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Summary The growth and development of root systems is influenced by mechanical properties of the substrate in which the plants grow. Mechanical impedance, such as by compacted soil, can reduce root elongation and limit crop productivity.To understand better the mechanisms involved in plant root responses to mechanical impedance stress, we investigated changes in the root transcriptome and hormone signalling responses of Arabidopsis to artificial root barrier systems in vitro.We demonstrate that upon encountering a barrier, reduced Arabidopsis root growth and a characteristic ‘step‐like’ growth pattern is due to a reduction in cell elongation associated with changes in signalling gene expression. Data from RNA‐sequencing combined with reporter line and mutant studies identified essential roles for reactive oxygen species, ethylene and auxin signalling during the barrier response.We propose a model in which early responses to mechanical impedance include reactive oxygen signalling integrated with ethylene and auxin responses to mediate root growth changes. Inhibition of ethylene responses allows improved growth in response to root impedance, an observation that may inform future crop breeding programmes., See also the Commentary on this article by Del Bianco & Kepinski 231: 8–10.

Flowering time, a key transition point from vegetative to reproductive growth, is regulated by an intrinsic complex of endogenous and exogenous signals including nutrient status. For hundreds of years, nitrogen has been well known to modulate flowering time, but the molecular genetic basis on how plants adapt to ever-changing nitrogen availability remains not fully explored. Here we explore how Arabidopsis natural variation in flowering time responds to nitrate fluctuation. Upon nitrate availability change, we detect accession- and photoperiod-specific flowering responses, which also feature a accession-specific dependency on growth traits. The flowering time variation correlates well with the expression of floral integrators, SOC1 and FT, in an accession-specific manner. We find that gene expression variation of key hub genes in the photoperiod-circadian-clock (GI), aging (SPLs) and autonomous (FLC) pathways associates with the expression change of these integrators, hence flowering time variation. Our results thus shed light on the molecular genetic mechanisms on regulation of accession- and photoperiod-specific flowering time variation in response to nitrate availability.

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits—tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.

Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding OKS of Polygonum cuspidatum was isolated using a homology-based cloning strategy. When produced in Escherichia coli, P. cuspidatum octaketide synthase (PcOKS) catalyzed the condensation of eight molecules of malonyl-CoA to yield a mixture of unphysiologically folded aromatic octaketides. However, when the ORF for PcOKS was expressed in Arabidopsis thaliana, the anthranoid emodin was detected in the roots of transgenic lines. No emodin was found in the roots of wild-type A. thaliana. This result indicated that OKS is the key enzyme of plant anthranoids biosynthesis. In addition, the root growth of the transgenic A. thaliana lines was inhibited to an extent that resembled the inhibitory effect of exogenous emodin on the root growth of wild-type A. thaliana. Immunochemical studies of P. cuspidatum plants detected PcOKS mainly in roots and rhizome, in which anthranoids accumulate. Co-incubation of E. coli - produced PcOKS and cell-free extract of wild-type A. thaliana roots did not form a new product, suggesting an alternative, physiological folding of PcOKS and its possible interaction with additional factors needed for anthranoids assembling in transgenic A. thaliana. Thus, transgenic A. thaliana plants producing PcOKS provide an interesting system for elucidating the route of plant anthranoid biosynthesis.

A reduction in cytosolic invertase in Arabidopsis roots changes the sucrose to hexose ratio, resulting in profound alterations of metabolism, growth, development, and patterns of gene expression., Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth, and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are involved in auxin signaling pathway and play an important role in plant growth and development. However, many studies focus on Aux/IAA gene families and much less known in Bletilla striata. In this study, a total of 27 Aux/IAA genes (BsIAA1-27) were cloned from the transcriptome of Bletilla striata. Based on a phylogenetic analysis of the Aux/IAA protein sequences from B. striata, Arabidopsis thaliana and Dendrobium officinale, the Aux/IAA genes of B. striata (BsIAAs) were categorized into 2 subfamilies and 9 groups. While BsIAAs were more closer to those of D. officinale compared to A. thaliana. EST-SSR marker mining test showed that 4 markers could be stably amplified with obvious polymorphisms among 4 landraces. Our results suggested that BsIAAs were involved in the process of tuber development and provided insights into functional roles of Aux/IAA genes in B. striata and other plants., Graphical Abstract In this paper, we identified the 27 members of the Aux/IAA gene family which can be divided into 2 groups and 9 subfamilies. The similar branches of Aux/IAA proteins had the same or similar motifs, such as genes between BsIAA10 and BsIAA12, BsIAA11 and BsIAA13, BsIAA18 and BsIAA20, indicating that Aux/IAA proteins were conserved. Among them, BsIAAs were more closer to those of D. officinale compared to A. thaliana.

Summary Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase‐promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin‐ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis.We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis.Meiocytes from apc8‐1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8‐1, including abnormal sister chromatid segregation and microtubule morphology.Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C‐mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with 15N and 13C for NMR analysis and obtained near complete 1H, 13C, and 15N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges., PDB Code(s): 6NU4

Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised. To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl2 using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15). The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana. These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling., ATP binding and hydrolysis, mediated by Walker A and B motifs, are important for Arabidopsis thaliana ABCE2 RNA silencing suppressor function and so is its FeS cluster domain.

Centro de Biotecnología y Genómica de Plantas (CBGP), The evolutionary success of plants relies to a large extent on their extraordinary ability to adapt to changes in their environment. These adaptations require that plants balance their growth with their stress responses. Plant hormones are crucial mediators orchestrating the underlying adaptive processes. However, whether and how the growth-related hormone auxin and the stress-related hormones jasmonic acid, salicylic acid, and abscisic acid (ABA) are coordinated remains largely elusive. Here, we analyse the physiological role of AMIDASE 1 (AMI1) in Arabidopsis plant growth and its possible connection to plant adaptations to abiotic stresses. AMI1 contributes to cellular auxin homeostasis by catalysing the conversion of indole-acetamide into the major plant auxin indole-3-acetic acid. Functional impairment of AMI1 increases the plant's stress status rendering mutant plants more susceptible to abiotic stresses. Transcriptomic analysis of ami1 mutants disclosed the reprogramming of a considerable number of stress-related genes, including jasmonic acid and ABA biosynthesis genes. The ami1 mutants exhibit only moderately repressed growth but an enhanced ABA accumulation, which suggests a role for AMI1 in the crosstalk between auxin and ABA. Altogether, our results suggest that AMI1 is involved in coordinating the trade-off between plant growth and stress responses, balancing auxin and ABA homeostasis., This research was supported by grants from the German Research Foundation (DFG, SFB480/A10) and the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, BFU2017-82826-R to SP and a grant from the Swedish Research Council (VR) to HA. JM was supported by the ‘Severo Ochoa Program for Centers of Excellence in R&D’ from the Agencia Estatal de Investigación of Spain, grant SEV-2016-0672 (2017-2021) to the CBGP., 17 Pág.

Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes., Induced cell death is a crucial component of plant defence. An extracellular downy mildew cysteine-rich protein effector inhibited plant cell death enabling host colonization.

Temperature response of stem elongation in wheat grown under field conditions indicated that temperature response is highly heritable and linked to the flowering pathway., In wheat, temperature affects the timing and intensity of stem elongation. Genetic variation for this process is therefore important for adaptation. This study investigates the genetic response to temperature fluctuations during stem elongation and its relationship to phenology and height. Canopy height of 315 wheat genotypes (GABI wheat panel) was scanned twice weekly in the field phenotyping platform (FIP) of ETH Zurich using a LIDAR. Temperature response was modelled using linear regressions between stem elongation and mean temperature in each measurement interval. This led to a temperature-responsive (slope) and a temperature-irresponsive (intercept) component. The temperature response was highly heritable (H2=0.81) and positively related to a later start and end of stem elongation as well as final height. Genome-wide association mapping revealed three temperature-responsive and four temperature-irresponsive quantitative trait loci (QTLs). Furthermore, putative candidate genes for temperature-responsive QTLs were frequently related to the flowering pathway in Arabidopsis thaliana, whereas temperature-irresponsive QTLs corresponded to growth and reduced height genes. In combination with Rht and Ppd alleles, these loci, together with the loci for the timing of stem elongation, accounted for 71% of the variability in height. This demonstrates how high-throughput field phenotyping combined with environmental covariates can contribute to a smarter selection of climate-resilient crops.

USB1, an exoribonuclease catalyzing U6 production for spliceosome assembly, negatively regulates, cooperatively with SOAR1, ABA signaling in early seedling development, probably by affecting spliceosome assembly., Signaling by the phytohormone abscisic acid (ABA) involves pre-mRNA splicing, a key process of post-transcriptional regulation of gene expression. However, the regulatory mechanism of alternative pre-mRNA splicing in ABA signaling remains largely unknown. We previously identified a pentatricopeptide repeat protein SOAR1 (suppressor of the ABAR-overexpressor 1) as a crucial player downstream of ABAR (putative ABA receptor) in ABA signaling. In this study, we identified a SOAR1 interaction partner USB1, which is an exoribonuclease catalyzing U6 production for spliceosome assembly. We reveal that together USB1 and SOAR1 negatively regulate ABA signaling in early seedling development. USB1 and SOAR1 are both required for the splicing of transcripts of numerous genes, including those involved in ABA signaling pathways, suggesting that USB1 and SOAR1 collaborate to regulate ABA signaling by affecting spliceosome assembly. These findings provide important new insights into the mechanistic control of alternative pre-mRNA splicing in the regulation of ABA-mediated plant responses to environmental cues.

In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein

Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs inArabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be “hot spots” for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant’s defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22., The transcription factor WRKY22 links the explant’s defense and osmotic stress response during Arabidopsis shoot tip cryo-stress.

N-hydroxypipecolic acid, which systemically accumulates in phloem sap of Pseudomonas syringae-inoculated cucumber, is synthesized and functions as an activator of systemic acquired resistance in diverse monocotyledonous and dicotyledonous plant species., Recent work has provided evidence for the occurrence of N-hydroxypipecolic acid (NHP) in Arabidopsis thaliana, characterized its pathogen-inducible biosynthesis by a three-step metabolic sequence from l-lysine, and established a central role for NHP in the regulation of systemic acquired resistance. Here, we show that NHP is biosynthesized in several other plant species in response to microbial attack, generally together with its direct metabolic precursor pipecolic acid and the phenolic immune signal salicylic acid. For example, NHP accumulates locally in inoculated leaves and systemically in distant leaves of cucumber in response to Pseudomonas syringae attack, in Pseudomonas-challenged tobacco and soybean leaves, in tomato inoculated with the oomycete Phytophthora infestans, in leaves of the monocot Brachypodium distachyon infected with bacterial (Xanthomonas translucens) and fungal (Magnaporthe oryzae) pathogens, and in M. oryzae-inoculated barley. Notably, resistance assays indicate that NHP acts as a potent inducer of acquired resistance to bacterial and fungal infection in distinct monocotyledonous and dicotyledonous species. Pronounced systemic accumulation of NHP in leaf phloem sap of locally inoculated cucumber supports a function for NHP as a phloem-mobile immune signal. Our study thus generalizes the existence and function of an NHP resistance pathway in plant systemic acquired resistance.

Plants are continuously exposed to different abiotic and biotic stresses; therefore, to protect themselves, they depend on the fast reprogramming of large gene repertoires to prioritize the expression of a given stress-induced gene set over normal cellular household genes. The activity of the proteasome, a large proteolytic complex that degrades proteins, is vital to coordinate the expression of such genes. Proteins are labeled for degradation by the action of E3 ligases that site-specifically alter their substrates by adding chains of ubiquitin. Recent publications have revealed an extensive role of ubiquitination in the utilization of nutrients. This study presents the transcriptomic profiles of sulfur-deficient rosettes and roots of Arabidopsis thaliana rpt2a mutant with proteasomal malfunction. We found that genes connected with sulfur metabolism are regulated to the lesser extent in rpt2a mutant while genes encoding transfer RNAs and small nucleolar RNAs are highly upregulated. Several genes encoding E3 ligases are specifically regulated by sulfur deficiency. Furthermore, we show that a key transcription factor of sulfur deficiency response, Sulfur LIMitation1, undergoes proteasomal degradation and is able to interact with F-box protein, EBF1.

PPC2, together with ABI5 and ABA, is involved in plant acclimation to low CO2 through linking photorespiratory metabolism with primary metabolism., Phosphoenolpyruvate carboxylase (PEPC) plays a pivotal role in the photosynthetic CO2 fixation of C4 plants. However, the functions of PEPCs in C3 plants are less well characterized, particularly in relation to low atmospheric CO2 levels. Of the four genes encoding PEPC in Arabidopsis, PPC2 is considered as the major leaf PEPC gene. Here we show that the ppc2 mutants suffered a growth arrest when transferred to low atmospheric CO2 conditions, together with decreases in the maximum efficiency of PSII (Fv/Fm) and lower levels of leaf abscisic acid (ABA) and carbohydrates. The application of sucrose, malate, or ABA greatly rescued the growth of ppc2 lines under low CO2 conditions. Metabolite profiling analysis revealed that the levels of glycine and serine were increased in ppc2 leaves, while the abundance of photosynthetic metabolites was decreased under these conditions. The transcript levels of encoding enzymes involved in glycine or serine metabolism was decreased in ppc2 in an ABI5-dependent manner. Like the ppc2 mutants, abi5-1 mutants had lower photosynthetic rates and Fv/Fm compared with the wild type under photorespiratory conditions (i.e. low CO2 availability). However, the growth of these mutants was similar to that of the wild type under non-photorespiratory (low O2) conditions. The constitutive expression of ABI5 prevented the growth arrest of ppc2 lines under low CO2 conditions. These findings demonstrate that PPC2 plays an important role in the acclimation of Arabidopsis plants to low CO2 availability by linking photorespiratory metabolism to primary metabolism, and that this is mediated, at least in part, through ABA- and ABI5-dependent processes.

Flavonoids are a major group of plant-specific metabolites that determine flower and seed coloration. In plant cells, flavonoids are synthesized at the cytosolic surface of the endoplasmic reticulum and are sequestered in the vacuole. It is possible that membrane trafficking, including vesicle trafficking and organelle dynamics, contributes to flavonoid transport and accumulation. However, the underlying mechanism has yet to be fully elucidated. Here we show that the Arabidopsis ECHIDNA protein plays a role in flavonoid accumulation in the vacuole and protein trafficking to the vacuole. We found defective pigmentation patterns in echidna seed, possibly caused by reduced levels of proanthocyanidins, which determine seed coloration. The echidna mutant has defects in protein sorting to the protein storage vacuole as well as vacuole morphology. These findings indicate that ECHIDNA is involved in the vacuolar trafficking pathway as well as the previously described secretory pathway. In addition, we found a genetic interaction between echidna and green fluorescent seed 9 (gfs9), a membrane trafficking factor involved in flavonoid accumulation. Our findings suggest that vacuolar trafficking and/or vacuolar development, both of which are collectively regulated by ECHIDNA and GFS9, are required for flavonoid accumulation, resulting in seed coat pigmentation., An Arabidopsis thaliana mutant lacking the trans-Golgi network-localized protein ECHIDNA shows vacuole-related phenotypes, including altered seed coloration, vacuolar protein sorting, and vacuolar morphology.

Use of genetically encoded fluorescent probes to monitor self-incompatibility (SI)-induced changes in pollen of the heterologous Arabidopsis ‘SI’ system has allowed multiparameter imaging and identified the involvement of clathrin-mediated endocytosis., Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS–PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS–green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.

Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.

A series of aryloxyacetic acid derivatives were designed and synthesized as 4-hydoxyphenylpyruvate dioxygenase (HPPD) inhibitors. Preliminary bioassay results reveal that these derivatives are promising Arabidopsis thaliana HPPD (AtHPPD) inhibitors, in particular compounds I12 (Ki = 0.011 µM) and I23 (Ki = 0.012 µM), which exhibit similar activities to that of mesotrione, a commercial HPPD herbicide (Ki = 0.013 µM). Furthermore, the newly synthesized compounds show significant greenhouse herbicidal activities against tested weeds at dosages of 150 g ai/ha. In particular, II4 exhibited high herbicidal activity for pre-emergence treatment that was slightly better than that of mesotrione. In addition, compound II4 was safe for weed control in maize fields at a rate of 150 g ai/ha, and was identified as the most potent candidate for a novel HPPD inhibitor herbicide. The compounds described herein may provide useful guidance for the design of new HPPD inhibiting herbicides and their modification.

Transcription factors (TFs) help plants respond to environmental stresses by regulating gene expression. Up till now, studies on the MYB family of TFs have mainly focused on the highly abundant R2R3-subtype. While the less well-known 1R-subtype has been generally shown to enhance abscisic acid (ABA) sensitivity by acting as transcriptional activators, the mechanisms of their functions are unclear. Here we identified an ABA sensitivity-associated gene from soybean, ABA-Sensitive 1 (GmABAS1), of the 1R-subtype of MYB. Using the GFP-GmABAS1 fusion protein, we demonstrated that GmABAS1 is localized in the nucleus, and with yeast reporter systems, we showed that it is a transcriptional repressor. We then identified the target gene of GmABAS1 to be Glyma.01G060300, an annotated ABI five-binding protein 3 and showed that GmABAS1 binds to the promoter of Glyma.01G060300 both in vitro and in vivo. Furthermore, Glyma.01G060300 and GmABAS1 exhibited reciprocal expression patterns under osmotic stress, inferring that GmABAS1 is a transcriptional repressor of Glyma.01G060300. As a further confirmation, AtAFP2, an orthologue of Glyma.01G060300, was down-regulated in GmABAS1-transgenic Arabidopsis thaliana, enhancing the plant’s sensitivity to ABA. This is the first time a 1R-subtype of MYB from soybean has been reported to enhance ABA sensitivity by acting as a transcriptional repressor., ABAS1 negatively regulates the expression of a soybean AFP3 homologue and enhances ABA sensitivity. This is the first report on the transcriptional repressor activity of a 1R-subtype MYB from soybean.

The CNGC19 calcium channel plays an important role in regulating the colonization of Arabidopsis roots by Piriformospora indica via modulation of systemic and defense-related pathways of MAMP-triggered immunity., The activation of calcium signaling is a crucial event for perceiving environmental stress. Colonization by Piriformospora indica, a growth-promoting root endosymbiont, activates cytosolic Ca2+ in Arabidopsis roots. In this study, we examined the role and functional relevance of calcium channels responsible for Ca2+ fluxes. Expression profiling revealed that CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) is an early-activated gene, induced by unidentified components in P. indica cell-wall extract. Functional analysis showed that loss-of-function of CNGC19 resulted in growth inhibition by P.indica, due to increased colonization and loss of controlled fungal growth. The cngc19 mutant showed reduced elevation of cytosolic Ca2+ in response to P. indica cell-wall extract in comparison to the wild-type. Microbe-associated molecular pattern-triggered immunity was compromised in the cngc19 lines, as evidenced by unaltered callose deposition, reduced cis-(+)-12-oxo-phytodienoic acid, jasmonate, and jasmonoyl isoleucine levels, and down-regulation of jasmonate and other defense-related genes, which contributed to a shift towards a pathogenic response. Loss-of-function of CNGC19 resulted in an inability to modulate indole glucosinolate content during P. indica colonization. CNGC19-mediated basal immunity was dependent on the AtPep receptor, PEPR. CNGC19 was also crucial for P. indica-mediated suppression of AtPep-induced immunity. Our results thus demonstrate that Arabidopsis CNGC19 is an important Ca2+ channel that maintains a robust innate immunity and is crucial for growth-promotion signaling upon colonization by P. indica.

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

The presence of varied numbers of CALCINEURIN B-LIKE10 (CBL10) calcium sensor genes in species across the Brassicaceae and the demonstrated role of CBL10 in salt tolerance in Arabidopsis thaliana and Eutrema salsugineum provided a unique opportunity to determine if CBL10 function is modified in different species and linked to salt tolerance. Salinity effects on species growth and cross-species complementation were used to determine the extent of conservation and divergence of CBL10 function in four species representing major lineages within the core Brassicaceae (A. thaliana, E. salsugineum, Schrenkiella parvula, and Sisymbrium irio) as well as the first diverging lineage (Aethionema arabicum). Evolutionary and functional analyses indicate that CBL10 duplicated within expanded lineage II of the Brassicaceae and that, while portions of CBL10 function are conserved across the family, there are species-specific variations in CBL10 function. Paralogous CBL10 genes within a species diverged in expression and function probably contributing to the maintenance of the duplicated gene pairs. Orthologous CBL10 genes diverged in function in a species-specific manner, suggesting that functions arose post-speciation. Multiple CBL10 genes and their functional divergence may have expanded calcium-mediated signaling responses and contributed to the ability of certain members of the Brassicaceae to maintain growth in salt-affected soils., Duplication of the CBL10 calcium sensor within expanded lineage II of the Brassicaceae led to both conservation of and species-specific variations in CBL10 function across the family.

Summary Flowering time is a key adaptive and agronomic trait. In Arabidopsis, natural variation in expression levels of the floral repressor FLOWERING LOCUS C (FLC) leads to differences in vernalization. In Brassica napus there are nine copies of FLC. Here, we study how these multiple FLC paralogues determine vernalization requirement as a system.We collected transcriptome time series for Brassica napus spring, winter, semi‐winter, and Siberian kale crop types. Modelling was used to link FLC expression dynamics to floral response following vernalization.We show that relaxed selection pressure has allowed expression of FLC paralogues to diverge, resulting in variation of FLC expression during cold treatment between paralogues and accessions. We find that total FLC expression dynamics best explains differences in cold requirement between cultivars, rather than expression of specific FLC paralogues.The combination of multiple FLC paralogues with different expression dynamics leads to rich behaviour in response to cold and a wide range of vernalization requirements in B. napus. We find evidence for different strategies to determine the response to cold in existing winter rapeseed accessions.

Summary Low oxygen availability often is associated with soil waterlogging or submergence, but may occur also as hypoxic niches in otherwise aerobic tissues. Experimental evidence assigns a role in Botrytis cinerea resistance to a group of oxygen‐unstable Ethylene Response Factors (ERF‐VII). Given that infection by B. cinerea often occurs in aerobic organs such as leaves, where ERF‐VII stability should be compromised, we explored the possibility of local leaf hypoxia at the site of infection.We analyzed the expression of hypoxia‐responsive genes in infected leaves. Confocal microscopy was utilized to verify the localization of the ERF‐VII protein RAP2.12. Oxygen concentration was measured to evaluate the availability of oxygen (O2).We discovered that infection by B. cinerea induces increased respiration, leading to a drastic drop in the O2 concentration in an otherwise fully aerobic leaf. The establishment of a local hypoxic area results in stabilization and nuclear relocalization of RAP2.12. The possible roles of defence elicitors, ABA and ethylene were evaluated.Local hypoxia at the site of B. cinerea infection allows the stabilization of ERF‐VII proteins. Hypoxia at the site of pathogen infection generates a nearly O2‐free environment that may affect the stability of other N‐degron‐regulated proteins as well as the metabolism of elicitors., See also the Editorial on this article by Sasidharan et al., 229: 5–7.

Summary Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear.Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains.Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane.This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Iron (Fe) is an essential micronutrient that plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence was on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.

Summary Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells.Here, direct incubation of leaf explants from the non‐medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High‐throughput LC‐MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells.A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA‐treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin‐dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis‐(+)‐12‐oxophytodienoic acid, in synergy, could act as anticancer compounds.Our work suggests a universal role for MeJA‐treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti‐cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments

Summary The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development. The establishment of auxin gradients requires the asymmetric distribution of members of the auxin efflux carrier PIN‐FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localisation. N‐Ethylmaleimide‐sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q‐ or R‐SNAREs based on their sequence. We analysed gain‐ and loss‐of‐function mutants, dominant‐negative transgenics and localisation of the Arabidopsis R‐SNARE VAMP714 protein to understand its function.We demonstrate that VAMP714 is essential for the insertion of PINs into the plasma membrane, for polar auxin transport, root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co‐localises with endoplasmic reticulum and Golgi vesicles and with PIN proteins at the plasma membrane.It is proposed that VAMP714 mediates the delivery of PIN‐carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714‐dependent PIN/auxin transport system to control development.

Summary Plant pathogens use effector proteins to promote host colonisation. The mode of action of effectors from root‐invading pathogens, such as Fusarium oxysporum (Fo), is poorly understood. Here, we investigated whether Fo effectors suppress pattern‐triggered immunity (PTI), and whether they enter host cells during infection.Eight candidate effectors of an Arabidopsis‐infecting Fo strain were expressed with and without signal peptide for secretion in Nicotiana benthamiana and their effect on flg22‐triggered and chitin‐triggered reactive oxidative species (ROS) burst was monitored. To detect uptake, effector biotinylation by an intracellular Arabidopsis‐produced biotin ligase was examined following root infection.Four effectors suppressed PTI signalling; two acted intracellularly and two apoplastically. Heterologous expression of a PTI‐suppressing effector in Arabidopsis enhanced bacterial susceptibility. Consistent with an intracellular activity, host cell uptake of five effectors, but not of the apoplastically acting ones, was detected in Fo‐infected Arabidopsis roots.Multiple Fo effectors targeted PTI signalling, uncovering a surprising overlap in infection strategies between foliar and root pathogens. Extracellular targeting of flg22 signalling by a microbial effector provides a new mechanism on how plant pathogens manipulate their host. Effector translocation appears independent of protein size, charge, presence of conserved motifs or the promoter driving its expression.

Summary The nucellus tissue in flowering plants provides nutrition for the development of the female gametophyte (FG) and young embryo. The nucellus degenerates as the FG develops, but the mechanism controlling the coupled process of nucellar degeneration and FG expansion remains largely unknown.The degeneration process of the nucellus and spatiotemporal auxin distribution in the developing ovule before fertilization were investigated in Arabidopsis thaliana.Nucellar degeneration before fertilization occurs through vacuolar cell death and in an ordered degeneration fashion. This sequential nucellar degeneration is controlled by the signalling molecule auxin.Auxin efflux plays the core role in precisely controlling the spatiotemporal pattern of auxin distribution in the nucellus surrounding the FG. The auxin efflux carrier PIN1 transports maternal auxin into the nucellus while PIN3/PIN4/PIN7 further delivers auxin to degenerating nucellar cells and concurrently controls FG central vacuole expansion. Notably, auxin concentration and auxin efflux are controlled by the maternal tissues, acting as a key communication from maternal to filial tissue.

The multinational Arabidopsis research community is highly collaborative and over the past thirty years these activities have been documented by the Multinational Arabidopsis Steering Committee (MASC). Here, we (a) highlight recent research advances made with the reference plantArabidopsis thaliana; (b) provide summaries from recent reports submitted by MASC subcommittees, projects and resources associated with MASC and from MASC country representatives; and (c) initiate a call for ideas and foci for the "fourth decadal roadmap," which will advise and coordinate the global activities of the Arabidopsis research community., UKRI-BBSRC grant [BB/M004376/1]; HHMI Faculty Scholar Fellowship; Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [118Z137], UKRI-BBSRC grant, Grant/Award Number: BB/M004376/1; HHMI Faculty Scholar Fellowship; the Scientific and Technological Research Council of Turkey, Grant/Award Number: 118Z137

Summary Plants respond to phosphorus (P) limitation through an array of morphological, physiological and metabolic changes which are part of the phosphate (Pi) starvation response (PSR). This response influences the establishment of the arbuscular mycorrhizal (AM) symbiosis in most land plants. It is, however, unknown to what extent available P and the PSR redefine plant interactions with the fungal microbiota in soil.Using amplicon sequencing of the fungal taxonomic marker ITS2, we examined the changes in root‐associated fungal communities in the AM nonhost species Arabidopsis thaliana in response to soil amendment with P and to genetic perturbations in the plant PSR.We observed robust shifts in root‐associated fungal communities of P‐replete plants in comparison with their P‐deprived counterparts, while bulk soil communities remained unaltered. Moreover, plants carrying mutations in the phosphate signaling network genes, phr1, phl1 and pho2, exhibited similarly altered root fungal communities characterized by the depletion of the chytridiomycete taxon Olpidium brassicae specifically under P‐replete conditions.This study highlights the nutritional status and the underlying nutrient signaling network of an AM nonhost plant as previously unrecognized factors influencing the assembly of the plant fungal microbiota in response to P in nonsterile soil.

SummaryThe epidermal cells of leaves lend themselves readily to observation and display many shapes and types: tabular pavement cells, complex trichomes, and stomatal complexes1. Pavement cells fromZea mays(maize) andArabidopsis thaliana(arabidopsis) both have highly undulate anticlinal walls and are held as representative of monocots and eudicots, respectively. In these two model species, we have a nuanced understanding of the molecular mechanisms that generate undulating pavement cell shape2–9. This model-system dominance has led to two common assumptions: first, that particular plant lineages are characterized by particular pavement cell shapes; and second, that undulatory pavement cell shapes are common enough to be model shapes. To test these assumptions, we quantified pavement cell shape in the leaves of 278 vascular plant taxa and assessed cell shape metrics across large taxonomic groups. We settled on two metrics that described cell shape diversity well in this dataset: aspect ratio (degree of cell elongation) and solidity (a proxy for margin undulation). We found that pavement cells in the monocots tended to have weakly undulating margins, pavement cells in ferns had strongly undulating margins, and pavement cells in the eudicots showed no particular degree of undulation. Indeed, we found that cells with strongly undulating margins, like those of arabidopsis and maize, were in the minority in seed plants. At the organ level, we found a trend towards cells with more undulating margins on the abaxial leaf surface vs. the adaxial surface. We also detected a correlation between cell and leaf aspect ratio: highly elongated leaves tended to have highly elongated cells (low aspect ratio), but not in the eudicots. This indicates that while plant anatomy and plant morphology can be connected, superficially similar leaves can develop through very different underlying growth dynamics (cell expansion and division patterns). This work reveals the striking diversity of pavement cell shapes across vascular plants, and lays the quantitative groundwork for testing hypotheses about pavement cell form and function.

Summary Plant‐specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY‐YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators.Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana.Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET‐specific differentially expressed genes and mostly independently characteristic, ET‐specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects.The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

Soil water‐logging and flooding are common environmental stress conditions that can impair plant fitness. Roots are the first organs to be confronted with reduced oxygen tension as a result of flooding. While anatomical and morphological adaptations of roots are extensively studied, the root system architecture is only now becoming a focus of flooding research. Adventitious root (AR) formation shifts the root system higher up the plant, thereby facilitating supply with oxygen, and thus improving root and plant survival.We used Arabidopsis knockout mutants and overexpressors of ERFVII transcription factors to study their role in AR formation under hypoxic conditions and in response to ethylene.Results show that ethylene inhibits AR formation. Hypoxia mainly promotes AR elongation rather than formation mediated by ERFVII transcription factors, as indicated by reduced AR elongation in erfVII seedlings. Overexpression of HRE2 induces AR elongation to the same degree as hypoxia, while ethylene overrides HRE2‐induced AR elongation.The ERFVII transcription factors promote establishment of an AR system that is under negative control by ethylene. Inhibition of growth of the main root system and promotion of AR elongation under hypoxia strengthens the root system in upper soil layers where oxygen shortage may last for shorter time periods.

The polar transport of auxin controls many aspects of plant development. However, the molecular mechanisms underlying auxin tranport regulation remain to be further elucidated. We identified a mutant named as usl1 (unflattened and small leaves) in a genetic screen in Arabidopsis thaliana. The usl1 displayed multiple aspects of developmental defects in leaves, embryogenesis, cotyledons, silique phyllotaxy and lateral roots in addition to abnormal leaves. USL1 encodes a protein orthologous to the yeast vacuolar protein sorting (Vps) 38p and human UV RADIATION RESISTANCE‐ASSOCIATED GENE (UVRAG). Cell biology, Co‐IP/MS and yeast two‐hybrid were used to identify the function of USL1. USL1 colocalizes at the subcellular level with VPS29, a key factor of the retromer complex that controls auxin transport. The morphology of the VPS29‐associated late endosomes (LE) is altered from small dots in the wild‐type to aberrant enlarged circles in the usl1 mutants. The usl1 mutant synergistically interacts with vps29. We also found that USL1 forms a complex with AtVPS30 and AtVPS34. We propose that USL1 controls multiple aspects of plant development by affecting late endosome morphology and by regulating the PIN1 polarity. Our findings provide a new layer of the understanding on the mechanisms of plant development regulation.

Summary Susceptibility to the root‐knot nematode Meloidogyne incognita in plants is thought to be a complex trait based on multiple genes involved in cell differentiation, growth and defence. Previous genetic analyses of susceptibility to M. incognita have mainly focused on segregating dominant resistance genes in crops. It is not known if plants harbour significant genetic variation in susceptibility to M. incognita independent of dominant resistance.To study the genetic architecture of susceptibility to M. incognita, we analysed nematode reproduction on a highly diverse set of 340 natural inbred lines of Arabidopsis thaliana with genome‐wide association mapping. We observed a surprisingly large variation in nematode reproduction among these lines.Genome‐wide association mapping revealed four quantitative trait loci (QTLs) located on chromosomes 1 and 5 of A. thaliana significantly associated with reproductive success of M. incognita, none of which harbours typical resistance gene homologues. Mutant analysis of three genes located in two QTLs showed that the transcription factor BRASSINAZOLE RESISTANT1 and an F‐box family protein may function as (co‐)regulators of susceptibility to M. incognita in Arabidopsis.Our data suggest that breeding for loss‐of‐susceptibility, based on allelic variants critically involved in nematode feeding, could be used to make crops more resilient to root‐knot nematodes.

Plant response to light is a complex and diverse phenomenon. Several studies have elucidated the mechanisms via which light and hormones regulate hypocotyl growth. However, the hormone-dependent ultraviolet-B (UV-B) response in plants remains obscure. Involvement of gibberellins (GAs) in UV-B-induced hypocotyl inhibition and its mechanisms in Arabidopsis thaliana were investigated in the present research. UV-B exposure remarkably decreased the endogenous GA(3) content through the UV RESISTANCE LOCUS 8 (UVR8) receptor pathway, and exogenous GA(3) partially restored the hypocotyl growth. UV-B irradiation affected the expression levels of GA metabolism-related genes (GA20ox1, GA2ox1 and GA3ox1) in the hy5-215 mutant, resulting in increased GA content.ELONGATED HYPOCOTYL 5 (HY5) promoted the accumulation of DELLA proteins under UV-B radiation; HY5 appeared to regulate the abundance of DELLAs at the transcriptional level under UV-B. As a result, the GA(3) content decreased, which eventually led to the shortening of the hypocotyl. To conclude, the present study provides new insight into the regulation of plant photomorphogenesis under UV-B.