NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Flavin-containing monooxygenases FMOGS-OXs integrate flowering transition and salt tolerance in Arabidopsis thaliana.

Salt stress substantially leads to flowering delay. The regulation of salt-induced late flowering has been studied at the transcriptional and protein levels; however, the involvement of secondary metabolites has rarely been investigated. Here, we report that FMOGS-OXs (EC 1.14.13.237), the enzymes that catalyze the biosynthesis of glucosinolates (GSLs), promote flowering transition in Arabidopsis thaliana. It has been reported that WRKY75 is a positive regulator, and MAF4 is a negative regulator of flowering transition. The products of FMOGS-OXs, methylsulfinylalkyl GSLs (MS GSLs), facilitate flowering by inducing WRKY75 and repressing the MAS-MAF4 module. We further show that the degradation of MS GSLs is involved in salt-induced late flowering and salt tolerance. Salt stress induces the expression of myrosinase genes, resulting in the degradation of MS GSLs, thereby relieving the promotion of WRKY75 and inhibition of MAF4, leading to delayed flowering. In addition, the degradation products derived from MS GSLs enhance salt tolerance. Previous studies have revealed that FMOGS-OXs exhibit alternative catalytic activity to form trimethylamine N-oxide (TMAO) under salt stress, which activates multiple stress-related genes to promote salt tolerance. Therefore, FMOGS-OXs integrate flowering transition and salt tolerance in various ways. Our study shed light on the functional diversity of GSLs and established a connection between flowering transition, salt resistance, and GSL metabolism.

Complex genetic interaction between glucose sensor HXK1 and E3 SUMO ligase SIZ1 in regulating plant morphogenesis.

Sugar signaling forms the basis of metabolic activities crucial for an organism to perform essential life activities. In plants, sugars like glucose, mediate a wide range of physiological responses ranging from seed germination to cell senescence. This has led to the elucidation of cell signaling pathways involving glucose and its counterparts and the mechanism of how these sugars take control over major hormonal pathways such as auxin, ethylene, abscisic acid and cytokinin in Arabidopsis. Plants use HXK1(Hexokinase) as a glucose sensor to modulate changes in photosynthetic gene expression in response to high glucose levels. Other proteins such as SIZ1, a major SUMO E3 ligase have recently been implicated in controlling sugar responses via transcriptional and translational regulation of a wide array of sugar metabolic genes. Here, we show that these two genes work antagonistically and are epistatic in controlling responsiveness toward high glucose conditions.

Understanding megasporogenesis through model plants: contemporary evidence and future insights.

The megasporangium serves as a model system for understanding the concept of individual cell identity, and cell-to-cell communication in angiosperms. As development of the ovule progresses, three distinct layers, the epidermal (L1), the subepidermal or the hypodermal (L2) and the innermost layers (L3) are formed along the MMC (megaspore mother cell). The MMC, which is the primary female germline cell, is initiated as a single subepidermal cell amongst several somatic cells. MMC development is governed by various regulatory pathways involving intercellular signaling, small RNAs and DNA methylation. The programming and reprograming of a single nucellar cell to enter meiosis is governed by 'permissive' interacting processes and factors. Concomitantly, several nucellar sister cells are prevented from germline fate also by a set of 'repressive' factors. However, in certain angiosperms, anomalies in development of the female gametophyte have been observed. The sporophytic tissue surrounding the female gametophyte affects the gametophyte in multiple ways. The role of genes and transcription factors in the development of the MMC and in the regulation of various processes studied in selected model plants such as Arabidopsis is explained in detail in this paper. However, as angiosperms display enormous diversity, it is important to investigate early stages of megasporogenesis in other plant systems as well. Such studies provide valuable insights in understanding the regulation of megasporogenesis and the evolution of the female gametophyte from gymnosperms to flowering plants.

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.

Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.

Characterization of Thermoresponsive Photobody Dynamics.

Photobodies (PBs) are subnuclear membraneless organelles that self-assemble via the condensation of the plant photoreceptor and thermosensor phytochrome B (phyB). Changes in the light and temperature environment directly modulate PB formation and maintenance by altering the number and size of PBs. In thermomorphogenesis, increases in the ambient temperature incrementally reduce the number of PBs, suggesting that individual PBs possess distinct thermostabilities. Here, we describe a detailed protocol for characterizing cell type-specific PB dynamics induced by warm temperatures in Arabidopsis.

Characterization of PIF4 Phosphorylation by SPA1.

The PHYTOCHROME INTERACTING FACTORs (PIFs) play pivotal roles in regulating thermo- and photo-morphogenesis in Arabidopsis. One of the main hubs in thermomorphogenesis is PIF4, which regulates plant development under high ambient temperature along with other PIFs. PIF4 enhances its own transcription and PIF4 protein is stabilized under high ambient temperature. However, the mechanisms of thermo-stabilization of PIF4 are less understood. Recently, it was shown that SUPPRESSOR OF PHYA-105 1 (SPA1) can function as a serine/threonine kinase to phosphorylate PIF4 in vitro, and the phosphorylated form of PIF4 is more stable under high ambient temperature conditions. In this chapter, we describe the in vitro kinase assay of PIF4 by SPA1. In principle, this protocol can be applied for other putative substrates and kinases.

GST Pull-Down Assay to Study PIF4 Binding In Vitro.

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

RuBisCO Depletion and Subcellular Fractionation for Enhanced Florigen Detection in Arabidopsis.

In plants, complex signaling networks monitor and respond to environmental cues to determine the optimal time for the transition from the vegetative to reproductive phase. Understanding these networks requires robust tools to examine the levels and subcellular localization of key factors. The florigen FLOWERING LOCUS T (FT) is a crucial regulator of flowering time and occurs in soluble and membrane-bound forms. At low ambient temperatures, the ratio of these forms of FT undergoes a significant shift, which leads to a delay in the onset of flowering. To investigate these changes in FT localization, epitope-tagged FT protein can be isolated from plants by subcellular fractionation and its localization examined by immunoblot analysis of the resulting fractions. However, the highly abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) can interfere with methods to detect and characterize low-abundance proteins such as FT. In this chapter, we present a method for analyzing the ratio of HA-tagged FT (HA:FT) in different subcellular fractions while mitigating the interference from RuBisCO by using protamine sulfate (PS) to deplete RuBisCO during protein purification, thereby enhancing HA:FT detection in fractionated samples.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

The small iron-deficiency-induced protein OLIVIA and its relation to the bHLH transcription factor POPEYE.

Iron (Fe) is a crucial micronutrient needed in many metabolic processes. To balance needs and potential toxicity, plants control the amount of Fe they take up and allocate to leaves and seeds during their development. One important regulator of this process is POPEYE (PYE). PYE is a Fe deficiency-induced key bHLH transcription factor (TF) for allocation of internal Fe in plants. In the absence of PYE, there is altered Fe translocation and plants develop a leaf chlorosis. NICOTIANAMINE SYNTHASE4 (NAS4), FERRIC-REDUCTION OXIDASE3 (FRO3), and ZINC-INDUCED FACILITATOR1 (ZIF1) genes are expressed at higher level in pye-1 indicating that PYE represses these genes. PYE activity is controlled in a yet unknown manner. Here, we show that a small Fe deficiency-induced protein OLIVIA (OLV) can interact with PYE. OLV has a conserved C-terminal motif, that we named TGIYY. Through deletion mapping, we pinpointed that OLV TGIYY and several regions of PYE can be involved in the protein interaction. An OLV overexpressing (OX) mutant line exhibited an enhanced NAS4 gene expression. This was a mild Fe deficiency response phenotype that was related to PYE function. Leaf rosettes of olv mutants remained smaller than those of wild type, indicating that OLV promotes plant growth. Taken together, our study identified a small protein OLV as a candidate that may connect aspects of Fe homeostasis with regulation of leaf growth.

Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids.

KEY MESSAGE: Two peanut LEC1-type genes exhibit partial functional redundancy. AhNFYB10 could complement almost all the defective phenotypes of lec1-2 in terms of embryonic morphology, while AhNF-YB1 could partially affect these phenotypes. LEAFY COTYLEDON1 (LEC1) is a member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two LEC1-type genes from Arachis hypogeae were identified and designated as AhNF-YB1 and AhNF-YB10; these genes belong to subgenome A and subgenome B, respectively. The functions of AhNF-YB1 and AhNF-YB10 were investigated by complementation analysis of their defective phenotypes of the Arabidopsis lec1-2 mutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, AhNF-YB10 complemented almost all the defective phenotypes of lec1-2 in terms of embryonic morphology and hypocotyl length, while AhNF-YB1 had only a partial effect. In addition, 30-40% of the seeds of the AhNF-YB1 transformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing AhNF-YB10-overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content.

Characterization and expression analysis of GLABRA3 (GL3) genes in cotton: insights into trichome development and hormonal regulation.

BACKGROUND: GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) genes encode a typical helix-loop-helix (bHLH) transcription factors that primarily regulate trichome branching and root hair development, DNA endoreduplication, trichoblast size, and stomatal formation. The functions of GL3 genes in cotton crop have been poorly characterized. In this study, we performed comprehensive genome-wide scans for GL3 and EGL3 homologs to enhance our comprehension of their potential roles in trichome and fiber development in cotton crop. METHODS AND RESULTS: Our findings paraded that Gossypium hirsutum and G. barbadense have 6 GL3s each, unevenly distributed on 4 chromosomes whereas, G. arboreum, and G. raimondii have 3 GL3s each, unevenly distributed on 2 chromosomes. Gh_A08G2088 and Gb_A09G2187, despite having the same bHLH domain as the other GL3 genes, were excluded due to remarkable short sequences and limited number of motifs, indicating a lack of potential functional activity. The phylogenetic analysis categorized remaining 16 GL3s into three subfamilies (Group I-III) closely related to A. thaliana. The 16 GL3s have complete bHLH domain, encompassing 590-631 amino acids, with molecular weights (MWs) ranging from 65.92 to 71.36 kDa. Within each subfamily GL3s depicted shared similar gene structures and motifs, indicating conserved characteristics within respective groups. Promoter region analysis revealed 27 cis-acting elements, these elements were responsive to salicylic acid, abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellin. The expression of GL3 genes was analyzed across 12 tissues in both G. barbadense and G. hirsutum using the publicly available RNA-seq data. Among GL3s, Gb_D11G0219, Gb_D11G0214, and Gb_D08G2182, were identified as relatively highly expressed across different tissues, consequently selected for hormone treatment and expression validation in G. barbadense. RT-qPCR results demonstrated significant alterations in the expression levels of Gb_D11G0219 and Gb_D11G0214 following MeJA, GA, and ABA treatment. Subcellular localization prediction revealed that most GL3 proteins were predominantly expressed in the nucleus, while a few were localized in the cytoplasm and chloroplasts. CONCLUSIONS: In summary, this study lays the foundation for subsequent functional validation of GL3 genes by identifying hormonal regulation patterns and probable sites of action in cotton trichome formation and fiber development. The results stipulate a rationale to elucidate the roles and regulatory mechanisms of GL3 genes in the intricate process of cotton fibre and trichome development.

WOX2 functions redundantly with WOX1 and WOX4 to positively regulate seed germination in Arabidopsis.

MAIN CONCLUSION: WOX family gene WOX2 is highly expressed during seed development, which functions redundantly with WOX1 and WOX4 to positively regulate seed germination. WOX (WUSCHEL-related homeobox) is a family of transcription factors in plants. They play essential roles in the regulation of plant growth and development, but their function in seed germination is not well understood. In this report, we show that WOX1, WOX2, and WOX4 are close homologues in Arabidopsis. WOX2 has a redundant function with WOX1 and WOX4, respectively, in seed germination. WOX2 is highly expressed during seed development, from the globular embryonic stage to mature dry seeds, and its expression is decreased after germination. Loss of function single mutant wox2, and double mutants wox1 wox2 and wox2 wox4-1 show decreased germination speed. WOX2 and WOX4 are essential for hypocotyl-radicle zone elongation during germination, potentially by promoting the expression of cell wall-related genes. We also found that WOX2 and WOX4 regulate germination through the gibberellin (GA) pathway. These results suggest that WOX2 and WOX4 integrate the GA pathway and downstream cell wall-related genes during germination.

Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.