A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate inositol hexaphosphate accumulation.

Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.

Structures and ion transport mechanisms of plant high-affinity potassium transporters.

Plant high-affinity K+ transporters (HKTs) mediate Na+ and K+ uptake, maintain Na+/K+ homeostasis, and therefore play crucial roles in plant salt tolerance. In this study, we present cryoelectron microscopy structures of HKTs from two classes, class I HKT1;1 from Arabidopsis thaliana (AtHKT1;1) and class II HKT2;1 from Triticum aestivum (TaHKT2;1), in both Na+- and K+-bound states at 2.6- to 3.0-Å resolutions. Both AtHKT1;1 and TaHKT2;1 function as homodimers. Each HKT subunit consists of four tandem domain units (D1-D4) with a repeated K+-channel-like M-P-M topology. In each subunit, D1-D4 assemble into an ion conduction pore with a pseudo-four-fold symmetry. Although both TaHKT2;1 and AtHKT1;1 have only one putative Na+ ion bound in the selectivity filter with a similar coordination pattern, the two HKTs display different K+ binding modes in the filter. TaHKT2;1 has three K+ ions bound in the selectivity filter, but AtHKT1;1 has only two K+ ions bound in the filter, which has a narrowed external entrance due to the presence of a Ser residue in the first filter motif. These structures, along with computational, mutational, and electrophysiological analyses, enable us to pinpoint key residues that are critical for the ion selectivity of HKTs. The findings provide new insights into the ion selectivity and ion transport mechanisms of plant HKTs and improve our understanding about how HKTs mediate plant salt tolerance and enhance crop growth.

Structures and mechanisms of the Arabidopsis cytokinin transporter AZG1.

Cytokinins are essential for plant growth and development, and their tissue distributions are regulated by transmembrane transport. Recent studies have revealed that members of the 'Aza-Guanine Resistant' (AZG) protein family from Arabidopsis thaliana can mediate cytokinin uptake in roots. Here we present 2.7 to 3.3 Å cryo-electron microscopy structures of Arabidopsis AZG1 in the apo state and in complex with its substrates trans-zeatin (tZ), 6-benzyleaminopurine (6-BAP) or kinetin. AZG1 forms a homodimer and each subunit shares a similar topology and domain arrangement with the proteins of the nucleobase/ascorbate transporter (NAT) family. These structures, along with functional analyses, reveal the molecular basis for cytokinin recognition. Comparison of the AZG1 structures determined in inward-facing conformations and predicted by AlphaFold2 in the occluded conformation allowed us to propose that AZG1 may carry cytokinins across the membrane through an elevator mechanism.

The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

An LRH-RSL4 feedback regulatory loop controls the determinate growth of root hairs in Arabidopsis.

Root hairs are tubular-shaped outgrowths of epidermal cells essential for plants acquiring water and nutrients from the soil. Despite their importance, the growth of root hairs is finite. How this determinate growth is precisely regulated remains largely unknown. Here we identify LONG ROOT HAIR (LRH), a GYF domain-containing protein, as a unique repressor of root hair growth. We show that LRH inhibits the association of eukaryotic translation initiation factor 4Es (eIF4Es) with the mRNA of ROOT HAIR DEFECTIVE6-LIKE4 (RSL4) that encodes the master regulator of root hair growth, repressing RSL4 translation and thus root hair elongation. RSL4 in turn directly transactivates LRH expression to maintain a proper LRH gradient in the trichoblasts. Our findings reveal a previously uncharacterized LRH-RSL4 feedback regulatory loop that limits root hair growth, shedding new light on the mechanism underlying the determinate growth of root hairs.

Beyond iron-storage pool: functions of plant apoplastic iron during stress.

Iron (Fe) is an essential micronutrient for plants, and its storage in the apoplast represents an important Fe pool. Plants have developed various strategies to reutilize this apoplastic Fe pool to adapt to Fe deficiency. In addition, growing evidence indicates that the dynamic changes in apoplastic Fe are critical for plant adaptation to other stresses, including ammonium stress, phosphate deficiency, and pathogen attack. In this review, we discuss and scrutinize the relevance of apoplastic Fe for plant behavior changes in response to stress cues. We mainly focus on the relevant components that modulate the actions and downstream events of apoplastic Fe in stress signaling networks.

ART1 and putrescine contribute to rice aluminum resistance via OsMYB30 in cell wall modification.

Cell wall is the first physical barrier to aluminum (Al) toxicity. Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance; nevertheless, how it is regulated in rice remains largely unknown. In this study, we show that exogenous application of putrescines (Put) could significantly restore the Al resistance of art1, a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1 (ART1), and reduce its Al accumulation particularly in the cell wall of root tips. Based on RNA-sequencing, yeast-one-hybrid and electrophoresis mobility shift assays, we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance. Furthermore, transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30, and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation, hence reducing the Al-binding capacity of cell wall and enhancing Al resistance. Additionally, Put repressed OsMYB30 expression by eliminating Al-induced H2 O2 accumulation, while exogenous H2 O2 promoted OsMYB30 expression. We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Abscisic acid-dependent PMT1 expression regulates salt tolerance by alleviating abscisic acid-mediated reactive oxygen species production in Arabidopsis.

Phosphocholine (PCho) is an intermediate metabolite of nonplastid plant membranes that is essential for salt tolerance. However, how PCho metabolism modulates response to salt stress remains unknown. Here, we characterize the role of phosphoethanolamine N-methyltransferase 1 (PMT1) in salt stress tolerance in Arabidopsis thaliana using a T-DNA insertional mutant, gene-editing alleles, and complemented lines. The pmt1 mutants showed a severe inhibition of root elongation when exposed to salt stress, but exogenous ChoCl or lecithin rescued this defect. pmt1 also displayed altered glycerolipid metabolism under salt stress, suggesting that glycerolipids contribute to salt tolerance. Moreover, pmt1 mutants exhibited altered reactive oxygen species (ROS) accumulation and distribution, reduced cell division activity, and disturbed auxin distribution in the primary root compared with wild-type seedlings. We show that PMT1 expression is induced by salt stress and relies on the abscisic acid (ABA) signaling pathway, as this induction was abolished in the aba2-1 and pyl112458 mutants. However, ABA aggravated the salt sensitivity of the pmt1 mutants by perturbing ROS distribution in the root tip. Taken together, we propose that PMT1 is an important phosphoethanolamine N-methyltransferase participating in root development of primary root elongation under salt stress conditions by balancing ROS production and distribution through ABA signaling.

RING-box proteins regulate leaf senescence and stomatal closure via repression of ABA transporter gene ABCG40.

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.

Ethylene signaling modulates Arabidopsis thaliana nitrate metabolism.

MAIN CONCLUSION: Genetic analysis reveals a previously unknown role for ethylene signaling in regulating Arabidopsis thaliana nitrogen metabolism. Nitrogen (N) is essential for plant growth, and assimilation of soil nitrate (NO3-) and ammonium ions is an important route of N acquisition. Although N import and assimilation are subject to multiple regulatory inputs, the extent to which ethylene signaling contributes to this regulation remains poorly understood. Here, our analysis of Arabidopsis thaliana ethylene signaling mutants advances that understanding. We show that the loss of CTR1 function ctr1-1 mutation confers resistance to the toxic effects of the NO3- analogue chlorate (ClO3-), and reduces the activity of the nitrate reductase (NR) enzyme of NO3- assimilation. Our further analysis indicates that the lack of the downstream EIN2 component (conferred by novel ein2 mutations) suppresses the effect of ctr1-1, restoring ClO3- sensitivity and NR activity to normal. Collectively, our observations indicate an important role for ethylene signaling in regulating Arabidopsis thaliana NO3- metabolism. We conclude that ethylene signaling enables environmentally responsive coordination of plant growth and N metabolism.

Potential Role of Domains Rearranged Methyltransferase7 in Starch and Chlorophyll Metabolism to Regulate Leaf Senescence in Tomato.

Deoxyribonucleic acid (DNA) methylation is an important epigenetic mark involved in diverse biological processes. Here, we report the critical function of tomato (Solanum lycopersicum) Domains Rearranged Methyltransferase7 (SlDRM7) in plant growth and development, especially in leaf interveinal chlorosis and senescence. Using a hairpin RNA-mediated RNA interference (RNAi), we generated SlDRM7-RNAi lines and observed pleiotropic developmental defects including small and interveinal chlorosis leaves. Combined analyses of whole genome bisulfite sequence (WGBS) and RNA-seq revealed that silencing of SlDRM7 caused alterations in both methylation levels and transcript levels of 289 genes, which are involved in chlorophyll synthesis, photosynthesis, and starch degradation. Furthermore, the photosynthetic capacity decreased in SlDRM7-RNAi lines, consistent with the reduced chlorophyll content and repression of genes involved in chlorophyll biosynthesis, photosystem, and photosynthesis. In contrast, starch granules were highly accumulated in chloroplasts of SlDRM7-RNAi lines and associated with lowered expression of genes in the starch degradation pathway. In addition, SlDRM7 was activated by aging- and dark-induced senescence. Collectively, these results demonstrate that SlDRM7 acts as an epi-regulator to modulate the expression of genes related to starch and chlorophyll metabolism, thereby affecting leaf chlorosis and senescence in tomatoes.

Phloem iron remodels root development in response to ammonium as the major nitrogen source.

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH4+ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

Structural basis of ALMT1-mediated aluminum resistance in Arabidopsis.

The plant aluminum (Al)-activated malate transporter ALMT1 mediates the efflux of malate to chelate the Al in acidic soils and underlies the plant Al resistance. Here we present cryo-electron microscopy (cryo-EM) structures of Arabidopsis thaliana ALMT1 (AtALMT1) in the apo, malate-bound, and Al-bound states at neutral and/or acidic pH at up to 3.0 Å resolution. The AtALMT1 dimer assembles an anion channel and each subunit contains six transmembrane helices (TMs) and six cytosolic α-helices. Two pairs of Arg residues are located in the center of the channel pore and contribute to malate recognition. Al binds at the extracellular side of AtALMT1 and induces conformational changes of the TM1-2 loop and the TM5-6 loop, resulting in the opening of the extracellular gate. These structures, along with electrophysiological measurements, molecular dynamic simulations, and mutagenesis study in Arabidopsis, elucidate the structural basis for Al-activated malate transport by ALMT1.

STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity.

Protons (H+) in acidic soils arrest plant growth. However, the mechanisms by which plants optimize their biological processes to diminish the unfavorable effects of H+ stress remain largely unclear. Here, we showed that in the roots of Arabidopsis thaliana, the C2H2-type transcription factor STOP1 in the nucleus was enriched by low pH in a nitrate-independent manner, with the spatial expression pattern of NITRATE TRANSPORTER 1.1 (NRT1.1) established by low pH required the action of STOP1. Additionally, the nrt1.1 and stop1 mutants, as well as the nrt1.1 stop1 double mutant, had a similar hypersensitive phenotype to low pH, indicating that STOP1 and NRT1.1 function in the same pathway for H+ tolerance. Molecular assays revealed that STOP1 directly bound to the promoter of NRT1.1 to activate its transcription in response to low pH, thus upregulating its nitrate uptake. This action improved the nitrogen use efficiency (NUE) of plants and created a favorable rhizospheric pH for root growth by enhancing H+ depletion in the rhizosphere. Consequently, the constitutive expression of NRT1.1 in stop1 mutants abolished the hypersensitive phenotype to low pH. These results demonstrate that STOP1-NRT1.1 is a key module for plants to optimize NUE and ensure better plant growth in acidic media.

A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis.

Phosphorus (P) is an indispensable macronutrient required for plant growth and development. Natural phosphate (Pi) reserves are finite, and a better understanding of Pi utilization by crops is therefore vital for worldwide food security. Ammonium has long been known to enhance Pi acquisition efficiency in agriculture; however, the molecular mechanisms coordinating Pi nutrition and ammonium remains unclear. Here, we reveal that ammonium is a novel initiator that stimulates the accumulation of a key regulatory protein, STOP1, in the nuclei of Arabidopsis root cells under Pi deficiency. We show that Pi deficiency promotes ammonium uptake mediated by AMT1 transporters and causes rapid acidification of the root surface. Rhizosphere acidification-triggered STOP1 accumulation activates the excretion of organic acids, which help to solubilize Pi from insoluble iron or calcium phosphates. Ammonium uptake by AMT1 transporters is downregulated by a CIPK23 protein kinase whose expression is directly modulated by STOP1 when ammonium reaches toxic levels. Taken together, we have identified a STOP1-centered regulatory network that links external ammonium with efficient Pi acquisition from insoluble phosphate sources. These findings provide a framework for developing possible strategies to improve crop production by enhancing the utilization of non-bioavailable nutrients in soil.

Restriction of iron loading into developing seeds by a YABBY transcription factor safeguards successful reproduction in Arabidopsis.

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression.

Tease out the future: How tea research might enable crop breeding for acid soil tolerance.

Unlike most crops, in which soil acidity severely limits productivity, tea (Camellia sinensis) actually prefers acid soils (pH 4.0-5.5). Specifically, tea is very tolerant of acidity-promoted aluminum (Al) toxicity, a major factor that limits the yield of most other crops, and it even requires Al for optimum growth. Understanding tea Al tolerance and Al-stimulatory mechanisms could therefore be fundamental for the future development of crops adapted to acid soils. Here, we summarize the Al-tolerance mechanisms of tea plants, propose possible mechanistic explanations for the stimulation of tea growth by Al based on recent research, and put forward ideas for future crop breeding for acid soils.

Thermal stress accelerates Arabidopsis thaliana mutation rate.

Mutations are the source of both genetic diversity and mutational load. However, the effects of increasing environmental temperature on plant mutation rates and relative impact on specific mutational classes (e.g., insertion/deletion [indel] vs. single nucleotide variant [SNV]) are unknown. This topic is important because of the poorly defined effects of anthropogenic global temperature rise on biological systems. Here, we show the impact of temperature increase on Arabidopsis thaliana mutation, studying whole genome profiles of mutation accumulation (MA) lineages grown for 11 successive generations at 29°C. Whereas growth of A. thaliana at standard temperature (ST; 23°C) is associated with a mutation rate of 7 × 10-9 base substitutions per site per generation, growth at stressful high temperature (HT; 29°C) is highly mutagenic, increasing the mutation rate to 12 × 10-9 SNV frequency is approximately two- to threefold higher at HT than at ST, and HT-growth causes an ∼19- to 23-fold increase in indel frequency, resulting in a disproportionate increase in indels (vs. SNVs). Most HT-induced indels are 1-2 bp in size and particularly affect homopolymeric or dinucleotide A or T stretch regions of the genome. HT-induced indels occur disproportionately in nucleosome-free regions, suggesting that much HT-induced mutational damage occurs during cell-cycle phases when genomic DNA is packaged into nucleosomes. We conclude that stressful experimental temperature increases accelerate plant mutation rates and particularly accelerate the rate of indel mutation. Increasing environmental temperatures are thus likely to have significant mutagenic consequences for plants growing in the wild and may, in particular, add detrimentally to mutational load.