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Summary The endoplasmic reticulum‐associated degradation (ERAD) system eliminates misfolded and short‐lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain‐of‐function mutant, doa10a‐1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono‐ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING‐type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono‐ubiquitination. [ABSTRACT FROM AUTHOR]

SUMMARY Tartary buckwheat is known for its ability to adapt to intricate growth conditions and to possess robust stress‐resistant properties. Nevertheless, it remains vulnerable to drought stress, which can lead to reduced crop yield. To identify potential genes involved in drought resistance, a genome‐wide association study on drought tolerance in Tartary buckwheat germplasm was conducted. A gene encoding pectin methylesterase inhibitors protein (FtPMEI13) was identified, which is not only associated with drought tolerance but also showed induction during drought stress and abscisic acid (ABA) treatment. Further analysis revealed that overexpression of FtPMEI13 leads to improved drought tolerance by altering the activities of antioxidant enzymes and the levels of osmotically active metabolites. Additionally, FtPMEI13 interacts with pectin methylesterase (PME) and inhibits PME activity in response to drought stress. Our results suggest that FtPMEI13 may inhibit the activity of FtPME44/FtPME61, thereby affecting pectin methylesterification in the cell wall and modulating stomatal closure in response to drought stress. Yeast one‐hybrid, dual‐luciferase assays, and electrophoretic mobility shift assays demonstrated that an ABA‐responsive transcription factor FtbZIP46, could bind to the FtPMEI13 promoter, enhancing FtPMEI13 expression. Further analysis indicated that Tartary buckwheat accessions with the genotype resulting in higher FtPMEI13 and FtbZIP46 expression exhibited higher drought tolerance compared to the others. This suggests that this genotype has potential for application in Tartary buckwheat breeding. Furthermore, the natural variation of FtPMEI13 was responsible for decreased drought tolerance during Tartary buckwheat domestication. Taken together, these results provide basic support for Tartary buckwheat breeding for drought tolerance. [ABSTRACT FROM AUTHOR]

SUMMARY: Wheat (Triticum aestivum L.) is a globally staple crop vulnerable to various fungal diseases, significantly impacting its yield. Plant cell surface receptors play a crucial role in recognizing pathogen‐associated molecular patterns (PAMPs) and activating PAMP‐triggered immunity, boosting resistance against a wide range of plant diseases. Although the role of plant chitin receptor CERK1 in immune recognition and defense has been established in Arabidopsis and rice, its function and potential agricultural applications in enhancing resistance to crop diseases remain largely unexplored. Here, we identify and characterize TaCERK1 in Triticeae crop wheat, uncovering its involvement in chitin recognition, immune regulation, and resistance to fungal diseases. By a comparative analysis of CERK1 homologs in Arabidopsis and monocot crops, we demonstrate that AtCERK1 in Arabidopsis elicits the most robust immune response. Moreover, we show that overexpressing TaCERK1 and AtCERK1 in wheat confers resistance to multiple fungal diseases, including Fusarium head blight, stripe rust, and powdery mildew. Notably, transgenic wheat lines with moderately expressed AtCERK1 display superior disease resistance and heightened immune responses without adversely affecting growth and yield, compared to TaCERK1 overexpression transgenics. Our findings highlight the significance of plant chitin receptors across diverse plant species and suggest potential strategies for bolstering crop resistance against broad‐spectrum diseases in agricultural production through the utilization of plant immune receptors. Significance Statement: CERK1 acts as a chitin immune receptor for plant defense and survival, but its role and applied potential in Triticeae crops remain unknown. We identify TaCERK1 in wheat, revealing distinct characteristics of CERK1 homologs in chitin recognition and immune activation between cereals and the dicot Arabidopsis. Notably, overexpressing CERK1s from both cereal and Arabidopsis in wheat confers broad‐spectrum resistance to fungal diseases. Our findings highlight the potential of plant immune receptors in controlling crop diseases. [ABSTRACT FROM AUTHOR]

SUMMARY: Hydathodes are small organs found on the leaf margins of vascular plants which release excess xylem sap through a process called guttation. While previous studies have hinted at additional functions of hydathode in metabolite transport or auxin metabolism, experimental support is limited. We conducted comprehensive transcriptomic, metabolomic and physiological analyses of mature Arabidopsis hydathodes. This study identified 1460 genes differentially expressed in hydathodes compared to leaf blades, indicating higher expression of most genes associated with auxin metabolism, metabolite transport, stress response, DNA, RNA or microRNA processes, plant cell wall dynamics and wax metabolism. Notably, we observed differential expression of genes encoding auxin‐related transcriptional regulators, biosynthetic processes, transport and vacuolar storage supported by the measured accumulation of free and conjugated auxin in hydathodes. We also showed that 78% of the total content of 52 xylem metabolites was removed from guttation fluid at hydathodes. We demonstrate that NRT2.1 and PHT1;4 transporters capture nitrate and inorganic phosphate in guttation fluid, respectively, thus limiting the loss of nutrients during this process. Our transcriptomic and metabolomic analyses unveil an organ with its specific physiological and biological identity. Significance Statement: Hydathodes are vascular discontinuities at leaf margins of all vascular plants which allow the release of excess water during a process known as guttation. The physiology of this organ is poorly characterised. We conducted comprehensive transcriptomic, metabolomic and physiological analyses of mature hydathodes of Arabidopsis and demonstrated that those organs are sites of auxin accumulation and active nutrient scavenging. [ABSTRACT FROM AUTHOR]

SUMMARY Rhythmic oscillation of biological processes helps organisms adapt their physiological responses to the most appropriate time of the day. Chromatin remodeling has been described as one of the molecular mechanisms controlling these oscillations. The importance of these changes in transcriptional activation as well as in the maintenance of heterochromatic regions has been widely demonstrated. However, little is still known on how diurnal changes can impact the global status of chromatin modifications and, hence, control gene expression. In plants, the repressive mark H3K27me1, deposited by ARABIDOPSIS TRITHORAX‐RELATED PROTEIN 5 and 6 (ATXR5 and 6) methyltransferases, is largely associated with transposable elements but also covers lowly expressed genes. Here we show that this histone modification is preferentially deposited during the night. In euchromatic regions, it is found along the bodies of DNA damage response genes (DDR), where it is needed for their proper expression. The absence of H3K27me1 translates into an enhanced expression of DDR genes that follows a rhythmic oscillation pattern. This evidences a link between chromatin modifications and their synchronization with the diurnal cycle in order to accurately modulate the activation of biological processes to the most appropriate time of the day. [ABSTRACT FROM AUTHOR]

• A machine learning model, AtML, was constructed to predict the stages of Arabidopsis root cells and identify biomarkers. • Performance testing revealed that AtML achieved very high accuracy and recall. • The AtML model identified 160 important marker genes for annotating cell type. Arabidopsis thaliana synthesizes various medicinal compounds, and serves as a model plant for medicinal plant research. Single-cell transcriptomics technologies are essential for understanding the developmental trajectory of plant roots, facilitating the analysis of synthesis and accumulation patterns of medicinal compounds in different cell subpopulations. Although methods for interpreting single-cell transcriptomics data are rapidly advancing in Arabidopsis, challenges remain in precisely annotating cell identity due to the lack of marker genes for certain cell types. In this work, we trained a machine learning system, AtML, using sequencing datasets from six cell subpopulations, comprising a total of 6000 cells, to predict Arabidopsis root cell stages and identify biomarkers through complete model interpretability. Performance testing using an external dataset revealed that AtML achieved 96.50% accuracy and 96.51% recall. Through the interpretability provided by AtML, our model identified 160 important marker genes, contributing to the understanding of cell type annotations. In conclusion, we trained AtML to efficiently identify Arabidopsis root cell stages, providing a new tool for elucidating the mechanisms of medicinal compound accumulation in Arabidopsis roots. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results. [ABSTRACT FROM AUTHOR]

Summary: Leaf senescence is a developmental program regulated by both endogenous and environmental cues. Abiotic stresses such as nutrient deprivation can induce premature leaf senescence, which profoundly impacts plant growth and crop yield. However, the molecular mechanisms underlying stress‐induced senescence are not fully understood.In this work, employing a carbon deprivation (C‐deprivation)‐induced senescence assay in Arabidopsis seedlings, we identified PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, as a negative regulator of C‐deprivation‐induced senescence.Furthermore, we demonstrated that PRL1 directly interacts with the RPA2A subunit of the single‐stranded DNA‐binding Replication Protein A (RPA) complex. Consistently, the loss of RPA2A leads to premature senescence, while increased expression of RPA2A inhibits senescence. Moreover, overexpression of RPA2A reverses the accelerated senescence in prl1 mutants, and the interaction with PRL1 stabilizes RPA2A under C‐deprivation.In summary, our findings reveal the involvement of the PRL1‐RPA2A functional module in C‐deprivation‐induced plant senescence. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis, the enzymatically active lysin motif‐containing receptor‐like kinase (LysM‐RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and the pseudokinases LYSIN MOTIF‐CONTAINING RECEPTOR‐LIKE KINASE 5 (LYK5) and LYK4 are the core components of the canonical chitin receptor complex. CERK1 dimerizes and autophosphorylates upon chitin binding, resulting in activation of chitin signaling.In this study, we clarified and further elucidated the individual contributions of LYK4 and LYK5 to chitin‐dependent signaling using mutant (combination)s and stably transformed Arabidopsis plants expressing fluorescence‐tagged LYK5 and LYK4 variants from their endogenous promoters.Our analyses revealed that LYK5 interacts with CERK1 upon chitin treatment, independently of LYK4 and vice versa. We show that chitin‐induced autophosphorylation of CERK1 is predominantly dependent on LYK5, whereas chitin‐triggered ROS generation is almost exclusively mediated by LYK4. This suggests specific signaling functions of these two co‐receptor proteins apart from their redundant function in mitogen‐activated protein kinase (MAPK) signaling and transcriptional reprogramming.Moreover, we demonstrate that LYK5 is subject to chitin‐induced and CERK1‐dependent ubiquitination, which serves as a signal for chitin‐induced internalization of LYK5. Our experiments provide evidence that a combination of phosphorylation and ubiquitination events controls LYK5 removal from the plasma membrane via endocytosis, which likely contributes to receptor complex desensitization. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome‐dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown.We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein–protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP‐qPCR at C‐REPEAT BINDING FACTOR1 (CBF1) and COLD‐REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants.HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity.We propose a novel mechanism whereby HOS15‐mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance. [ABSTRACT FROM AUTHOR]

Flooding events are highly detrimental to most terrestrial plant species. However, there is an impressive diversity of plant species that thrive in flood‐prone regions and represent a treasure trove of unexplored flood‐resilience mechanisms. Here we surveyed a panel of four species from the Cardamineae tribe representing a broad tolerance range. This included the flood‐tolerant Cardamine pratensis, Rorippa sylvestris and Rorippa palustris and the flood‐sensitive species Cardamine hirsuta. All four species displayed a quiescent strategy, evidenced by the repression of shoot growth underwater. Comparative transcriptomics analyses between the four species and the sensitive model species Arabidopsis thaliana were facilitated via de novo transcriptome assembly and identification of 16 902 universal orthogroups at a high resolution. Our results suggest that tolerance likely evolved separately in the Cardamine and Rorippa species. While the Rorippa response was marked by a strong downregulation of cell‐cycle genes, Cardamine minimized overall transcriptional regulation. However, a weak starvation response was a universal trait of tolerant species, potentially achieved in multiple ways. It could result from a strong decline in cell‐cycle activity, but is also intertwined with autophagy, senescence, day‐time photosynthesis and night‐time fermentation capacity. Our data set provides a rich source to study adaptational mechanisms of flooding tolerance. Summary Statement: This collection of submergence‐induced transcriptome responses of Brassicaceae species with strong variation in tolerance provides a treasure trove for the study of adaptational mechanisms of flooding tolerance. [ABSTRACT FROM AUTHOR]

Presence of cadmium in soil affects the growth status of plants; research on the underlying mechanism has focused on its lethal and sublethal consequences in plants, ignoring the impacts of soil organisms. Therefore, this study designed a microorganism – earthworm – plant ecosystem, investigated the influence of Cd (0, 0.3, 1.0, and 3.0 mg kg−1) on the growth of Arabidopsis thaliana in the presence of earthworms (Eisenia fetida), and revealed the mechanisms from the perspective of altered relationships among different soil organisms. In this study, there was no significant difference in the death rate of earthworms with low Cd addition concentration, but the microorganisms in earthworms and soil microorganisms changed, abundance of Actinomycetes in soil increased, while Planctomycetes decreased, Mycobacterium in the gut of Eisenia fetida decreased under Cd stress. Microorganisms affect soil pH, which in turn affects soil physicochemical properties and the form of cadmium in soil. Compared with the control, soil pH significantly decreases under cadmium stress, Under 0.3 mg kg−1 treatment, soil total nitrogen, available phosphorus and total potassium are 181.11, 5.09, 51.90, and 7.55 mg kg−1, respectively, They were significantly higher than control group. Improved the growth environment of Arabidopsis thaliana and promoted the absorption of Cd in soil which affected the proportion of available Cd in the soil (when Cd was added at 1.0 mg kg−1, the proportion of available Cd in the soil decreased significantly from 95.05% to 49.40%). A. thaliana also absorbed more Cd, which increased Cd toxicity, and the reproductive growth of A. thaliana was enhanced, while vegetative growth was weakened.Compared with the control group (278.11 mg), the biomass of A. thaliana decreased significantly to 189.83 mg when Cd was supplemented at 0.3 mg kg−1. Therefore, the activities of and interactions among earthworms, plants, and microorganisms play important roles in reducing soil heavy metals and improving soil properties. HIGHLIGHTS: Under cadmium (Cd) stress, the relative abundance of Actinomycetes in soil increased, while Planctomycetes decreased. The relative abundance of Mycobacterium in the gut of Eisenia fetida decreased under Cd stress. Microbial changes and E. fetida activity changed soil physical and chemical properties and reduced the ratio of soil available Cd. Microorganisms and E. fetida enhanced the reproductive growth and weakened the vegetative growth of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Main conclusion: PATOL1 contributes to increasing biomass not only by effective stomatal movement but also by root meristematic activity. PATROL1 (PROTON ATPase TRANSLOCATION CONTROL 1), a protein with a MUN domain, is involved in the intercellular trafficking of AHA1 H+-ATPase to the plasma membrane in guard cells. This allows for larger stomatal opening and more efficient photosynthesis, leading to increased biomass. Although PATROL1 is expressed not only in stomata but also in other tissues of the shoot and root, the role in other tissues than stomata has not been determined yet. Here, we investigated PATROL1 functions in roots using a loss-of-function mutant and an overexpressor. Cytological observations revealed that root meristematic size was significantly smaller in the mutant resulting in the short primary root. Grafting experiments showed that the shoot biomass of the mutant scion was increased when it grafted onto wild-type or overexpressor rootstocks. Conversely, grafting of the overexpressor scion shoot enhanced the growth of the mutant rootstock. The leaf temperatures of the grafted plants were consistent with those of their respective genotypes, indicating cell-autonomous behavior of stomatal movement and independent roles of PATROL1 in plant growth. Moreover, plasma membrane localization of AHA1 was not altered in root epidermal cells in the patrol1 mutant implying existence of a different mode of PATROL1 action in roots. Thus PATROL1 plays a role in root meristem and contributes to increase shoot biomass. [ABSTRACT FROM AUTHOR]

Phytochrome B (phyB) and phytochrome-interacting factors (PIFs) constitute a well-established signaling module critical for plants adapting to ambient light. However, mechanisms underlying phyB photoactivation and PIF binding for signal transduction remain elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of the photoactivated phyB or the constitutively active phyBY276H mutant in complex with PIF6, revealing a similar trimer. The light-induced configuration switch of the chromophore drives a conformational transition of the nearby tongue signature within the phytochrome-specific (PHY) domain of phyB. The resulting α-helical PHY tongue further disrupts the head-to-tail dimer of phyB in the dark-adapted state. These structural remodelings of phyB facilitate the induced-fit recognition of PIF6, consequently stabilizing the N-terminal extension domain and a head-to-head dimer of activated phyB. Interestingly, the phyB dimer exhibits slight asymmetry, resulting in the binding of only one PIF6 molecule. Overall, our findings solve a key question with respect to how light-induced remodeling of phyB enables PIF signaling in phytochrome research. [Display omitted] • Red light activates phyB by remodeling the head-to-tail dimer in the dark-adapted state • PIF6-APB binding stabilizes the NTE domain and a head-to-head dimer of activated phyB • The asymmetric phyB dimer can accommodate only one PIF6-APB molecule • The constitutively active phyBY276H assembles a similar trimeric complex with PIF6-APB Cryo-EM structures of Arabidopsis photoactivated phyB and its constitutively active phyBY276H mutant in complex with the N terminus of PIF6 provide insights into light-induced photoreceptor remodeling and signal transduction. [ABSTRACT FROM AUTHOR]

Stomatal pores in leaves mediate CO2 uptake into the plant and water loss via transpiration. Most plants are hypostomatous with stomata present only in the lower leaf surface (abaxial epidermis). Many herbs, including the model plant Arabidopsis, have substantial numbers of stomata also on the upper (adaxial) leaf surface. Studies of stomatal development have mostly focused on abaxial stomata and very little is known of adaxial stomatal formation. We analysed the role of leaf number in determining stomatal density and stomatal ratio, and studied adaxial and abaxial stomatal patterns in Arabidopsis mutants deficient in known abaxial stomatal development regulators. We found that stomatal density in some genetic backgrounds varies between different fully expanded leaves, and thus we recommend using defined leaves for analyses of stomatal patterning. Our results indicate that stomatal development is at least partly independently regulated in adaxial and abaxial epidermis, as (i) plants deficient in ABA biosynthesis and perception have increased stomatal ratios, (ii) the epf1epf2 , tmm , and sdd1 mutants have reduced stomatal ratios, (iii) erl2 mutants have increased adaxial but not abaxial stomatal index, and (iv) stomatal precursors preferentially occur in abaxial epidermis. Further studies of adaxial stomata can reveal new insights into stomatal form and function. [ABSTRACT FROM AUTHOR]

Both the pollen tube and hyphae of filamentous pathogens penetrate the outer layer of the host and then grow within host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla in the stigma of Arabidospis, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcome pollen tube. We showed that Phytophtora parasitica , a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cell, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganization of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion. [ABSTRACT FROM AUTHOR]

The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K 0.5 G A B A ~8 mM. l -Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2 -overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress. [ABSTRACT FROM AUTHOR]

SUMMARY Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26‐overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain‐fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very‐long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought‐inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed. [ABSTRACT FROM AUTHOR]

ABSTRACT Nitrate (NO3−) deficiency decreases root water uptake and root hydraulic conductance. This adaptive response is correlated with reduced abundance and activity of plasma membrane intrinsic protein (PIP) aquaporins. We therefore screened changes in the root architecture of a complete set of Arabidopsis pip loss‐of‐function mutants grown under NO3− deficiency to systematically approach the impact of PIPs under these conditions. NO3− deprivation led to attenuated responses of specific pip single mutants compared to the strongly altered LR parameters of wild‐type plants. In particular, pip1;1 exhibited a lower relative reduction in LR length and LR density, revealing that PIP1;1 represses LR development when NO3− is scarce. Indeed, PIP1;1 compromises root and shoot NO3− accumulation during early developmental stages. A fluorescent VENUS‐PIP1;1 fusion revealed that PIP1;1 is specifically repressed in the pericycle, endodermis and at the flanks of emerging LRs upon NO3− deficiency. Thus, LR plasticity and NO3− uptake are affected by an interactive mechanism involving aquaporins (PIP1;1) and nitrate accumulation during seedling development under NO3−‐deficient conditions. [ABSTRACT FROM AUTHOR]

The addition of non-templated nucleotides at the 3' terminus of RNA is a pervasive and evolutionarily conserved posttranscriptional modification in eukaryotes. Apart from canonical poly(A) polymerases (PAPs), which are responsible for catalyzing polyadenylation of messenger RNAs in the nucleus, a distinct group of non-canonical PAPs (ncPAPs), also known as nucleotidyl transferase proteins (NTPs), mediate the addition of uridine and adenosine or of more intricate combinations of nucleotides. Among these, HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1) are the two most extensively studied NTPs responsible for the addition of uridine to the 3' ends of RNAs (RNA uridylation). Recent discoveries have improved our understanding of the functions and mechanisms of uridylation mediated by HESO1 and URT1 in RNA metabolism. Furthermore, more NTPs have been identified to function in the 3' tailing of RNA and not solely through uridylation. Accumulating evidence indicates that RNA tailing plays important roles in plant growth and development, stress responses, and disease resistance. In this review, we examined the latest developments in RNA tailing by NTPs, with a focus on RNA uridylation and metabolism in plants. We also discussed the essential aspects for future research in this field. [ABSTRACT FROM AUTHOR]

Developmental reprogramming allows for flexibility in growth and adaptation to changing environmental conditions. In plants, wounding events can result in new stem cell niches and lateral organs. Adventitious roots develop from aerial parts of the plant and are regulated by multiple stimuli, including wounding. Here, we find that Arabidopsis thaliana seedlings wounded at the hypocotyl-root junction reprogram certain pericycle cells to produce adventitious roots proximal to the wound site. We have determined that competence for this reprogramming is controlled; basal cells close to the wound site can produce adventitious roots, whereas cells distal from the wound site mostly cannot. We found that altering cytokinin response or indole-3-butyric acid (IBA)-to-(indole-3-acetic acid) IAA conversion resulted in an expanded adventitious root competence zone and delineated the connection between these pathways. Our work highlights the importance of endogenous IBA-derived auxin and its interaction with cytokinin in adventitious root formation and the regenerative properties of plants. [Display omitted] • Excision-induced adventitious roots (ARs) form proximal to the wound site • Endogenous IBA-derived auxin promotes AR formation • IBA-derived auxin regulates the zone of AR formation • Cytokinin regulates IBA-derived auxin to control the AR competence zone Damodaran and Strader determine that, in Arabidopsis, adventitious roots created in response to wounding occur proximal to the wound site, suggesting that the wound site sets a zone of competence for cellular reprogramming. Cytokinin and IBA-derived auxin dictate this competence zone. [ABSTRACT FROM AUTHOR]

N 6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. It governs RNA fate and is essential for development and stress responses. However, the dynamic, context-dependent m6A methylomes across tissues and in response to various stimuli remain largely unknown in multicellular organisms. Here, we generate a comprehensive census that identifies m6A methylomes in 100 samples during development or following exposure to various external conditions in Arabidopsis thaliana. We demonstrate that m6A is a suitable biomarker to reflect the developmental lineage, and that various stimuli rapidly affect m6A methylomes that constitute the regulatory network required for an effective response to the stimuli. Integrative analyses of the census and its correlation with m6A regulators identify multiple layers of regulation on highly context-dependent m6A modification in response to diverse developmental and environmental stimuli, providing insights into m6A modification dynamics in the myriad contexts of multicellular organisms. [Display omitted] • m6A dynamics of Arabidopsis development and response to stimuli are surveyed • m6A is a suitable biomarker to reflect the developmental lineage • Reversible reprogramming of m6A occurs during stress treatment and recovery • Multiple layers of regulation determine highly context-dependent functions of m6A N 6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. Zhang et al. report a comprehensive m6A census of Arabidopsis development and response to stimuli, which offers insights into the highly context-dependent nature of m6A signatures and their underlying modulatory mechanisms in multicellular organisms. [ABSTRACT FROM AUTHOR]

We review the newly classified ascorbate peroxidase-related (APX-R) proteins, which do not use ascorbate as electron donor to scavenge H2O2. We summarize recent discoveries on the function and the characterization of the APX-R protein of the green unicellular alga Chlamydomonas reinhardtii and the land plant Arabidopsis thaliana. Additionally, we conduct in silico analyses on the conserved MxxM motif, present in most of the APX-R protein in different organisms, which is proposed to bind copper. Based on these analyses, we discuss the similarities between the APX-R and the class III peroxidases. [ABSTRACT FROM AUTHOR]

SUMMARY: Cotton fiber (Gossypium hirsutum) serves as an ideal model for investigating the molecular mechanisms of plant cell elongation at the single‐cell level. Brassinosteroids (BRs) play a crucial role in regulating plant growth and development. However, the mechanism by which BR influences cotton fiber elongation remains incompletely understood. In this study, we identified EXORDIUM‐like (GhEXL3) through transcriptome analysis of fibers from BR‐deficient cotton mutant pagoda 1 (pag1) and BRI1‐EMS‐SUPPRESSOR 1 (GhBES1.4, encoding a central transcription factor of BR signaling) overexpression cotton lines. Knockout of GhEXL3 using CRISPR/Cas9 was found to impede cotton fiber elongation, while its overexpression promoted fiber elongation, suggesting a positive regulatory function for GhEXL3 in fiber elongation. Furthermore, in vitro ovule culture experiments revealed that the overexpression of GhEXL3 partially counteracted the inhibitory effects of brassinazole (BRZ) on cotton fiber elongation, providing additional evidence of GhEXL3 involvement in BR signaling pathways. Moreover, our findings demonstrate that GhBES1.4 directly binds to the E‐box (CACGTG) motif in the GhEXL3 promoter region and enhances its transcription. RNA‐seq analysis revealed that overexpression of GhEXL3 upregulated the expression of EXPs, XTHs, and other genes associated with fiber cell elongation. Overall, our study contributes to understanding the mechanism by which BR regulates the elongation of cotton fibers through the direct modulation of GhEXL3 expression by GhBES1.4. Significance Statement: Understanding the molecular mechanisms of cotton fiber elongation is crucial for improving fiber yield. This study reveals that the EXORDIUM‐like gene (GhEXL3) positively regulates fiber elongation in response to brassinosteroids (BRs). By demonstrating that GhBES1.4, a key BR signaling transcription factor, directly activates GhEXL3, we provide insights into how BR signaling pathways influence fiber growth. These findings enhance our understanding of BR‐mediated fiber development and offer potential targets for genetic improvement in cotton. [ABSTRACT FROM AUTHOR]

Summary: During Arabidopsis embryogenesis, the transition of the embryo's symmetry from radial to bilateral between the globular and heart stage is a crucial event, involving the formation of cotyledon primordia and concurrently the establishment of a shoot apical meristem (SAM). However, a coherent framework of how this transition is achieved remains to be elucidated.In this study, we investigated the function of DELAYED GREENING 1 (DG1) in Arabidopsis embryogenesis using a newly identified dg1‐3 mutant. The absence of chloroplast‐localized DG1 in the mutants led to embryos being arrested at the globular or heart stage, accompanied by an expansion of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) expression. This finding pinpoints the essential role of DG1 in regulating the transition to bilateral symmetry. Furthermore, we showed that this regulation of DG1 may not depend on its role in plastid RNA editing.Nevertheless, we demonstrated that the DG1 function in establishing bilateral symmetry is genetically mediated by GENOMES UNCOUPLED 1 (GUN1), which represses the transition process in dg1‐3 embryos.Collectively, our results reveal that DG1 functionally antagonizes GUN1 to promote the transition of the Arabidopsis embryo's symmetry from radial to bilateral and highlight the role of plastid signals in regulating pattern formation during plant embryogenesis. [ABSTRACT FROM AUTHOR]

Summary: Aberrant RNA modifications can lead to dysregulated gene expression and impeded growth in plants. Ribosomal RNA (rRNA) constitutes a substantial portion of total RNA, while the precise functions and molecular mechanisms underlying rRNA modifications in plants remain largely elusive.Here, we elucidated the exclusive occurrence of the canonical RNA modification N6‐methyladenosine (m6A) solely 18S rRNA, but not 25S rRNA. We identified a completely uncharacterized protein, ATMETTL5, as an Arabidopsis m6A methyltransferase responsible for installing m6A methylation at the 1771 site of the 18S rRNA. ATMETTL5 is ubiquitously expressed and localized in both nucleus and cytoplasm, mediating rRNA m6A methylation.Mechanistically, the loss of ATMETTL5‐mediated methylation results in attenuated translation. Furthermore, we uncovered the role of ATMETTL5‐mediated methylation in coordinating blue light‐mediated hypocotyl growth by regulating the translation of blue light‐related messenger RNAs (mRNAs), specifically HYH and PRR9.Our findings provide mechanistic insights into how rRNA modification regulates ribosome function in mRNA translation and the response to blue light, thereby advancing our understanding of the role of epigenetic modifications in precisely regulating mRNA translation in plants. [ABSTRACT FROM AUTHOR]

Summary: Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well‐established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored.Using LC–MS/MS‐based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots.AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton‐26S proteasome‐YUC8‐auxin' axis that controls auxin levels.Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome. [ABSTRACT FROM AUTHOR]

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's β-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4. Synopsis: The biogenesis and maintenance of photosynthetic complexes requires precise coordination of cotranslational maturation, folding, targeting and insertion of their plastid-encoded subunits. This work offers insight into the interactome of photosystem II reaction center-specific ribosome-nascent chain complexes, and identifies a role for STIC2 in their cotranslational protein sorting. Proteins from metabolic, biogenesis, and protein processing pathways associate with D1-translating ribosomes. Knock-out of STIC2 and the targeting factor cpSRP54 in Arabidopsis causes sensitivity to high light and low accumulation of photosystem subunits. STIC2 partially associates with ribosomes and affects chloroplast translation. The C-terminal motif III of the thylakoid membrane proteins Alb3 and Alb4 mediates their interaction within the β-sheet region of STIC2. The chloroplast protein STIC2 associates with ribosomes translating the photosystem II reaction center protein D1 and supports insertion into the thylakoid membrane. [ABSTRACT FROM AUTHOR]

SUMMARY E2 ubiquitin‐conjugating enzymes play a crucial role in the ubiquitination process by catalyzing ubiquitin transfer. Although the function of ubiquitin‐protein ligases (E3s) in plants response to diverse abiotic stress by targeting specific substrates has been well studied, the involvement of E2s in environmental responses and their downstream targets are not well understood. In this study, we demonstrated that the E2 ubiquitin‐conjugating enzyme 18 (UBC18) influences the stability of FREE1 to modulate iron deficiency stress. UBC18 affects the ubiquitination of FREE1 and promotes its degradation, and overexpression of UBC18 decreases plants’ sensitivity to iron deficiency by reducing FREE1 level, whereas the ubc18 mutant exhibits sensitivity due to elevated FREE1 accumulation. This study also identified that lysine residues K227, K295, K315, and K540 are required for FREE1 ubiquitination and stability regulation. Mutating these lysine residues in FREE1 resulted in plants’ sensitivity to iron starvation. Taken together, our findings shed light on the mechanism of UBC18 in responding to iron deficiency stress by modulating the abundance of FREE1, and further elucidate the role of ubiquitination sites in FREE1 stability regulation and the plant iron deficiency response. [ABSTRACT FROM AUTHOR]

Different convergent evolutionary strategies adopted by angiosperm fruits lead to diverse functional seed dispersal units. Dry dehiscent fruits are a common type of fruit, characterized by their lack of fleshy pericarp and the release of seeds at maturity through openings (dehiscence zones, DZs) in their structure. In previous decades, a set of core players in DZ formation have been intensively characterized in Arabidopsis and integrated in a gene regulatory network (GRN) that explains the morphogenesis of these tissues. In this work, we compile all the experimental data available to date to build a discrete Boolean model as a mechanistic approach to validate the network and, if needed, to identify missing components of the GRN and/or propose new hypothetical regulatory interactions, but also to provide a new formal framework to feed further work in Brassicaceae fruit development and the evolution of seed dispersal mechanisms. Hence, by means of exhaustive in-silico validations and experimental evidence, we are able to incorporate both the NO TRANSMITTING TRACT (NTT) transcription factor as a new additional node, and a new set of regulatory hypothetical rules to uncover the dynamics of Arabidopsis DZ specification. [ABSTRACT FROM AUTHOR]

Plant cell walls are essential for growth. The cell wall hemicellulose xyloglucan (XyG) is produced in the Golgi apparatus before secretion. Loss of the Arabidopsis galactosyltransferase MURUS3 (MUR3) decreases XyG d -galactose side chains and causes intracellular aggregations and dwarfism. It is unknown how changing XyG synthesis can broadly impact organelle organization and growth. We show that intracellular aggregations are not unique to mur3 and are found in multiple mutant lines with reduced XyG D -galactose side chains. mur3 aggregations disrupt subcellular trafficking and induce formation of intracellular cell-wall-like fragments. Addition of d -galacturonic acid onto XyG can restore growth and prevent mur3 aggregations. These results indicate that the presence, but not the composition, of XyG side chains is essential, likely by ensuring XyG solubility. Our results suggest that XyG polysaccharides are synthesized in a highly substituted form for efficient secretion and then later modified by cell-wall-localized enzymes to fine-tune cell wall properties. [Display omitted] • A reduction in xyloglucan (XyG) side chains causes intracellular aggregations • Intracellular aggregations form during cell expansion and disrupt secretion • An alternate XyG side chain restores secretion but not cell wall composition Hoffmann and McFarlane show that the plant cell wall polysaccharide xyloglucan requires side chains for effective secretion following its biosynthesis in the Golgi apparatus, as production of side-chain-deficient xyloglucan causes intracellular aggregations of proteins and polysaccharides. These results highlight the importance of polysaccharide structure for efficient secretion to the cell wall. [ABSTRACT FROM AUTHOR]

Identifying the essential factors and underlying mechanisms regulating plant heat stress (HS) responses is crucial for mitigating the threat posed by HS on plant growth, development, distribution, and productivity. In this study, we found that the Arabidopsis (Arabidopsis thaliana) super-killer2 (ski2) dicer-like4 (dcl4) mutant, characterized by RNA processing defects and the accumulation of abundant 22-nt small interfering RNAs derived from protein-coding transcripts, displayed significantly increased expression levels of HS-responsive genes and enhanced thermotolerance. These traits primarily resulted from the suppression of SMAX1-LIKE4 (SMXL4) and SMXL5, which encode 2 putative transcriptional regulators that belong to the SMXL protein family. While smxl4 and smxl5 single mutants were similar to wild type, the smxl4 smxl5 double mutant displayed substantially heightened seedling thermotolerance. Further investigation demonstrated that SMXL4 and SMXL5 repressed the transcription of HEAT-SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), encoding a master regulator of thermotolerance, independently of ethylene-response factor–associated amphiphilic repression motifs. Moreover, SMXL4 and SMXL5 interacted with HSFA1d and HSFA1e, central regulators sensing and transducing HS stimuli, and antagonistically affected their transactivation activity. In addition, HSFA2 directly bound to the SMXL4 and SMXL5 promoters, inducing their expression during recovery from HS. Collectively, our findings elucidate the role of the SMXL4/SMXL5–HSFA2 regulatory module in orchestrating plant thermotolerance under HS. The elevated temperatures inhibit 2 suppressor proteins, leading to the activation of 2 crucial regulators that detect and relay heat stress (HS) signals, ultimately triggering plant responses to HS. [ABSTRACT FROM AUTHOR]

Following whole-genome duplication (WGD), duplicate gene pairs (homoeologs) can evolve varying degrees of expression divergence. However, the determinants influencing these relative expression level differences (RFPKM) between homoeologs remain elusive. In this study, we analyzed the RFPKM between homoeologs in 3 angiosperms, Nymphaea colorata, Nelumbo nucifera, and Acorus tatarinowii, all having undergone a single WGD since the origin of angiosperms. Our results show significant positive correlations in RFPKM of homoeologs among tissues within the same species, and among orthologs across these 3 species, indicating convergent expression balance/bias between homoeologous gene copies following independent WGDs. We linked RFPKM between homoeologs to gene attributes associated with dosage-balance constraints, such as protein–protein interactions, lethal-phenotype scores in Arabidopsis (Arabidopsis thaliana) orthologs, domain numbers, and expression breadth. Notably, homoeologs with lower RFPKM often had more interactions and higher lethal-phenotype scores, indicating selective pressures favoring balanced expression. Also, homoeologs with lower RFPKM were more likely to be retained after WGDs in angiosperms. Within Nelumbo, greater RFPKM between homoeologs correlated with increased cis- and trans-regulatory differentiation between species, highlighting the ongoing escalation of gene expression divergence. We further found that expression degeneration in 1 copy of homoeologs is inclined toward nonfunctionalization. Our research highlights the importance of balanced expression, shaped by dosage-balance constraints, in the evolutionary retention of homoeologs in plants. Following whole-genome duplication, gene expression divergence of homoeologs is influenced by dosage-balance constraints, with similarly expressed homoeologs more likely to be evolutionarily retained. [ABSTRACT FROM AUTHOR]

SO2/H2SO3 can damage plants. However, its toxic mechanism has still been controversial. Two models have been proposed, cytosolic acidification model and cellular oxidation model. Here, we assessed the toxic mechanism of H2SO3 in three cell types of Arabidopsis thaliana , mesophyll cells, guard cells (GCs), and petal cells. The sensitivity of GCs of Chloride channel a (CLC a)-knockout mutants to H2SO3 was significantly lower than those of wildtype plants. Expression of other CLC genes in mesophyll cells and petal cells were different from GCs. Treatment with antioxidant, disodium 4,5-dihydroxy-1,3-benzenedisulfonate (tiron), increased the median lethal concentration (LC50) of H2SO3 in GCs indicating the involvement of cellular oxidation, while the effect was negligible in mesophyll cells and petal cells. These results indicate that there are two toxic mechanisms of SO2 to Arabidopsis cells: cytosolic acidification and cellular oxidation, and the toxic mechanism may vary among cell types. [ABSTRACT FROM AUTHOR]

Seed dormancy corresponds to a reversible blockage of germination. Primary dormancy is established during seed maturation, while secondary dormancy is set up on the dispersed seed, following an exposure to unfavorable factors. Both dormancies are relieved in response to environmental factors, such as light, nitrate, and coldness. Quantitive Trait Locus (QTL) analyses for preharvest sprouting identified MKK3 kinase in cereals as a player in dormancy control. Here, we showed that MKK3 also plays a role in secondary dormancy in Arabidopsis within a signaling module composed of MAP3K13/14/19/20, MKK3, and clade-C MAPKs. Seeds impaired in this module acquired heat-induced secondary dormancy more rapidly than wild-type (WT) seeds, and this dormancy is less sensitive to nitrate, a signal able to release dormancy. We also demonstrated that MPK7 was strongly activated in the seed during dormancy release, especially in response to light and nitrate. This activation was greatly reduced in map3k13/14/19/20 and mkk3 mutants. Finally, we showed that the module was not regulated and apparently did not regulate the genes controlling abscisic acid/gibberellin acid hormone balance, one of the crucial mechanisms of seed dormancy control. Overall, our work identified a MAPK module controlling seed germination and enlarged the panel of functions of the MKK3-related modules in plants. [ABSTRACT FROM AUTHOR]

Summary Phosphate (Pi) plays a key role in plant growth and development. Hence, plants display a range of adaptations to acquire it, including changes in root system architecture (RSA). Whether Pi triggers directional root growth is unknown. We investigated whether Arabidopsis roots sense Pi and grow towards it, that is whether they exhibit phosphotropism. While roots did exhibit a clear Pi‐specific directional growth response, it was, however, always to the left, independent of the direction of the Pi gradient. We discovered that increasing concentrations of KH2PO4, trigger a dose‐dependent skewing response, in both primary and lateral roots. This phenomenon is Pi‐specific – other nutrients do not trigger this – and involves the reorganisation of the microtubule cytoskeleton in epidermal cells of the root elongation zone. Higher Pi levels promote left‐handed cell file rotation that results in right‐handed, clockwise, root growth and leftward skewing as a result of the helical movement of roots (circumnutation). Our results shed new light on the role of Pi in root growth, and may provide novel insights for crop breeding to optimise RSA and P‐use efficiency. [ABSTRACT FROM AUTHOR]

Key message: The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein–protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling. [ABSTRACT FROM AUTHOR]

C1-FDX (Complex I-ferredoxin) has been defined as a component of CI in a ferredoxin bridge in Arabidopsis mitochondria. However, its full function remains to be addressed. We created two c1-fdx mutants in Arabidopsis using the CRISPR-Cas9 methodology. The mutants show delayed seed germination. Over-expression of C1-FDX rescues the phenotype. Molecular analyses showed that loss of the C1-FDX function decreases the abundance and activity of both CI and subcomplexes of CV. In contrast, the over-expression of C1-FDX-GFP enhances the CI* (a sub-complex of CI) and CV assembly. Immunodetection reveals that the stoichiometric ratio of the α:β subunits in the F1 module of CV is altered in the c1-fdx mutant. In the complemented mutants, C1-FDX-GFP was found to be associated with the F' and α/β sub-complexes of CV. Protein interaction assays showed that C1-FDX could interact with the β, γ, δ, and ε subunits of the F1 module, indicating that C1-FDX, a structural component of CI, also functions as an assembly factor in the assembly of F' and α/β sub-complexes of CV. These results reveal a new role of C1-FDX in the CI and CV assembly and seed germination in Arabidopsis. Author summary: The mitochondrial oxidative phosphorylation (OXPHOS) system is composed of five large multiprotein complexes, complex I to V (CI to CV). However, their assembly is not fully understood in plants. C1-FDX, a ferredoxin protein, was found to form a ferredoxin bridge in CI. Here, we show that C1-FDX is also involved in the assembly of CI* and CV. CI* is an important intermedate of CI. Mutation of C1-FDX affects the assembly of these two complexes and delays the germination of Arabidopsis. C1-FDX interacts with several CV subunits, including the β, γ, δ, and ε subunits of the F1 sub-complex, and physically associates with the CV assembly intermediates, sub-complexes F' and α/β, but not with the mature CV, indicating that C1-FDX is dissociated when the CV is fully assembled. This study provides insights into the dual-function of CI-FDX in CI and CV assembly, facilitating the understanding of the CI and CV assembly mechanism in plants. [ABSTRACT FROM AUTHOR]

Key message: Two plant U-box E3 ligases, PUB5 and PUB44, antagonistically regulate the NLR receptor SUMM2-mediated autoimmunity in Arabidopsis, indicating a new regulatory mechanism for fine-tuning plant immunity. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor‐like kinase (RLK) family at the cell surface. Various signaling peptide‐RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN‐FORMED (PIN)‐regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1‐INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen‐activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response. Significance Statement: Multiple phytocytokines are involved in the plant immune response. Here, we report that the SCOOP‐MIK2‐MPK6 pathway modulates Arabidopsis root growth and development by regulating PIN abundance at the PM and subsequent auxin transport. [ABSTRACT FROM AUTHOR]

SUMMARY: Among the three active aldehyde oxidases in Arabidopsis thaliana leaves (AAO1‐3), AAO3, which catalyzes the oxidation of abscisic‐aldehyde to abscisic‐acid, was shown recently to function as a reactive aldehyde detoxifier. Notably, aao2KO mutants exhibited less senescence symptoms and lower aldehyde accumulation, such as acrolein, benzaldehyde, and 4‐hydroxyl‐2‐nonenal (HNE) than in wild‐type leaves exposed to UV‐C or Rose‐Bengal. The effect of AAO2 expression absence on aldehyde detoxification by AAO3 and/or AAO1 was studied by comparing the response of wild‐type plants to the response of single‐functioning aao1 mutant (aao1S), aao2KO mutants, and single‐functioning aao3 mutants (aao3Ss). Notably, aao3Ss exhibited similar aldehyde accumulation and chlorophyll content to aao2KO treated with UV‐C or Rose‐Bengal. In contrast, wild‐type and aao1S exhibited higher aldehyde accumulation that resulted in lower remaining chlorophyll than in aao2KO leaves, indicating that the absence of active AAO2 enhanced AAO3 detoxification activity in aao2KO mutants. In support of this notion, employing abscisic‐aldehyde as a specific substrate marker for AAO3 activity revealed enhanced AAO3 activity in aao2KO and aao3Ss leaves compared to wild‐type treated with UV‐C or Rose‐Bengal. The similar abscisic‐acid level accumulated in leaves of unstressed or stressed genotypes indicates that aldehyde detoxification by AAO3 is the cause for better stress resistance in aao2KO mutants. Employing the sulfuration process (known to activate aldehyde oxidases) in wild‐type, aao2KO, and molybdenum‐cofactor sulfurase (aba3‐1) mutant plants revealed that the active AAO2 in WT employs sulfuration processes essential for AAO3 activity level, resulting in the lower AAO3 activity in WT than AAO3 activity in aao2KO. Significance Statement: The current study demonstrates that impairment of AAO2 causes enhancement of AAO3 activity and carbonyl aldehyde detoxification increase in Arabidopsis. [ABSTRACT FROM AUTHOR]

SUMMARY: Maintaining an optimal redox status is essential for plant growth and development, particularly when the plants are under stress. AT‐hook motif nuclear localized (AHL) proteins are evolutionarily conserved transcription factors in plants. Much of our understanding about this gene family has been derived from studies on clade A members. To elucidate the functions of clade B genes, we first analyzed their spatial expression patterns using transgenic plants expressing a nuclear localized GFP under the control of their promoter sequences. AHL1, 2, 6, 7, and 10 were further functionally characterized owing to their high expression in the root apical meristem. Through mutant analyses and transgenic studies, we showed that these genes have the ability to promote root growth. Using yeast one‐hybrid and dual luciferase assays, we demonstrated that AHL1, 2, 6, 7, and 10 are transcription regulators and this activity is required for their roles in root growth. Although mutants for these genes did not showed obvious defects in root growth, transgenic plants expressing their fusion proteins with the SRDX repressor motif exhibited a short‐root phenotype. Through transcriptome analysis, histochemical staining and molecular genetics experiments, we found that AHL10 maintains redox homeostasis via direct regulation of glutathione transferase (GST) genes. When the transcript level of GSTF2, a top‐ranked target of AHL10, was reduced by RNAi, the short‐root phenotype in the AHL10‐SRDX expressing plant was largely rescued. These results together suggest that AHL genes function redundantly in promoting root growth through direct regulation of redox homeostasis. [ABSTRACT FROM AUTHOR]

SUMMARY: The accessory proteins Hyponastic‐like 1 (HYL1) and Serrated (SE) enhance the precise and efficient processing of miRNAs by Dicer‐like 1 (DCL1), which is important for proper miRNA function. However, other factors determining the precision and efficiency of miRNA biogenesis are not well‐known. Here, we found that an asymmetric bulge (AB) at the 3′ end of miR‐5p (produced from the 5′ arm of the pre‐miRNA) reduced the precision of the second cleavage, whereas an AB at other sites of miR‐5p mainly affected the accumulation level of miR‐5p in transient expression in Nicotiana benthamiana. In contrast, many ABs in miR‐3p (produced from the 3′ arm of the pre‐miRNA) impose strong negative impact on the processing precision and the accumulation level of miR‐5p in N. benthamiana. Arabidopsis DCL1/SE/HYL1 complex‐mediated miRNA processing was reconstituted in Saccharomyces cerevisiae to further investigate AB‐mediated interference with DCL1 processing. With this system, the positional effect of AB on miRNA processing was tested. The results showed that ABs on the middle of miR‐5p have less of an impact on DCL1 cleavage efficiency and precision, whereas those on miR‐3p or near the ends of miR‐5p strongly reduce DCL1 cleavage activity, precision or both. Studies using the yeast miRNA processing system and transgenic Arabidopsis also revealed the importance of the interaction between the 2‐nt 3′ overhang of pre‐miRNA and the 3′ overhang binding pocket (3′BP) on the precision of the second cleavage reaction for many endogenous miRNAs. These findings provide new insights into the mechanism of miRNA biogenesis. Significance Statement: Factors determining the precision and efficiency of miRNA biogenesis are not well‐known. This study found that an asymmetric bulge at different positions of miR‐5p and miR‐3p had different impacts on miRNA levels and processing precision and revealed the importance of the interaction between the 2‐nt 3′ overhang of pre‐miRNA and the 3′ overhang binding pocket (3'BP) on the precision of the second cleavage reaction for many endogenous miRNAs. [ABSTRACT FROM AUTHOR]

Sequence‐specific endonucleases have been key to the study of the mechanisms and control of DNA double‐strand break (DSB) repair and recombination, and the availability of CRISPR‐Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5′ overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much‐studied Cas9 nuclease cleaves DNA to generate blunt‐ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5′‐overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end‐joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut‐sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single‐cut and double‐cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single‐strand annealing recombination. [ABSTRACT FROM AUTHOR]

The Cysteine‐rich secretory proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis‐related 1 (PR‐1) protein (CAP) superfamily members are found in multiple eukaryotic organisms, including yeasts, animals, and plants. Although one of the plant CAP family genes, PR‐1 is known to respond to pathogen infection in plants, the functions of other CAP family genes in Arabidopsis remain largely unknown. In this study, we conducted a comprehensive analysis of the similarities, loci, and expression patterns of 22 Arabidopsis CAP genes/proteins, providing a clue to elucidate their molecular functions. According to the promoter‐β‐glucuronidase (GUS) analysis, members of the Arabidopsis CAP family were expressed in various young tissues or organs, such as root and shoot meristems, reproductive tissues, and particularly at the lateral root initiation site before the formation of the lateral root primordium, with distinct expression specificity. In particular, CAP51, CAP52, and CAP53 were specifically expressed in the cortical cells at the lateral root developing regions, suggesting that these genes may function in lateral root development. Thus, the expression patterns of Arabidopsis CAP family genes suggest that CAP family proteins may have certain function in the expressed organs or tissues in Arabidopsis plant. [ABSTRACT FROM AUTHOR]

We assessed the flowering Chinese cabbage (Brassica rapa var. parachinensis), a specialty vegetable found in southern China. The sugar content of the stem tip is closely related to bolting and flowering. Sugar Will Eventually be Exported Transporters (SWEETs) are bidirectional sugar transporter proteins involved in numerous plant growth and development processes. The expression of BraSWEET12 is positively correlated with sugar content. However, it is unclear whether BraSWEET12 is involved in bolting and flowering. In this study, we identified and characterized BraSWEET12. BraSWEET12 in flowering Chinese cabbage contains 288 amino acids and is located on the cell membrane as a sucrose transporter protein. BraSWEET12 is highly expressed in the petals and stem tips of flowering Chinese cabbage and is upregulated by gibberellin and low temperatures. Overexpression of BraSWEET12 in Arabidopsis can increase sucrose content at the stem tip, upregulate the expression of AtAP1 and AtLFY, and advance the flowering time. Subsequently, our results indicate that BraSWEET12 is involved in sucrose accumulation at the stem tip of flowering Chinese cabbage and plays a crucial role in flowering regulation. These results provide a reference for elucidating the regulatory mechanisms underlying flowering Chinese cabbage bolting and flowering. [ABSTRACT FROM AUTHOR]

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 (AtGRP7) and AtGRP8 that promote the transition to flowering. AtGRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for AtGRP7's physiological role in flowering time control. We constructed AtGRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type AtGRP7. Furthermore, overexpression of AtGRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of AtGRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of AtGRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized AtGRP7, suggesting that additional methyltransferases may be responsible for modification of AtGRP7. [ABSTRACT FROM AUTHOR]

Research has demonstrated that strigolactones (SLs) mediate plant disease resistance; however, the basal mechanism is unclear. Here, we provide key genetic evidence supporting how SLs mediate plant disease resistance. Exogenous application of the SL analog, rac-GR24, increased Arabidopsis thaliana resistance to virulent Pseudomonas syringae. SL-biosynthetic mutants and overexpression lines of more axillary growth 1 (MAX1, an SL-biosynthetic gene) enhanced and reduced bacterial susceptibility, respectively. In addition, rac-GR24 promoted bacterial pattern flg22-induced callose deposition and hydrogen peroxide production. SL-biosynthetic mutants displayed reduced callose deposition but not hydrogen peroxide production under flg22 treatment. Moreover, rac-GR24 did not affect avirulent effector-induced cell death between Col-0 and SL-biosynthetic mutants. Furthermore, rac-GR24 increased the free salicylic acid (SA) content and significantly promoted the expression of pathogenesis-related gene 1 related to SA signaling. Importantly, rac-GR24- and MAX1-induced bacterial resistance disappeared completely in Arabidopsis plants lacking both callose synthase and SA. Taken together, our data revealed that callose and SA are two important determinants in SL-mediated plant disease resistance, at least in Arabidopsis. [ABSTRACT FROM AUTHOR]

The Defective in Induced Resistance 1 (DIR1) gene, a member of the lipid transferase proteins (LTPs), plays a crucial role in plant defense against pathogens. While previous transcriptomic studies have highlighted the significant expression of citrus LTPs during biotic stress, functional annotations of LTPs in the Citrus genera remain limited. In this study, we cloned the Murraya paniculata DIR1 (MpDIR1(t)) gene and overexpressed it in Arabidopsis thaliana to evaluate its stress response mechanisms against biotic stress. The transgenic Arabidopsis lines showed fewer disease symptoms in response to Pseudomonas syringae (Pst DC3000) compared to wild-type Arabidopsis. Defense and pathogenesis-responsive genes such as PR1, PR4, PR5, and WRKY12 were significantly induced, showing a 2- to 12-fold increase in all transgenic lines compared to the wild type. In addition, the Pst DC3000-infected transgenic Arabidopsis lines demonstrated elevated levels of flavonoids and salicylic acid (SA), along with higher expression of SA-related genes, compared to the wild type. Moreover, all transgenic lines possessed lower reactive oxygen species levels and higher activity of antioxidant defense enzymes such as superoxide dismutase, peroxidase, and catalase under Pst DC3000 stress compared to the wild type. The up-regulation of defense genes, activation of the SA pathway, accumulation of flavonoids, and reinforcement of antioxidant defense mechanisms in transgenic Arabidopsis lines in response to Pst DC3000 underscore the critical role of MpDIR1(t) in fortifying plant immunity. Thus, MpDIR1(t) constitutes a promising candidate gene for improving bacterial disease resistance in commercial citrus cultivars. [ABSTRACT FROM AUTHOR]

This study investigated the germination response to temperature of seeds of nine Arabidopsis thaliana ecotypes. They are characterized by a similar temperature dependency of seed germination, and 10 °C and 29 °C were found to be suboptimal low and high temperatures for all nine ecotypes, even though they originated from regions with diverse climates. We tested the potential of four PGPR strains from the genera Pseudomonas and Bacillus to stimulate seed germination in the two ecotypes under these suboptimal conditions. Biopriming of seeds with only the biofilm-forming strain Pseudomonas putida KT2440 significantly increased the germination of Cape Verde Islands (Cvi-0) seeds at 10 °C. However, biopriming did not significantly improve the germination of seeds of the widely utilized ecotype Columbia 0 (Col-0) at any of the two tested temperatures. To functionally investigate the role of KT2440's biofilm formation in the stimulation of seed germination, we used mutants with compromised biofilm-forming abilities. These bacterial mutants had a reduced ability to stimulate the germination of Cvi-0 seeds compared to wild-type KT2440, highlighting the importance of biofilm formation in promoting germination. These findings highlight the potential of PGPR-based biopriming for enhancing seed germination at low temperatures. [ABSTRACT FROM AUTHOR]