance of root growth plasticity under ever-changing environmental conditions. Here, we demonstrate that arsenate (AsV), the most prevalent form of arsenic (As) in nature, restrains elongation of the primary root through transcriptional regulation of local auxin biosynthesis genes in the root tips of Arabidopsis (Arabidopsis thaliana) plants. The ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) and BETA SUBUNIT 1 (ASB1) genes encode enzymes that catalyze the conversion of chorismate to anthranilate (ANT) via the tryptophan-dependent auxin biosynthesis pathway. Our results showed that AsV upregulates ASA1 and ASB1 expression in root tips, and ASA1- and ASB1-mediated auxin biosynthesis is involved in AsV-induced root growth inhibition. Further investigation confirmed that AsV activates cytokinin signaling by stabilizing the type-B ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) protein, which directly promotes the transcription of ASA1 and ASB1 genes by binding to their promoters. Genetic analysis revealed that ASA1 and ASB1 are epistatic to ARR1 in the AsV-induced inhibition of primary root elongation. Overall, the results of this study illustrate a molecular framework that explains AsV-induced root growth inhibition via crosstalk between two major plant growth regulators, auxin and cytokinin.

Strigolactones (SLs), first identified as germination stimulants for root parasitic weeds, act as endogenous phytohormones regulating shoot branching and as root-derived signal molecules mediating symbiotic communications in the rhizosphere. Canonical SLs typically have an ABCD ring system and can be classified into orobanchol- and strigol-type based on the C-ring stereochemistry. Their simplest structures are 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS), respectively. Diverse canonical SLs are chemically modified with one or more hydroxy or acetoxy groups introduced into the A- and/or B-ring of these simplest structures, but the biochemical mechanisms behind this structural diversity remain largely unexplored. Sorgomol in sorghum (Sorghum bicolor [L.] Moench) is a strigol-type SL with a hydroxy group at C-9 of 5DS. In this study, we characterized sorgomol synthase. Microsomal fractions prepared from a high-sorgomol-producing cultivar of sorghum, Sudax, were shown to convert 5DS to sorgomol. A comparative transcriptome analysis identified SbCYP728B subfamily as candidate genes encoding sorgomol synthase. Recombinant SbCYP728B35 catalyzed the conversion of 5DS to sorgomol in vitro. Substrate specificity revealed that the C-8bS configuration in the C-ring of 5DS stereoisomers was essential for this reaction. The overexpression of SbCYP728B35 in Lotus japonicus hairy roots, which produce 5DS as an endogenous SL, also resulted in the conversion of 5DS to sorgomol. Furthermore, SbCYP728B35 expression was not detected in nonsorgomol-producing cultivar, Abu70, suggesting that this gene is responsible for sorgomol production in sorghum. Identification of the mechanism modifying parental 5DS of strigol-type SLs provides insights on how plants biosynthesize diverse SLs.

Erratum in Plant Physiol. 2021 Nov 3;187(3):1834.

Membranes are essential for cells and organelles to function. As membranes are impermeable to most polar and charged molecules, they provide electrochemical energy to transport molecules across and create compartmentalized microenvironments for specific enzymatic and cellular processes. Membranes are also responsible for guided transport of cargoes between organelles and during endo- and exocytosis. In addition, membranes play key roles in cell signaling by hosting receptors and signal transducers and as substrates and products of lipid second messengers. Anionic lipids and their specific interaction with target proteins play an essential role in these processes, which are facilitated by specific lipid-binding domains. Protein crystallography, lipid-binding studies, subcellular localization analyses, and computer modeling have greatly advanced our knowledge over the years of how these domains achieve precision binding and what their function is in signaling and membrane trafficking, as well as in plant development and stress acclimation.

Fruit set is established during and soon after fertilization of the ovules inside the quiescent ovary, but the signaling pathways involved remain obscure. The tomato (Solanum lycopersicum) CRISPR loss-of-function mutant of the transcription factor gene AGAMOUS-like6 (SlAGL6; slagl6CR-sg1) is capable of fertilization-independent setting of normal, yet seedless (parthenocarpic), fruit. To gain insight into the mechanism of fleshy fruit set, in this study, we investigated how slagl6CR-sg1 uncouples fruit set from fertilization. We found that mutant ovules were enlarged due to integument over-proliferation and failed to differentiate an endothelium, the integument's innermost layer, upon maturation. A causal relationship between slagl6 loss-of-function and these abnormal phenotypes is inferred from the observation that SlAGL6 is predominantly expressed in the immature ovule integument, and upon ovule maturation, its expression shifts to the endothelium. The transcriptome of unfertilized mutant ovules profoundly differs from that of wild-type and exhibits substantial overlap with the transcriptomes of fertilized ovules sporophytic tissues. One prominent upregulated gene was the fertilization-induced cytochrome P450 cell proliferation regulator SlKLUH. Indeed, ectopic overexpression of SlKLUH stimulated both integument growth in unfertilized ovules and parthenocarpy, suggesting that its suppression by SlAGL6 is paramount for preventing fertilization-independent fruit set. Taken together, our study informs on the transcriptional programs that are regulated by SlAGL6 and demonstrates that it acts from within the ovule integument to inhibit ovary growth beyond anthesis. That by suppressing components of the fertilization-induced ovule reprogramming underlying fruit set.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by β-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine β-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks β-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of β-glucosidase multimerization, an organization common to many defensive GH1 members.

The gynoecium is the most complex organ formed by the flowering plants. It encloses the ovules, provides a surface for pollen contact and self-incompatibility reactions, allows pollen tube growth, and, post fertilization, develops into the fruit. Consequently, the regulation of gynoecium morphogenesis is complex and appropriate timing of this process in part determines reproductive success. However, little is known about the global control of gynoecium development, even though many regulatory genes have been characterized. Here, we characterized dynamic gene expression changes using laser-microdissected gynoecium tissue from four developmental stages in Arabidopsis. We provide a high-resolution map of global expression dynamics during gynoecium morphogenesis and link these to the gynoecium interactome. We reveal groups of genes acting together early and others acting late in morphogenesis. Clustering of co-expressed genes enables comparisons between the leaf, shoot apex, and gynoecium transcriptomes, allowing the dissection of common and distinct regulators. Furthermore, our results lead to the discovery of genes with putative transcription factor activity (B3LF1, -2, DOFLF1), which, when mutated, lead to impaired gynoecium expansion, illustrating that global transcriptome analyses reveal yet unknown developmental regulators. Our data show that genes encoding highly interacting proteins, such as SEPALLATA3, AGAMOUS, and TOPLESS, are expressed evenly during development but switch interactors over time, whereas stage-specific proteins tend to have fewer interactors. Our analysis connects specific transcriptional regulator activities, protein interactions, and underlying metabolic processes, contributing toward a dynamic network model for gynoecium development.

Plant growth and development rely on sugar transport between source and sink cells and between different organelles. The plastid-localized sugar transporter GLUCOSE-6-PHOSPHATE TRANSLOCATER1 (GPT1) is an essential gene in Arabidopsis (Arabidopsis thaliana). Using a partially rescued gpt1 mutant and cell-specific RNAi suppression of GPT1, we demonstrated that GPT1 is essential to the function of the embryo suspensor and the development of the embryo. GPT1 showed a dynamic expression/accumulation pattern during embryogenesis. Inhibition of GPT1 accumulation via RNAi using a suspensor-specific promoter resulted in embryos and seedlings with defects similar to auxin mutants. Loss of function of GPT1 in the suspensor also led to abnormal/ectopic cell division in the lower part of the suspensor, which gave rise to an ectopic embryo, resulting in twin embryos in some seeds. Furthermore, loss of function of GPT1 resulted in vacuolar localization of PIN-FORMED1 (PIN1) and altered DR5 auxin activity. Proper localization of PIN1 on the plasma membrane is essential to polar auxin transport and distribution, a key determinant of pattern formation during embryogenesis. Our findings suggest that the function of GPT1 in the embryo suspensor is linked to sugar and/or hormone distribution between the embryo proper and the maternal tissues, and is important for maintenance of suspensor identity and function during embryogenesis.

Plants perceive dynamic light conditions and optimize their growth and development accordingly by regulating gene expression at multiple levels. Alternative splicing (AS), a widespread mechanism in eukaryotes that post-transcriptionally generates two or more messenger RNAs (mRNAs) from the same pre-mRNA, is rapidly controlled by light. However, a detailed mechanism of light-regulated AS is still not clear. In this study, we demonstrate that histone 3 lysine 36 trimethylation (H3K36me3) rapidly and differentially responds to light at specific gene loci with light-regulated intron retention (IR) of their transcripts in the moss Physcomitrella patens. However, the level of H3K36me3 following exposure to light is inversely related to that of IR events. Physcomitrella patens MORF-related gene 1 (PpMRG1), a chromatin adaptor, bound with higher affinity to H3K36me3 in light conditions than in darkness and was differentially targeted to gene loci showing light-responsive IR. Transcriptome analysis indicated that PpMRG1 functions in the regulation of light-mediated AS. Furthermore, PpMRG1 was also involved in red light-mediated phototropic responses. Our results suggest that light regulates histone methylation, which leads to alterations of AS patterns. The chromatin adaptor PpMRG1 potentially participates in light-mediated AS, revealing that chromatin-coupled regulation of pre-mRNA splicing is an important aspect of the plant's response to environmental changes.

The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

The potassium ion (K+) is vital for plant growth and development, and K+-deprivation leads to reduced crop yields. Here we describe phenotypic, transcriptomic, and mutant analyses to investigate the signaling mechanisms mediating root architectural changes in Arabidopsis (Arabidopsis thaliana) Columbia. We showed effects on root architecture are mediated through a reduction in cell division in the lateral root (LR) meristems, the rate of LR initiation is reduced but LR density is unaffected, and primary root growth is reduced only slightly. This was primarily regulated through gibberellic acid (GA) signaling, which leads to the accumulation of growth-inhibitory DELLA proteins. The short LR phenotype was rescued by exogenous application of GA but not of auxin or by the inhibition of ethylene signaling. RNA-seq analysis showed upregulation by K+-deprivation of the transcription factors JUNGBRUNNEN1 (JUB1) and the C-repeat-binding factor (CBF)/dehydration-responsive element-binding factor 1 regulon, which are known to regulate GA signaling and levels that regulate DELLAs. Transgenic overexpression of JUB1 and CBF1 enhanced responses to K+ stress. Attenuation of the reduced LR growth response occurred in mutants of the CBF1 target gene SFR6, implicating a role for JUB1, CBF1, and SFR6 in the regulation of LR growth in response to K+-deprivation via DELLAs. We propose this represents a mechanism to limit horizontal root growth in conditions where K+ is available deeper in the soil.

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl-trimethyl-homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A3, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

A vast majority of cellular processes take root at the surface of biological membranes. By providing a two-dimensional platform with limited diffusion, membranes are, by nature, perfect devices to concentrate signaling and metabolic components. As such, membranes often act as "key processors" of cellular information. Biological membranes are highly dynamic and deformable and can be shaped into curved, tubular, or flat conformations, resulting in differentiated biophysical properties. At membrane contact sites, membranes from adjacent organelles come together into a unique 3D configuration, forming functionally distinct microdomains, which facilitate spatially regulated functions, such as organelle communication. Here, we describe the diversity of geometries of contact site-forming membranes in different eukaryotic organisms and explore the emerging notion that their shape, 3D architecture, and remodeling jointly define their cellular activity. The review also provides selected examples highlighting changes in membrane contact site architecture acting as rapid and local responses to cellular perturbations, and summarizes our current understanding of how those structural changes confer functional specificity to those cellular territories.

The identification of functional elements encoded in plant genomes is necessary to understand gene regulation. Although much attention has been paid to model species like Arabidopsis (Arabidopsis thaliana), little is known about regulatory motifs in other plants. Here, we describe a bottom-up approach for de novo motif discovery using peach (Prunus persica) as an example. These predictions require pre-computed gene clusters grouped by their expression similarity. After optimizing the boundaries of proximal promoter regions, two motif discovery algorithms from RSAT::Plants (http://plants.rsat.eu) were tested (oligo and dyad analysis). Overall, 18 out of 45 co-expressed modules were enriched in motifs typical of well-known transcription factor (TF) families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, and WRKY) and a few uncharacterized motifs. Our results indicate that small modules and promoter window of [-500 bp, +200 bp] relative to the transcription start site (TSS) maximize the number of motifs found and reduce low-complexity signals in peach. The distribution of discovered regulatory sites was unbalanced, as they accumulated around the TSS. This approach was benchmarked by testing two different expression-based clustering algorithms (network-based and hierarchical) and, as control, genes grouped for harboring ChIPseq peaks of the same Arabidopsis TF. The method was also verified on maize (Zea mays), a species with a large genome. In summary, this article presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to other species. A Docker software container is released to facilitate the reproduction of these analyses.

The past 50 years has been the greatest era of plant science discovery, and most of the discoveries have emerged from or been facilitated by our knowledge of plant chromosomes. At last we have descriptive and mechanistic outlines of the information in chromosomes that programs plant life. We had almost no such information 50 years ago when few had isolated DNA from any plant species. The important features of genes have been revealed through whole genome comparative genomics and testing of variants using transgenesis. Progress has been enabled by the development of technologies that had to be invented and then become widely available. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have played extraordinary roles as model species. Unexpected evolutionary dramas were uncovered when learning that chromosomes have to manage constantly the vast numbers of potentially mutagenic families of transposons and other repeated sequences. The chromatin-based transcriptional and epigenetic mechanisms that co-evolved to manage the evolutionary drama as well as gene expression and 3-D nuclear architecture have been elucidated these past 20 years. This perspective traces some of the major developments with which I have become particularly familiar while seeking ways to improve crop plants. I draw some conclusions from this look-back over 50 years during which the scientific community has (i) exposed how chromosomes guard, readout, control, recombine, and transmit information that programs plant species, large and small, weed and crop, and (ii) modified the information in chromosomes for the purposes of genetic, physiological, and developmental analyses and plant improvement.

The lipid bilayer of biological membranes has a complex composition, including high chemical heterogeneity, the presence of nanodomains of specific lipids, and asymmetry with respect to lipid composition between the two membrane leaflets. In membrane trafficking, membrane vesicles constantly bud off from one membrane compartment and fuse with another, and both budding and fusion events have been proposed to require membrane lipid asymmetry. One mechanism for generating asymmetry in lipid bilayers involves the action of the P4 ATPase family of lipid flippases; these are biological pumps that use ATP as an energy source to flip lipids from one leaflet to the other. The model plant Arabidopsis (Arabidopsis thaliana) contains 12 P4 ATPases (AMINOPHOSPHOLIPID ATPASE1-12; ALA1-12), many of which are functionally redundant. Studies of P4 ATPase mutants have confirmed the essential physiological functions of these pumps and pleiotropic mutant phenotypes have been observed, as expected when genes required for basal cellular functions are disrupted. For instance, phenotypes associated with ala3 (dwarfism, pollen defects, sensitivity to pathogens and cold, and reduced polar cell growth) can be related to membrane trafficking problems. P5 ATPases are evolutionarily related to P4 ATPases, and may be the counterpart of P4 ATPases in the endoplasmic reticulum. The absence of P4 and P5 ATPases from prokaryotes and their ubiquitous presence in eukaryotes make these biological pumps a defining feature of eukaryotic cells. Here, we review recent advances in the field of plant P4 and P5 ATPases.

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Comment in Plant Physiol. 2021 Apr 2;185(3):754-756.

Comment in Plant Physiol. 2021 Apr 2;185(3):757-758.

Anionic phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), and its phosphorylated derivatives the phosphoinositides (e.g. phosphatidylinositol-4-phosphate [PI4P] and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]). Although anionic phospholipids are low-abundant lipids, they are particularly important for membrane functions. In particular, anionic lipids act as biochemical and biophysical landmarks that contribute to the establishment of membrane identity, signaling activities, and compartment morphodynamics. Each anionic lipid accumulates in different endomembranes according to a unique subcellular pattern, where they locally provide docking platforms for proteins. As such, they are mostly believed to act in the compartments in which they accumulate. However, mounting evidence throughout eukaryotes suggests that anionic lipids are not as compartment-specific as initially thought and that they are instead organized as concentration gradients across different organelles. In this update, we review the evidence for the existence of anionic lipid gradients in plants. We then discuss the possible implication of these gradients in lipid dynamics and homeostasis, and also in coordinating subcellular activities. Finally, we introduce the notion that anionic lipid gradients at the cellular scale may translate into gradients at the tissue level, which could have implications for plant development.

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

Stomata are small pores on the surface of land plants that facilitate gas exchange for photosynthesis while minimizing water loss. The function of stomata is pivotal for plant growth and survival. Intensive research on the model plant Arabidopsis (Arabidopsis thaliana) has discovered key peptide signaling pathways, transcription factors, and polarity components that together drive proper stomatal development and patterning. In this review, we focus on recent findings that have revealed co-option of peptide-receptor kinase signaling modules-utilized for diverse developmental processes and immune response. We further discuss an emerging connection between extrinsic signaling and intrinsic polarity modules. These findings have further enlightened our understanding of this fascinating developmental process.

Endoplasmic reticulum (ER) type I signal peptidases (ER SPases I) are vital proteases that cleave signal peptides from secreted proteins. However, the specific function of ER SPase I in plants has not been genetically characterized, and the substrate is largely unknown. Here, we report the identification of a maize (Zea mays) miniature seed6 (mn6) mutant. The loss-of-function mn6 mutant exhibited severely reduced endosperm size. Map-based cloning and molecular characterization indicated that Mn6 is an S26-family ER SPase I, with Gly102 (box E) in Mn6 critical for protein function during processing. Mass spectrometric and immunoprecipitation analyses revealed that Mn6 is predominantly involved in processing carbohydrate synthesis-related proteins, including the cell wall invertase miniature seed1 (Mn1), which is specifically expressed in the basal endosperm transfer layer. RNA and protein expression levels of Mn1 were both significantly downregulated in the mn6 mutant. Due to the significant reduction in cell wall invertase activity in the transfer cell layer, mutation of Mn6 caused dramatic defects in endosperm development. These results suggest that proper maturation of Mn1 by Mn6 may be a crucial step for proper seed filling and maize development.

REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.

The emergence of type III polyketide synthases (PKSs) was a prerequisite for the conquest of land by the green lineage. Within the PKS superfamily, chalcone synthases (CHSs) provide the entry point reaction to the flavonoid pathway, while LESS ADHESIVE POLLEN 5 and 6 (LAP5/6) provide constituents of the outer exine pollen wall. To study the deep evolutionary history of this key family, we conducted phylogenomic synteny network and phylogenetic analyses of whole-genome data from 126 species spanning the green lineage including Arabidopsis thaliana, tomato (Solanum lycopersicum), and maize (Zea mays). This study thereby combined study of genomic location and context with changes in gene sequences. We found that the two major clades, CHS and LAP5/6 homologs, evolved early by a segmental duplication event prior to the divergence of Bryophytes and Tracheophytes. We propose that the macroevolution of the type III PKS superfamily is governed by whole-genome duplications and triplications. The combined phylogenetic and synteny analyses in this study provide insights into changes in the genomic location and context that are retained for a longer time scale with more recent functional divergence captured by gene sequence alterations.

Flavonoids are secondary metabolites that play important roles in fruit and vegetable development. Here, we examined the function of hyperoside, a unique flavonoid in okra (Abelmoschus esculentus), known to promote both flowering and seed set. We showed that the exogenous application of hyperoside significantly improved pollen germination rate and pollen tube growth by almost 50%, resulting in a 42.7% increase in the seed set rate. Of several genes induced by the hyperoside treatment, AeUF3GaT1, which encodes an enzyme that catalyzes the last step of hyperoside biosynthesis, was the most strongly induced. The transcription factor AeMYB30 enhanced AeUFG3aT1 transcription by directly binding to the AeUFG3aT1 promoter. We studied the effect of the hyperoside application on the expression of 10 representative genes at four stages of reproductive development, from pollination to seed maturity. We firstly developed an efficient transformation system that uses seeds as explants to study the roles of AeMYB30 and AeUFG3aT1. Overexpression of AeMYB30 or AeUF3GaT1 promoted seed development. Moreover, exogenous application of hyperoside partially restored the aberrant phenotype of AeUF3GaT1 RNA-interference plants. Thus, hyperoside promotes seed set in okra via a pathway involving AeUF3GaT and AeMYB30, and the exogenous application of this flavonoid is a simple method that can be used to improve seed quality and yield in okra.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Microalgae accumulate triacylglycerol (TAG) during nutrient deprivation and break it down after nutrient resupply, and these processes involve dramatic shifts in cellular carbon allocation. Due to the importance of algae in the global carbon cycle, and the potential of algal lipids as feedstock for chemical and fuel production, these processes are of both ecophysiological and biotechnological importance. However, the metabolism of TAG is not well understood, particularly the contributions of fatty acids (FAs) from different membrane lipids to TAG accumulation and the fate of TAG FAs during degradation. Here, we used isotopic labeling time course experiments on Chlamydomonas reinhardtii to track FA synthesis and transfer between lipid pools during nitrogen (N)-deprivation and resupply. When cells were labeled before N-deprivation, total levels of label in cellular FAs were unchanged during subsequent N-deprivation and later resupply, despite large fluxes into and out of TAG and membrane lipid pools. Detailed analyses of FA levels and labeling revealed that about one-third of acyl chains accumulating in TAG during N-deprivation derive from preexisting membrane lipids, and in total, at least 45% of TAG FAs passed through membrane lipids at one point. Notably, most acyl chains in membrane lipids during recovery after N-resupply come from TAG. Fluxes of polyunsaturated FAs from plastidic membranes into TAG during N-deprivation were particularly noteworthy. These findings demonstrate a high degree of integration of TAG and membrane lipid metabolism and highlight a role for TAG in storage and supply of membrane lipid components.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.

Unlike ovary-derived botanical fruits, strawberry (Fragaria x ananassa) is an accessory fruit derived from the receptacle, the stem tip subtending floral organs. Although both botanical and accessory fruits initiate development in response to auxin and gibberellic acid (GA) released from seeds, the downstream auxin and GA signaling mechanisms underlying accessory fruit development are presently unknown. We characterized GA and auxin signaling mutants in wild strawberry (Fragaria vesca) during early stage fruit development. While mutations in FveRGA1 and FveARF8 both led to the development of larger fruit, only mutations in FveRGA1 caused parthenocarpic fruit formation, suggesting FveRGA1 is a key regulator of fruit set. FveRGA1 mediated fertilization-induced GA signaling during accessory fruit initiation by repressing the expression of cell division and expansion genes and showed direct protein-protein interaction with FveARF8. Further, fvearf8 mutant fruits exhibited an enhanced response to auxin or GA application, and the increased response to GA was due to increased expression of FveGID1c coding for a putative GA receptor. The work reveals a crosstalk mechanism between FveARF8 in auxin signaling and FveGID1c in GA signaling. Together, our work provides functional insights into hormone signaling in an accessory fruit, broadens our understanding of fruit initiation in different fruit types, and lays the groundwork for future improvement of strawberry fruit productivity and quality.

In a crowded environment, establishing interactions between different molecular partners can take a long time. Biological membranes have solved this issue, as they simultaneously are fluid and possess compartmentalized domains. This nanoscale organization of the membrane is often based on weak, local, and multivalent interactions between lipids and proteins. However, from local interactions at the nanoscale, different functional properties emerge at the higher scale, and these are critical to regulate and integrate cellular signaling. Rho of Plant (ROP) proteins are small guanosine triphosphate hydrolase enzymes (GTPases) involved in hormonal, biotic, and abiotic signaling, as well as fundamental cell biological properties such as polarity, vesicular trafficking, and cytoskeleton dynamics. Association with the membrane is essential for ROP function, as well as their precise targeting within micrometer-sized polar domains (i.e. microdomains) and nanometer-sized clusters (i.e. nanodomains). Here, we review our current knowledge about the formation and the maintenance of the ROP domains in membranes. Furthermore, we propose a model for ROP membrane targeting and discuss how the nanoscale organization of ROPs in membranes could determine signaling parameters like signal specificity, amplification, and integration.

Heat shock proteins (HSPs) function as molecular chaperones and are key components responsible for protein folding, assembly, translocation, and degradation under stress conditions. However, little is known about how HSPs stabilize proteins and membranes in response to different hormonal or environmental cues in plants. Here, we combined molecular, biochemical, and genetic approaches to elucidate the involvement of cytosolic HSP70-3 in plant stress responses and the interplay between HSP70-3 and plasma membrane (PM)-localized phospholipase Dδ (PLDδ) in Arabidopsis (Arabidopsis thaliana). Analysis using pull-down, coimmunoprecipitation, and bimolecular fluorescence complementation revealed that HSP70-3 specifically interacted with PLDδ. HSP70-3 bound to microtubules, such that it stabilized cortical microtubules upon heat stress. We also showed that heat shock induced recruitment of HSP70-3 to the PM, where HSP70-3 inhibited PLDδ activity to mediate microtubule reorganization, phospholipid metabolism, and plant thermotolerance, and this process depended on the HSP70-3-PLDδ interaction. Our results suggest a model whereby the interplay between HSP70-3 and PLDδ facilitates the re-establishment of cellular homeostasis during plant responses to external stresses and reveal a regulatory mechanism in regulating membrane lipid metabolism.

Comment in Plant Physiol. 2021 Apr 2;185(3):759-760.

The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5'-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.

Transcription initiation of the genes coding for small nuclear RNA (snRNA) has been extensively analyzed in humans and fruit fly, but only a single ortholog of a snRNA-activating protein complex (SNAPc) subunit has so far been characterized in plants. The genome of the model plant Arabidopsis thaliana encodes orthologs of all three core SNAPc subunits, including A. thaliana SNAP complex 4 (AtSNAPc4)-a 4R-MYB-type protein with four-and-a-half adjacent MYB repeat units. We report the conserved role of AtSNAPc4 as subunit of a protein complex involved in snRNA gene transcription and present genetic evidence that AtSNAPc4 is an essential gene in gametophyte and zygote development. We present experimental evidence that the three A. thaliana SNAPc subunits assemble into a SNAP complex and demonstrate the binding of AtSNAPc4 to snRNA promoters. In addition, co-localization studies show a link between AtSNAPc4 accumulation and Cajal bodies, known to aggregate at snRNA gene loci in humans. Moreover, we show the strong evolutionary conservation of single-copy 4R-MYB/SNAPc4 genes in a broad range of eukaryotes and present additional shared protein features besides the MYB domain, suggesting a conservation of the snRNA transcription initiation machinery along the course of the eukaryotic evolution.