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Summary: During arbuscular mycorrhizal (AM) symbiosis, plant innate immunity is modulated to a prime state to allow for fungal colonization. The underlying mechanisms remain to be further explored. In this study, two rice genes encoding LysM extracellular (LysMe) proteins were investigated.By obtaining OsLysMepro:GUS transgenic plants and generating oslysme1, oslysme2 and oslysme1oslysme2 mutants via CRISPR/Cas9 technique, OsLysMe genes were revealed to be specifically induced in the arbusculated cells and mutations in either gene caused significantly reduced root colonization rate by AM fungus Rhizophagus irregularis. Overexpression of OsLysMe1 or OsLysMe2 dramatically increased the colonization rates in rice and Medicago truncatula.The electrophoretic mobility shift assay and dual‐luciferase reporter assay supported that OsLysMe genes are regulated by OsWRI5a. Either OsLysMe1 or OsLysMe2 can efficiently rescue the impaired AM phenotype of the mtlysme2 mutant, supporting a conserved function of LysMe across monocotyledonous and dicotyledonous plants.The co‐localization of OsLysMe proteins with the apoplast marker SP‐OsRAmy3A implies their probable localization to the periarbuscular space (PAS) during symbiosis. Relative to the fungal biomass marker RiTEF, some defense‐related genes showed disproportionately high expression levels in the oslysme mutants. These data support that rice plants deploy two OsLysMe proteins to facilitate AM symbiosis, likely by diminishing plant defense responses. [ABSTRACT FROM AUTHOR]

Summary Plant homeodomain leucine zipper IV (HD‐Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)‐related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START‐dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid‐ and phosphate (Pi) starvation‐related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression. [ABSTRACT FROM AUTHOR]

Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX‐based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild‐type and the glycine decarboxylase T‐protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)‐dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C‐labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine‐tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions. Summary statement: Thioredoxins o1 and h2 concertedly adapt the performance of mtLPD1‐dependent pathways towards short‐ and long‐term environmental changes. Simultaneous adjustments of mitochondrial pathway fluxes are achieved through fine tuning of mtLPD1 activity via redox regulation and subcellular NADH/NAD+ ratios. [ABSTRACT FROM AUTHOR]

Chloroplast function is essential for growth, development, and plant adaptation to stress. Organelle stress and plant defence responses were examined here using noxy8 (nonresponding to oxylipins 8) from a series of Arabidopsis mutants. The noxy8 mutation was located at the CLPC2 gene, encoding a chloroplast chaperone of the protease complex CLP. Although its CLPC1 paralogue is considered to generate redundancy, our data reveal significant differences distinguishing CLPC2 and CLPC1 functions. As such, clpc1 mutants displayed a major defect in housekeeping chloroplast proteostasis, leading to a pronounced reduction in growth and pigment levels, enhanced accumulation of chloroplast and cytosol chaperones, and resistance to fosmidomycin. Conversely, clpc2 mutants showed severe susceptibility to lincomycin inhibition of chloroplast translation and resistance to Antimycin A inhibition of mitochondrial respiration. In the response to Pseudomonas syringae pv. tomato, clpc2 but not clpc1 mutants were resistant to bacterial infection, showing higher salicylic acid levels, defence gene expression and 9‐LOX pathway activation. Our findings suggest CLPC2 and CLPC1 functional specificity, with a preferential involvement of CLPC1 in housekeeping processes and of CLPC2 in stress responses. Summary statement: The CLPC1 and CLPC2 chaperones participate in protein unfolding prior degradation by the chloroplast CLP protease. In contrast to previous data indicating functional redundancy, our results suggest CLPC2 and CLPC1 specificity, with a preferential involvement of CLPC1 in housekeeping processes and of CLPC2 in stress responses. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis lamin analogs CROWDED NUCLEIs (CRWNs) are necessary to maintain nuclear structure, genome function, and proper plant growth. However, whether and how CRWNs impact reproduction and genome‐wide epigenetic modifications is unknown. Here, we investigate the role of CRWNs during the development of gametophytes, seeds, and endosperm, using genomic and epigenomic profiling methods.We observed defects in crwn mutant seeds including seed abortion and reduced germination rate. Quadruple crwn null genotypes were rarely transmitted through gametophytes. Because defects in seeds often stem from abnormal endosperm development, we focused on crwn1 crwn2 (crwn1/2) endosperm. These mutant seeds exhibited enlarged chalazal endosperm cysts and increased expression of stress‐related genes and the MADS‐box transcription factor PHERES1 and its targets.Previously, it was shown that PHERES1 expression is regulated by H3K27me3 and that CRWN1 interacts with the PRC2 interactor PWO1. Thus, we tested whether crwn1/2 alters H3K27me3 patterns. We observed a mild loss of H3K27me3 at several hundred loci, which differed between endosperm and leaves.These data indicate that CRWNs are necessary to maintain the H3K27me3 landscape, with tissue‐specific chromatin and transcriptional consequences. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis Col‐0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown.To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa‐Cala2 and Hpa‐Noks1. We identified ATR2Cala2 as a non‐canonical RxLR‐type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection.ATR2Cala2 is recognized in accession Col‐0 but not in Ler‐0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility.Two additional Col‐0 TIR‐NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa‐Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2‐recognizing accessions. [ABSTRACT FROM AUTHOR]

Summary: Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation.We tested whether mutating potential ubiquitin‐conjugation sites in a previously reported improved Sesamum indicum OLE (SiO) variant, o3‐3 Cys‐OLE (SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3.Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two‐fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5, containing the five predominant TAG‐increasing mutations from SiCOv3, in Arabidopsis along with mouse DGAT2 (mD) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD.Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC‐dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops. See also the Commentary on this article by Sadre, 243: 7–9. [ABSTRACT FROM AUTHOR]

Summary: N6‐methyladenonsine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m6A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m6A editing tools by fusing the m6A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m6A sites on mRNAs. We demonstrated that our m6A editors could efficiently and specifically deposit and remove m6A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT‐ROOT (SHR) transcripts with a methylation editor could significantly increase its m6A levels with limited off‐target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study the functions of individual m6A modifications in plants, and may also have potential applications for future crop improvement. [ABSTRACT FROM AUTHOR]

Cell‐specific transcriptional regulatory networks (TRNs) play vital roles in plant development and response to environmental stresses. However, traditional single‐cell mono‐omics techniques are unable to directly capture the relationships and dynamics between different layers of molecular information within the same cells. While advanced algorithm facilitates merging scRNA‐seq and scATAC‐seq datasets, accurate data integration remains a challenge, particularly when investigating cell‐type‐specific TRNs. By examining gene expression and chromatin accessibility simultaneously in 16,670 Arabidopsis root tip nuclei, the TRNs are reconstructed that govern root tip development under osmotic stress. In contrast to commonly used computational integration at cell‐type level, 12,968 peak‐to‐gene linkage is captured at the bona fide single‐cell level and construct TRNs at an unprecedented resolution. Furthermore, the unprecedented datasets allow to more accurately reconstruct the coordinated changes of gene expression and chromatin states during cellular state transition. During root tip development, chromatin accessibility of initial cells precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for subsequent differentiation steps. Pseudo‐time trajectory analysis reveal that osmotic stress can shift the functional differentiation of trichoblast. Candidate stress‐related gene‐linked cis‐regulatory elements (gl‐cCREs) as well as potential target genes are also identified, and uncovered large cellular heterogeneity under osmotic stress. [ABSTRACT FROM AUTHOR]

Summary Single‐cell proteomics (SCP) is an emerging approach to resolve cellular heterogeneity within complex tissues of multi‐cellular organisms. Here, we demonstrate the feasibility of SCP on plant samples using the model plant Arabidopsis thaliana. Specifically, we focused on examining isolated single cells from the cortex and endodermis, which are two adjacent root cell types derived from a common stem cell lineage. From 756 root cells, we identified 3763 proteins and 1118 proteins/cell. Ultimately, we focus on 3217 proteins quantified following stringent filtering. Of these, we identified 596 proteins whose expression is enriched in either the cortex or endodermis and are able to differentiate these closely related plant cell types. Collectivity, this study demonstrates that SCP can resolve neighboring cell types with distinct functions, thereby facilitating the identification of biomarkers and candidate proteins to enable functional genomics. [ABSTRACT FROM AUTHOR]

SUMMARY Verticillium dahliae, a soil‐borne fungal pathogen, compromises host innate immunity by secreting a plethora of effectors, thereby facilitating host colonization and causing substantial yield and quality losses. The mechanisms underlying the modulation of cotton immunity by V. dahliae effectors are predominantly unexplored. In this study, we identified that the V. dahliae effector Vd6317 inhibits plant cell death triggered by Vd424Y and enhances PVX viral infection in Nicotiana benthamiana. Attenuation of Vd6317 significantly decreased the virulence of V. dahliae, whereas ectopic expression of Vd6317 in Arabidopsis and cotton enhanced susceptibility to V. dahliae infection, underscoring Vd6317's critical role in pathogenicity. We observed that Vd6317 targeted the Arabidopsis immune regulator AtNAC53, thereby impeding its transcriptional activity on the defense‐associated gene AtUGT74E2. Arabidopsis nac53 and ugt74e2 mutants exhibited heightened sensitivity to V. dahliae compared to wild‐type plants. A mutation at the conserved residue 193L of Vd6317 abrogated its interaction with AtNAC53 and reduced the virulence of V. dahliae, which was partially attributable to a reduction in Vd6317 protein stability. Our findings unveil a hitherto unrecognized regulatory mechanism by which the V. dahliae effector Vd6317 directly inhibits the plant transcription factor AtNAC53 activity to suppress the expression of AtUGT74E2 and plant defense. [ABSTRACT FROM AUTHOR]

Background: Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gβ subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling's survival under high salinity remains unclear. Results: We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. Conclusions: Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression. [ABSTRACT FROM AUTHOR]

In this study, the phytoremediation efficiency of Arabidopsis halleri L. in response to mechanical injury were compared between those irrigated with magnetized water and those irrigated with normal water. Under normal irrigation treatment, wounding stress increased malondialdehyde (MDA) concentrations and hydrogen peroxide (H2O2) levels in A. halleri leaves significantly, by 46.7–86.1% and 39.4–77.4%, respectively, relative to those in the intact tissues. In addition, wounding stresses decreased the content of Cd in leaves by 26.8–52.2%, relative to the control, indicating that oxidative damage in plant tissues was induced by mechanical injury, rather than Cd accumulation. There were no significant differences in MDA and H2O2 between A. halleri irrigated with magnetized water and with normal water under wounding conditions; however, the activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) in the leaves of plants treated with magnetized water were significantly increased by 25.1–56.7%, 47.3–183.6%, and 44.2–109.4%, respectively. Notably, under the magnetic field, the phytoremediation effect of 30% wounded A. halleri nearly returned to normal levels. We find that irrigation with magnetized water is an economical pathway to improve the tolerance of A. halleri to inevitable mechanical injury and may recover its phytoremediation effect. [ABSTRACT FROM AUTHOR]

Summary: In plant species, anthocyanin accumulation is specifically regulated by light signaling. Although the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA‐105 (COP1/SPA) complex is known to control anthocyanin biosynthesis in response to light, the precise mechanism underlying this process remains largely unknown.Here, we report that Increase in BONSAI Methylation 1 (IBM1), a JmjC domain‐containing histone demethylase, participates in the regulation of light‐induced anthocyanin biosynthesis in Arabidopsis.The expression of IBM1 was induced by high light (HL) stress, and loss‐of‐function mutations in IBM1 led to accelerated anthocyanin accumulation under HL conditions. We further identified that IBM1 is directly associated with SPA1/3/4 chromatin in vivo to establish a hypomethylation status on H3K9 and DNA non‐CG at these loci under HL, thereby releasing their expression. Genetic analysis showed that quadruple mutants of IBM1 and SPA1/3/4 resemble spa134 mutants. Overexpression of SPA1 in ibm1 mutants complements the mutant phenotype.Our results elucidate the significance and mechanism of IBM1 histone demethylase in the epigenetic regulation of anthocyanin biosynthesis in Arabidopsis under HL conditions. [ABSTRACT FROM AUTHOR]

Summary: Nicotianamine (NA) plays a crucial role in transporting metal ions, including iron (Fe), in plants; therefore, NICOTIANAMINE SYNTHASE (NAS) genes, which control NA synthesis, are tightly regulated at the transcriptional level. However, the transcriptional regulatory mechanisms of NAS genes require further investigations.In this study, we determined the role of bZIP44 in mediating plant response to Fe deficiency stress by conducting transformation experiments and assays.bZIP44 positively regulated the response of Arabidopsis to Fe deficiency stress by interacting with MYB10 and MYB72 to enhance their abilities to bind at NAS2 and NAS4 promoters, thereby increasing NAS2 and NAS4 transcriptional levels and promote NA synthesis.In summary, the transcription activities of bZIP44, MYB10, and MYB72 were induced in response to Fe deficiency stress, which enhanced the interaction between bZIP44 and MYB10 or MYB72 proteins, synergistically activated the transcriptional activity of NAS2 and NAS4, promoted NA synthesis, and improved Fe transport, thereby enhancing plant tolerance to Fe deficiency stress. [ABSTRACT FROM AUTHOR]

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues. Synopsis: The Arabidopsis leucine-rich repeat (LRR) receptor kinase SCHENGEN3 is necessary for development of the Casparian strip, a watertight lining that surrounds the vascular cells in plant roots. This work reveals that, in sub-optimal and complex soil conditions, SCHENGEN3 also decodes information on soil quality for integration into above-ground growth programs. A semi-synthetic MYB36 positive feedback loop (MYB36Loop) induces earlier and wider Casparian strip establishment in roots when compared to wild-type plants. Casparian strip formation in MYB36Loop plants is independent of Schengen pathway signaling. MYB36Loop over-rides the endogenous signaling pathways which regulate Casparian strip permeability in response to soil nitrogen status. The Schengen pathway informs the shoot of soil nitrogen status via an RBOHF-dependent signal. Decoupling Casparian strip formation from SCHENGEN3 signaling reveals new downstream pathways for the receptor. [ABSTRACT FROM AUTHOR]

SUMMARY: In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase‐like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5‐1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5‐5 and pgr5‐6, using the CRISPR‐Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5‐1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5‐1 allele. In the pgr5‐1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5‐1 locus. Overexpression of the pgr5‐1 allele, encoding the glycine130‐to‐serine change, complemented the pgr5‐5 phenotype, suggesting that the pgr5‐1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity. Significance Statement: Arabidopsis double mutants defective in two pathways of cyclic electron transport were generated using knockout alleles of proton gradient regulation 5 (pgr5). The activity of photosystem I was severely impaired, resulting in a pronounced growth defect. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants have developed the ability to adjust to the day/night cycle through the expression of diel genes, which allow them to effectively respond to environmental changes and optimise their growth and development. Diel oscillations also have substantial implications in many physiological processes, including photosynthesis, floral development, and environmental stress responses. The expression of diel genes is regulated by a combination of the circadian clock and responses to environmental cues, such as light and temperature. A great deal of information is available on the transcriptional regulation of diel gene expression. However, the extent to which translational regulation is involved in controlling diel changes in expression is not yet clear. To investigate the impact of translational regulation on diel expression, we conducted Ribo‐seq and RNA‐seq analyses on a time‐series sample of Arabidopsis shoots cultivated under a 12 h light/dark cycle. Our results showed that translational regulation is involved in about 71% of the genes exhibiting diel changes in mRNA abundance or translational activity, including clock genes, many of which are subject to both translational and transcriptional control. They also revealed that the diel expression of glycosylation and ion‐transporter‐related genes is mainly established through translational regulation. The expression of several diel genes likely subject to translational regulation through upstream open‐reading frames was also determined. Significance Statement: For plants, adaptation to diel oscillations in the environment is important for many biological processes; however, the impact of translational regulation on diel gene expression is not fully understood. In this study, time‐series ribosome profiling (Ribo‐seq) revealed that many diel genes, including clock genes, are translationally regulated; moreover, genes exhibiting diel oscillation solely in terms of translational activity tend to be more highly expressed at night. [ABSTRACT FROM AUTHOR]

SUMMARY: Deficiency in chromatin assembly factor‐1 (CAF‐1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication‐dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild‐type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single‐strand annealing pathway of homology‐dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF‐1 dysfunction. Significance Statement: We explore the contribution of KU70 to the specific genome instability of plants dysfunctional in chromatin assembly factor‐1 (fas1 plants) expressed as a progressive loss of telomeres and rDNA repeats. While KU dysfunction does not markedly affect rDNA instability in fas1 plants, it fully suppresses the progressive loss of telomeres in fas1 plants through activation of alternative telomere lengthening. [ABSTRACT FROM AUTHOR]

SUMMARY: Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de‐etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho‐signaling responders to light. Here, we reported a large‐scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule‐associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1‐INTERACTING‐PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7‐YFP and the phospho‐mimetic CIP7S915D‐YFP, but not the phospho‐null CIP7S915A‐YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho‐regulation of MT‐based processes during skotomorphogenic hypocotyl growth. Significance Statement: In our large‐scale phosphoproteome assay to elucidate the initial phospho‐signaling components triggered by a short light pulse, we found an enrichment of microtubule (MTs)‐associated proteins. Among these proteins, CIP7 (COP1‐INTERATING‐PROTEIN‐7) localizes to MTs and affects hypocotyl growth and MT orientation in dark hypocotyls. Serine915 of CIP7 is dephosphorylated by a brief light pulse. Phosphorylation status of Serine915 regulates this response. Our work establishes a link among skotomorphogenic development, phosphorylation dynamics and MTs‐based processes. [ABSTRACT FROM AUTHOR]

SUMMARY: Histone 2B ubiquitination (H2Bub) and trimethylation of H3 at lysine 4 (H3K4me3) are associated with transcription activation. However, the function of these modifications in transcription in plants remains largely unknown. Here, we report that coordination of H2Bub and H3K4me3 deposition with the binding of the RNA polymerase‐associated factor VERNALIZATION INDEPENDENCE2 (VIP2) to FLOWERING LOCUS C (FLC) modulates flowering time in Arabidopsis. We found that RING domain protein HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (we refer as HUB1/2), which are responsible for H2Bub, interact with ARABIDOPSIS TRITHORAX1 (ATX1), which is required for H3K4me3 deposition, to promote the transcription of FLC and repress the flowering time. The atx1‐2 hub1‐10 hub2‐2 triple mutant in FRIGIDIA (FRI) background displayed early flowering like FRI hub1‐10 hub2‐2 and overexpression of ATX1 failed to rescue the early flowering phenotype of hub1‐10 hub2‐2. Mutations in HUB1 and HUB2 reduced the ATX1 enrichment at FLC, indicating that HUB1 and HUB2 are required for ATX1 recruitment and H3K4me3 deposition at FLC. We also found that the VIP2 directly binds to HUB1, HUB2, and ATX1 and that loss of VIP2 in FRI hub1‐10 hub2‐2 and FRI atx1‐2 plants resulted in early flowering like that observed in FRI vip2‐10. Loss of function of HUB2 and ATX1 impaired VIP2 enrichment at FLC, and reduced the transcription initiation and elongation of FLC. In addition, mutations in VIP2 reduced HUB1 and ATX1 enrichment and H2Bub and H3K4me3 levels at FLC. Together, our findings revealed that HUB1/2, ATX1, and VIP2 coordinately modulate H2Bub and H3K4me3 deposition, FLC transcription, and flowering time. Significance Statement: Flowering at appropriate time is a critical trait for environmental adaption for plant pollination, and seed production. Here, we show that H2Bub, H3K4me3, and VIP2 coordinately modulate the transcription process and flowering time in Arabidopsis. [ABSTRACT FROM AUTHOR]

SUMMARY: Abscisic acid (ABA) is a phytohormone that promotes leaf senescence in response to environmental stress. We previously identified methyl CpG‐binding domain 10 (MBD10) as a phosphoprotein that becomes differentially phosphorylated after ABA treatment in Arabidopsis. ABA‐induced leaf senescence was delayed in mbd10 knockout plants but accelerated in MBD10‐overexpressing plants, suggesting that MBD10 positively regulates ABA‐induced leaf senescence. ABA‐induced phosphorylation of MBD10 occurs in planta on Thr‐89, and our results demonstrated that Thr‐89 phosphorylation is essential for MBD10's function in leaf senescence. The in vivo phosphorylation of Thr‐89 in MBD10 was significantly downregulated in a quadruple mutant of group C MAPKs (mpk1/2/7/14), and group C MAPKs directly phosphorylated MBD10 in vitro. Furthermore, mpk1/2/7/14 showed a similar phenotype as seen in mbd10 for ABA‐induced leaf senescence, suggesting that group C MAPKs are the cognate kinases of MBD10 for Thr‐89. Because group C MAPKs have been reported to function downstream of SnRK2s, our results indicate that group C MAPKs and MBD10 constitute a regulatory pathway for ABA‐induced leaf senescence. [ABSTRACT FROM AUTHOR]

High temperatures are a major stress factor that limit the growth of Pinellia ternata. WRKY proteins widely distribute in plants with the important roles in plant growth and stress responses. However, WRKY genes have not been identified in P. ternata thus far. In this study, five PtWRKYs with four functional subgroups were identified in P. ternata. One group III WRKY transcription factor, PtWRKY2, was strongly induced by high temperatures, whereas the other four PtWRKYs were suppressed. Analysis of transcription factor characteristics revealed that PtWRKY2 localized to the nucleus and specifically bound to W-box elements without transcriptional activation activity. Overexpression of PtWRKY2 increased the heat tolerance of Arabidopsis, as shown by the higher percentage of seed germination and survival rate, and the longer root length of transgenic lines under high temperatures compared to the wild-type. Moreover, PtWRKY2 overexpression significantly decreased reactive oxygen species accumulation by increasing the catalase, superoxide dismutase, and peroxidase activities. Furthermore, the selected heat shock-associated genes, including five transcription factors (HSFA1A, HSFA7A, bZIP28, DREB2A, and DREB2B), two heat shock proteins (HSP70 and HSP17.4), and three antioxidant enzymes (POD34, CAT1, and SOD1), were all upregulated in transgenic Arabidopsis. The study identifies that PtWRKY2 functions as a key transcriptional regulator in the heat tolerance of P. ternata, which might provide new insights into the genetic improvement of P. ternata. [ABSTRACT FROM AUTHOR]

SUMMARY Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein–RNA, protein–DNA, and protein–protein interactions. Here, we reveal an integrated multi‐level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15‐inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING‐type E3 ligase MED25‐BINDING RING‐H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild‐type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2‐dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss‐of‐function and decreased in MBR2 overexpression backgrounds. Heat stress‐induced accumulation of HSC70.3 promoted MBR2‐mediated degradation of C3H15 protein, implying that an auto‐regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops. [ABSTRACT FROM AUTHOR]

T-DNA transformation is prevalent in Arabidopsis research and has expanded to a broad range of crops and model plants. While major progress has been made in optimizing the Agrobacterium -mediated transformation process for various species, a variety of pitfalls associated with the T-DNA insertion may lead to the misinterpretation of T-DNA mutant analysis. Indeed, secondary mutagenesis either on the integration site or elsewhere in the genome, together with epigenetic interactions between T-DNA inserts or frequent genomic rearrangements, can be tricky to differentiate from the effect of the knockout of the gene of interest. These are mainly the case for genomic rearrangements that become balanced in filial generations without consequential phenotypical defects, which may be confusing particularly for studies that aim to investigate fertility and gametogenesis. As a cautionary note to the plant research community studying gametogenesis, we here report an overview of the consequences of T-DNA-induced secondary mutagenesis with emphasis on the genomic imbalance on gametogenesis. Additionally, we present a simple guideline to evaluate the T-DNA-mutagenized transgenic lines to decrease the risk of faulty analysis with minimal experimental effort. [ABSTRACT FROM AUTHOR]

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell over proliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem. [ABSTRACT FROM AUTHOR]

Phospholipase C (PLC) has been implicated in several stress responses, including drought. Overexpression (OE) of PLC has been shown to improve drought tolerance in various plant species. Arabidopsis contains nine PLC genes, which are subdivided into four clades. Earlier, OE of PLC3, PLC5 or PLC7 was found to increase Arabidopsis ' drought tolerance. Here, we confirm this for three other PLCs: PLC2 , the only constitutively expressed AtPLC; PLC4 , reported to have reduced salt tolerance and PLC9 , of which the encoded enzyme was presumed to be catalytically inactive. To compare each PLC and to discover any other potential phenotype, two independent OE lines of six At PLC genes, representing all four clades, were simultaneously monitored with the GROWSCREEN-FLUORO phenotyping platform, under both control- and mild-drought conditions. To investigate which tissues were most relevant to achieving drought survival, we additionally expressed At PLC5 using 13 different cell- or tissue-specific promoters. While no significant differences in plant size, biomass or photosynthesis were found between PLC lines and wild-type (WT) plants, all PLC -OE lines, as well as those tissue-specific lines that promoted drought survival, exhibited a stronger decrease in 'convex hull perimeter' (= increase in 'compactness') under water deprivation compared to WT. Increased compactness has not been associated with drought or decreased water loss before although a hyponastic decrease in compactness in response to increased temperatures has been associated with water loss. We propose that the increased compactness could lead to decreased water loss and potentially provide a new breeding trait to select for drought tolerance. [ABSTRACT FROM AUTHOR]

Shoot branching affects plant architecture. In strawberry (Fragaria L.), short branches (crowns) develop from dormant axillary buds to form inflorescences and flowers. While this developmental transition contributes greatly to perenniality and yield in strawberry, its regulatory mechanism remains unclear and understudied. In the woodland strawberry (Fragaria vesca), we identified and characterized 2 independent mutants showing more crowns. Both mutant alleles reside in FveMYB117a, a R2R3-MYB transcription factor gene highly expressed in shoot apical meristems, axillary buds, and young leaves. Transcriptome analysis revealed that the expression of several cytokinin pathway genes was altered in the fvemyb117a mutant. Consistently, active cytokinins were significantly increased in the axillary buds of the fvemyb117a mutant. Exogenous application of cytokinin enhanced crown outgrowth in the wild type, whereas the cytokinin inhibitors suppressed crown outgrowth in the fvemyb117a mutant. FveMYB117a binds directly to the promoters of the cytokinin homeostasis genes FveIPT2 encoding an isopentenyltransferase and FveCKX1 encoding a cytokinin oxidase to regulate their expression. Conversely, the type-B Arabidopsis response regulators FveARR1 and FveARR2b can directly inhibit the expression of FveMYB117a, indicative of a negative feedback regulation. In conclusion, we identified FveMYB117a as a key repressor of crown outgrowth by inhibiting cytokinin accumulation and provide a mechanistic basis for bud fate transition in an herbaceous perennial plant. An MYB transcription factor directly regulates cytokinin homeostasis to repress axillary bud growth and consequently affect crown formation in woodland strawberry. [ABSTRACT FROM AUTHOR]

Plants have an astonishing ability to regenerate new organs after wounding. Here, we report that the wound-inducible transcription factor ENHANCER OF SHOOT REGENERATION1 (ESR1) has a dual mode of action in activating ANTHRANILATE SYNTHASE ALPHA SUBUNIT1 (ASA1) expression to ensure auxin-dependent de novo root organogenesis locally at wound sites of Arabidopsis (Arabidopsis thaliana) leaf explants. In the first mode, ESR1 interacts with HISTONE DEACETYLASE6 (HDA6), and the ESR1–HDA6 complex directly binds to the JASMONATE-ZIM DOMAIN5 (JAZ5) locus, inhibiting JAZ5 expression through histone H3 deacetylation. As JAZ5 interferes with the action of ETHYLENE RESPONSE FACTOR109 (ERF109), the transcriptional repression of JAZ5 at the wound site allows ERF109 to activate ASA1 expression. In the second mode, the ESR1 transcriptional activator directly binds to the ASA1 promoter to enhance its expression. Overall, our findings indicate that the dual biochemical function of ESR1, which specifically occurs near wound sites of leaf explants, maximizes local auxin biosynthesis and de novo root organogenesis in Arabidopsis. The dual mode of action of ENHANCER OF SHOOT REGENERATION1, which occurs near wound sites of leaf explants, maximizes local auxin biosynthesis and de novo root organogenesis in Arabidopsis. [ABSTRACT FROM AUTHOR]

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells. Abscisic acid activates CYCLIC NUCLEOTIDE-GATED CHANNELs via OST1 kinase-mediated phosphorylation to elicit and regulate cytosolic Ca2+ signaling for stomatal closure in Arabidopsis. [ABSTRACT FROM AUTHOR]

The dynamic changes in membrane phospholipids affect membrane biophysical properties and cell signaling, thereby influencing numerous biological processes. Nonspecific phospholipase C (NPC) enzymes hydrolyze common phospholipids to release diacylglycerol (DAG), which is converted to phosphatidic acid (PA) and other lipids. In this study, 2 Arabidopsis (Arabidopsis thaliana) tandemly arrayed genes, NPC3 and NPC4, were identified as critical factors modulating auxin-controlled plant growth and tropic responses. Moreover, NPC3 and NPC4 were shown to interact with the auxin efflux transporter PIN-FORMED2 (PIN2). The loss of NPC3 and NPC4 enhanced the endocytosis and vacuolar degradation of PIN2, which disrupted auxin gradients and slowed gravitropic and halotropic responses. Furthermore, auxin-triggered activation of NPC3 and NPC4 is required for the asymmetric PA distribution that controls PIN2 trafficking dynamics and auxin-dependent tropic responses. Collectively, our study reveals an NPC-derived PA signaling pathway in Arabidopsis auxin fluxes that is essential for fine-tuning the balance between root growth and environmental responses. Nonspecific phospholipases NPC3 and NPC4 interact with the transporter PIN-FORMED2 and regulate auxin transport and signaling. [ABSTRACT FROM AUTHOR]

Key message: EXPANSIN15 is involved in petal cell morphology and size, the fusion of the medial tissues in the gynoecium and expansion of fruit valve cells. It genetically interacts with SPATULA and FRUITFULL. Cell expansion is fundamental for the formation of plant tissues and organs, contributing to their final shape and size during development. To better understand this process in flower and fruit development, we have studied the EXPANSIN15 (EXPA15) gene, which showed expression in petals and in the gynoecium. By analyzing expa15 mutant alleles, we found that EXPA15 is involved in petal shape and size determination, by affecting cell morphology and number. EXPA15 also has a function in fruit size, by affecting cell size and number. Furthermore, EXPA15 promotes fusion of the medial tissues in the gynoecium. In addition, we observed genetic interactions with the transcription factors SPATULA (SPT) and FRUITFULL (FUL) in gynoecium medial tissue fusion, style and stigma development and fruit development in Arabidopsis. These findings contribute to the importance of EXPANSINS in floral and fruit development in Arabidopsis. [ABSTRACT FROM AUTHOR]

Key message: The miR822 together with of AGO9 protein, modulates monosporic development in Arabidopsis thaliana through the regulation of target genes encoding Cysteine/Histidine-Rich C1 domain proteins, revealing a new role of miRNAs in the control of megaspore formation in flowering plants. In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a megaspore mother cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana (Arabidopsis) undergoes a monosporic type of gametogenesis as three meiotically derived cells degenerate, and a single one—the functional megaspore (FM), divides mitotically to form the female gametophyte. The genetic basis and molecular mechanisms that control monosporic gametophyte development remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the miR822, give rise to extranumerary surviving megaspores that acquire a FM identity and divides without giving rise to differentiated female gametophytes. The overexpression of three miR822 putative target genes encoding cysteine/histidine-rich C1 (DC1) domain proteins, At5g02350, At5g02330 and At2g13900 results in defects equivalent to those found in mutant mir822 plants. The three miR822 targets genes are overexpressed in ago9 mutant ovules, suggesting that miR822 acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins and restricts the survival of meiotically derived cells to a single megaspore. Our results identify a mechanism mediated by the AGO9-miR822 complex that modulates monosporic female gametogenesis in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Most mechanistic details of chronologically ordered regulation of leaf senescence are unknown. Regulatory networks centered on AtWRKY53 are crucial for orchestrating and integrating various senescence‐related signals. Notably, AtWRKY53 binds to its own promoter and represses transcription of AtWRKY53, but the biological significance and mechanism underlying this self‐repression remain unclear. In this study, we identified the VQ motif‐containing protein AtVQ25 as a cooperator of AtWRKY53. The expression level of AtVQ25 peaked at mature stage and was specifically repressed after the onset of leaf senescence. AtVQ25‐overexpressing plants and atvq25 mutants displayed precocious and delayed leaf senescence, respectively. Importantly, we identified AtWRKY53 as an interacting partner of AtVQ25. We determined that interaction between AtVQ25 and AtWRKY53 prevented AtWRKY53 from binding to W‐box elements on the AtWRKY53 promoter and thus counteracted the self‐repression of AtWRKY53. In addition, our RNA‐sequencing data revealed that the AtVQ25‐AtWRKY53 module is related to the salicylic acid (SA) pathway. Precocious leaf senescence and SA‐induced leaf senescence in AtVQ25‐overexpressing lines were inhibited by an SA pathway mutant, atsid2, and NahG transgenic plants; AtVQ25‐overexpressing/atwrky53 plants were also insensitive to SA‐induced leaf senescence. Collectively, we demonstrated that AtVQ25 directly attenuates the self‐repression of AtWRKY53 during the onset of leaf senescence, which is substantially helpful for understanding the timing of leaf senescence onset modulated by AtWRKY53. [ABSTRACT FROM AUTHOR]

Volatile compounds, such as nitric oxide and ethylene gas, play a vital role as signaling molecules in organisms. Ethylene is a plant hormone that regulates a wide range of plant growth, development, and responses to stress and is perceived by a family of ethylene receptors that localize in the endoplasmic reticulum. Constitutive Triple Response 1 (CTR1), a Raf‐like protein kinase and a key negative regulator for ethylene responses, tethers to the ethylene receptors, but undergoes nuclear translocation upon activation of ethylene signaling. This ER‐to‐nucleus trafficking transforms CTR1 into a positive regulator for ethylene responses, significantly enhancing stress resilience to drought and salinity. The nuclear trafficking of CTR1 demonstrates that the spatiotemporal control of ethylene signaling is essential for stress adaptation. Understanding the mechanisms governing the spatiotemporal control of ethylene signaling elements is crucial for unraveling the system‐level regulatory mechanisms that collectively fine‐tune ethylene responses to optimize plant growth, development, and stress adaptation. [ABSTRACT FROM AUTHOR]

Rhizosphere microbial community assembly results from microbe‐microbe‐plant interactions mediated by small molecules of plant and microbial origin. Studies with Arabidopsis thaliana have indicated a critical role of glucosinolates in shaping the root and/or rhizosphere microbial community, likely through breakdown products produced by plant or microbial myrosinases inside or outside of the root. Plant nitrile‐specifier proteins (NSPs) promote the formation of nitriles at the expense of isothiocyanates upon glucosinolate hydrolysis with unknown consequences for microbial colonisation of roots and rhizosphere. Here, we generated the A. thaliana triple mutant nsp134 devoid of nitrile formation in root homogenates. Using this line and mutants lacking aliphatic or indole glucosinolate biosynthesis pathways or both, we found bacterial/archaeal alpha‐diversity of the rhizosphere to be affected only by the ability to produce aliphatic glucosinolates. In contrast, bacterial/archaeal community composition depended on functional root NSPs as well as on pathways of aliphatic and indole glucosinolate biosynthesis. Effects of NSP deficiency were strikingly distinct from those of impaired glucosinolate biosynthesis. Our results demonstrate that rhizosphere microbial community assembly depends on functional pathways of both glucosinolate biosynthesis and breakdown in support of the hypothesis that glucosinolate hydrolysis by myrosinases and NSPs happens before secretion of products to the rhizosphere. Summary statement: This research demonstrates differential effects of lacking root nitrile‐specifier proteins and glucosinolate biosynthesis pathways on Arabidopsis rhizosphere microbiota. The results strengthen the hypothesis that glucosinolate hydrolysis in planta precedes secretion of products to the rhizosphere. [ABSTRACT FROM AUTHOR]

Multiple organellar RNA editing factor (MORF) complex was shown to be highly associated with C‐to‐U RNA editing of vascular plant editosome. However, mechanisms by which MORF9‐dependent plastid RNA editing controls plant development and responses to environmental alteration remain obscure. In this study, we found that loss of MORF9 function impaired PSII efficiency, NDH activity, and carbohydrate production, rapidly promoted nuclear gene expression including sucrose transporter and sugar/energy responsive genes, and attenuated root growth under sugar starvation conditions. Sugar repletion increased MORF9 and MORF2 expression in wild‐type seedlings and reduced RNA editing of matK‐706, accD‐794, ndhD‐383 and ndhF‐290 in the morf9 mutant. RNA editing efficiency of ndhD‐383 and ndhF‐290 sites was diminished in the gin2/morf9 double mutants, and that of matK‐706, accD‐794, ndhD‐383 and ndhF‐290 sites were significantly diminished in the snrk1/morf9 double mutants. In contrast, overexpressing HXK1 or SnRK1 promoted RNA editing rate of matK‐706, accD‐794, ndhD‐383 and ndhF‐290 in leaves of morf9 mutants, suggesting that HXK1 partially impacts MORF9 mediated ndhD‐383 and ndhF‐290 editing, while SnRK1 may only affect MORF9‐mediated ndhF‐290 site editing. Collectively, these findings suggest that sugar and/or its intermediary metabolites impair MORF9‐dependent plastid RNA editing resulting in derangements of plant root development. Summary statement: Sugar supplementation alters MORF9 levels and MORF9‐dependent plastid RNA editing, which in turn affects nuclear gene expression and root growth in Arabidopsis, suggesting that plastid RNA editing regulates auxin‐related root growth and sugar starvation response via a retrograde signalling pathway. [ABSTRACT FROM AUTHOR]

Zinc is an essential micronutrient for all living organisms. When challenged by zinc‐limiting conditions, Arabidopsis thaliana plants use a strategy centered on two transcription factors, bZIP19 and bZIP23, to enhance the expression of several zinc transporters to improve their zinc uptake capacity. In the zinc and cadmium hyperaccumulator plant Arabidopsis halleri, highly efficient root‐to‐shoot zinc translocation results in constitutive local zinc deficiency in roots and in constitutive high expression of zinc deficiency‐responsive ZIP genes, supposedly boosting zinc uptake and accumulation. Here, to disrupt this process and to analyze the functions of AhbZIP19, AhbZIP23 and their target genes in hyperaccumulation, the genes encoding both transcriptional factors were knocked down using artificial microRNAs (amiRNA). Although AhbZIP19, AhbZIP23, and their ZIP target genes were downregulated, amiRNA lines surprisingly accumulated more zinc and cadmium compared to control lines in both roots and shoot driving to shoot toxicity symptoms. These observations suggested the existence of a substitute metal uptake machinery in A. halleri to maintain hyperaccumulation. We propose that the iron uptake transporter AhIRT1 participates in this alternative pathway in A. halleri. Summary statement: Reduced expression of the zinc deficiency transcription factors bZIP19 and bZIP23 increases zinc and cadmium accumulation in roots and shoot, causes zinc retention in roots and results in cadmium sensitivity in the zinc and cadmium hyperaccumulator plant Arabidopsis halleri. [ABSTRACT FROM AUTHOR]

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects. [ABSTRACT FROM AUTHOR]

Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While Arabidopsis and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of SiAAP9, both of which were induced by NO3− and showed similar expression patterns. The overexpression of SiAAP9L and SiAAP9S in Arabidopsis inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, SiAAP9-OX transgenic Arabidopsis showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of SiAAP9 suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of SiAAP9 have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding. [ABSTRACT FROM AUTHOR]

The NAC family of transcription factors includes no apical meristem (NAM), Arabidopsis thaliana transcription activator 1/2 (ATAF1/2), and cup-shaped cotyledon (CUC2) proteins, which are unique to plants, contributing significantly to their adaptation to environmental challenges. In the present study, we observed that the PvNAC52 protein is predominantly expressed in the cell membrane, cytoplasm, and nucleus. Overexpression of PvNAC52 in Arabidopsis strengthened plant resilience to salt, alkali, osmotic, and ABA stresses. PvNAC52 significantly (p < 0.05) reduced the degree of oxidative damage to cell membranes, proline content, and plant water loss by increasing the expression of MSD1, FSD1, CSD1, POD, PRX69, CAT, and P5CS2. Moreover, the expression of genes associated with abiotic stress responses, such as SOS1, P5S1, RD29A, NCED3, ABIs, LEAs, and DREBs, was enhanced by PvNAC52 overexpression. A yeast one-hybrid assay showed that PvNAC52 specifically binds to the cis-acting elements ABRE (abscisic acid-responsive elements, ACGTG) within the promoter. This further suggests that PvNAC52 is responsible for the transcriptional modulation of abiotic stress response genes by identifying the core sequence, ACGTG. These findings provide a theoretical foundation for the further analysis of the targeted cis-acting elements and genes downstream of PvNAC52 in the common bean. [ABSTRACT FROM AUTHOR]

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles. [ABSTRACT FROM AUTHOR]

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots—its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency. [ABSTRACT FROM AUTHOR]

Summary: The S‐domain‐type receptor‐like kinase (SD‐RLK) LIPOOLIGOSACCHARIDE‐SPECIFIC REDUCED ELICITATION (LORE) from Arabidopsis thaliana is a pattern recognition receptor that senses medium‐chain 3‐hydroxy fatty acids, such as 3‐hydroxydecanoic acid (3‐OH‐C10:0), to activate pattern‐triggered immunity. Here, we show that LORE homomerization is required to activate 3‐OH‐C10:0‐induced immune signaling.Fluorescence lifetime imaging in Nicotiana benthamiana demonstrates that AtLORE homomerizes via the extracellular and transmembrane domains. Co‐expression of AtLORE truncations lacking the intracellular domain exerts a dominant negative effect on AtLORE signaling in both N. benthamiana and A. thaliana, highlighting that homomerization is essential for signaling.Screening for 3‐OH‐C10:0‐induced reactive oxygen species production revealed natural variation within the Arabidopsis genus. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3‐OH‐C10:0, although both possess a putative LORE ortholog. Both LORE orthologs have defective extracellular domains that bind 3‐OH‐C10:0 to a similar level as AtLORE, but lack the ability to homomerize. Thus, ligand binding is independent of LORE homomerization. Analysis of AtLORE and AlyrLORE chimera suggests that the loss of AlyrLORE homomerization is caused by several amino acid polymorphisms across the extracellular domain.Our findings shed light on the activation mechanism of LORE and the loss of 3‐OH‐C10:0 perception within the Arabidopsis genus. [ABSTRACT FROM AUTHOR]

Summary: Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE‐INDUCIBLE GARP‐TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency‐inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency‐induced changes in gene expression is still limited.Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response‐related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency‐inducible expression of three high‐affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N‐deficient conditions.Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N‐sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N‐deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes.These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time‐dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency‐inducible genes. [ABSTRACT FROM AUTHOR]

Summary: Seed dormancy governs germination timing, with both evolutionary and applied consequences. Despite extensive studies on the hormonal and genetic control of these processes, molecular mechanisms directly linking dormancy and germination remain poorly understood.By screening a collection of lines overexpressing Arabidopsis transcription factors, we identified ERF50 as a key gene to control dormancy and germination. To study its regulation, we measured seed‐related physiological parameters in loss‐of‐function mutants and carried out transactivation, protein interaction and ChIP‐PCR analyses.We found direct ERF50‐mediated repression of DOG1 and activation of EXPA2 transcription, which results in enhanced seed germination. Although ERF50 expression is increased by DOG1 in dormant seeds, ERF50 germination‐promoting activity is blocked by RGL2.The physiological, genetic and molecular evidence gathered here supports that ERF50 controls germination timing by regulating DOG1 levels to leverage its role as enhancer of seed germination, via RGL2 antagonism on EXPA2 expression. Our results highlight the central role of ERF50 as a feedback regulator to couple and fine‐tune seed dormancy and germination. [ABSTRACT FROM AUTHOR]

SUMMARY: Potassium (K+), being an essential macronutrient in plants, plays a central role in many aspects. Root growth is highly plastic and is affected by many different abiotic stresses including nutrient deficiency. The Shaker‐type K+ channel Arabidopsis (Arabidopsis thaliana) K+ Transporter 1 (AKT1) is responsible for K+ uptake under both low and high external K+ conditions. However, the upstream transcription factor of AKT1 is not clear. Here, we demonstrated that the WRKY6 transcription factor modulates root growth to low potassium (LK) stress in Arabidopsis. WRKY6 showed a quick response to LK stress and also to many other abiotic stress treatments. The two wrky6 T‐DNA insertion mutants were highly sensitive to LK treatment, whose primary root lengths were much shorter, less biomass and lower K+ content in roots than those of wild‐type plants, while WRKY6‐overexpression lines showed opposite phenotypes. A further investigation showed that WRKY6 regulated the expression of the AKT1 gene via directly binding to the W‐box elements in its promoter through EMSA and ChIP‐qPCR assays. A dual luciferase reporter analysis further demonstrated that WRKY6 enhanced the transcription of AKT1. Genetic analysis further revealed that the overexpression of AKT1 greatly rescued the short root phenotype of the wrky6 mutant under LK stress, suggesting AKT1 is epistatic to WRKY6 in the control of LK response. Further transcriptome profiling suggested that WRKY6 modulates LK response through a complex regulatory network. Thus, this study unveils a transcription factor that modulates root growth under potassium deficiency conditions by affecting the potassium channel gene AKT1 expression. Significance Statement: This study has identified a WRKY‐type transcription factor WRKY6, which positively regulates potassium uptake, and mutants and overexpression lines showed opposite phenotypes. It exerts its role by directly binding to the promoter region of the potassium channel gene AKT1 and promoting its expression. [ABSTRACT FROM AUTHOR]

SUMMARY: The SID2 (SA INDUCTION‐DEFICIENT2) gene that encodes ICS1 (isochorismate synthase), plays a central role in salicylic acid biosynthesis in Arabidopsis. The sid2 and NahG (encoding a bacterial SA hydroxylase) overexpressing mutants (NahG‐OE) have currently been shown to outperform wild type, presenting delayed leaf senescence, higher plant biomass and better seed yield. When grown under sulfate‐limited conditions (low‐S), sid2 mutants exhibited early leaf yellowing compared to the NahG‐OE, the npr1 mutant affected in SA signaling pathway, and WT. This indicated that the hypersensitivity of sid2 to sulfate limitation was independent of the canonical npr1 SA‐signaling pathway. Transcriptomic and proteomic analyses revealed that major changes occurred in sid2 when cultivated under low‐S, changes that were in good accordance with early senescence phenotype and showed the exacerbation of stress responses. The sid2 mutants displayed a lower sulfate uptake capacity when cultivated under low‐S and lower S concentrations in their rosettes. Higher glutathione concentrations in sid2 rosettes under low‐S were in good accordance with the higher abundance of proteins involved in glutathione and ascorbate redox metabolism. Amino acid and lipid metabolisms were also strongly modified in sid2 under low‐S. Depletion of total fatty acids in sid2 under low‐S was consistent with the fact that S‐metabolism plays a central role in lipid synthesis. Altogether, our results show that functional ICS1 is important for plants to cope with S limiting conditions. [ABSTRACT FROM AUTHOR]

SUMMARY: The petals of rose (Rosa sp.) flowers determine the ornamental and industrial worth of this species. The number of petals in roses was previously shown to be subject to fluctuations in ambient temperature. However, the mechanisms by which rose detects and responds to temperature changes are not entirely understood. In this study, we identified short interstitial telomere motifs (telo boxes) in the second intron of AGAMOUS (RcAG) from China rose (Rosa chinensis) that play an essential role in precise temperature perception. The second intron of RcAG harbors two telo boxes that recruit telomere repeat binding factors (RcTRBs), which interact with CURLY LEAF (RcCLF) to compose a repressor complex. We show that this complex suppresses RcAG expression when plants are subjected to low temperatures via depositing H3K27me3 marks (trimethylation of lysine 27 on histone H3) over the RcAG gene body. This regulatory mechanism explains the low‐temperature‐dependent decrease in RcAG transcript levels, leading to the production of more petals under these conditions. Our results underscore an interesting intron‐mediated regulatory mechanism governing RcAG expression, enabling rose plants to perceive temperature cues and establish petal numbers. Significance Statement: Our research underscores a new intron‐mediated regulatory mechanism governing RcAG expression, enabling rose plants to perceive temperature cues and establish petal numbers. It might be of great interest to a broad range of readers including environmental response, epigenetic modification, floral organ development, and other researchers in related fields. [ABSTRACT FROM AUTHOR]