Takushi Hachiya

Nitrogen (N) is a major determinant of plant growth and productivity. Because soil N availability and internal N demand fluctuate, plants have evolved sophisticated mechanisms to coordinate N acquisition and utilization at the whole-plant level. However, how this coordination is achieved remains poorly understood. Here, we show that N-inducible LATERAL ORGAN BOUNDARIES DOMAIN transcription factors LBD37, LBD38, and LBD39 (LBDs) function as repressors of local N uptake and assimilation and systemic N-demand signaling in Arabidopsis. Triple mutants lacking these three LBDs displayed enhanced nitrate influx and increased accumulation of nitrate, amino acids, and total N. Transcriptome analysis identified an array of N-starvation- and nitrate-inducible genes derepressed in shoots and roots, including C-TERMINALLY ENCODED PEPTIDE ( CEP ) and CEP DOWNSTREAM ( CEPD ) genes, as well as genes involved in N uptake and assimilation. Grafting and genetic analyses revealed that LBDs gate the systemic N-demand signaling relay by repressing CEP and CEPD expression organ-autonomously. We also found that LBDs locally repress genes involved in N uptake and assimilation through a distinct regulatory mechanism. We propose that LBDs are key transcriptional repressors in a regulatory framework for optimizing N acquisition and utilization under fluctuating N conditions at the whole-plant level.

Trans-zeatin (tZ)-type cytokinins (CKs) are predominantly synthesized in roots, transported to the shoot via the xylem, and coordinate diverse physiological processes in aerial organs. Within this process, the regulation of CK biosynthesis by nitrate signaling via nodule inception-like protein 7, as well as the loading of tZ-type CKs into the xylem by ATP-binding cassette transporter G14, have been well studied. However, the roles of other components remain unclear. Here, we show that CK perception and degradation in roots, as mediated by Arabidopsis histidine kinase 3 (AHK3) and CK oxidase/dehydrogenase 4 (CKX4), modulate root-to-shoot tZ-type cytokinin transport in response to nitrate. Grafting experiments demonstrate that root-specific AHK3 deficiency systemically increases the leaf blade area through long-distance signals of root-derived tZ-type CKs, perceived by shoot-expressed AHK3. Transcriptome and hormonome analyses reveal that root-specific AHK3 deficiency reduces CKX4 expression in roots, elevating tZ-type CK levels in roots and xylem sap and thereby enhancing the leaf CK response. Transfer experiments manipulating root nitrate levels show that root-specific AHK3 deficiency promotes the leaf blade area in a manner dependent on both nitrate and root-derived tZ-type CK signaling. Moreover, both nitrate signals and root-expressed AHK3 are required for maximal CKX4 induction in roots, and root-specific CKX4 deficiency enhances the leaf blade area in a nitrate-dependent manner. These findings reveal a novel mechanism in which an AHK3–CKX4 module governs xylem transport of tZ-type CKs, fine-tuning leaf size according to root nitrate status.

Plants systemically regulate growth and gene expression according to their internal nitrate status. Our previous study reported that shoot nitrate accumulation increases shoot expression of adenosine phosphate‐ ISOPENTENYLTRANSFERASE 3 ( IPT3 ) and shoot levels of N ⁶ ‐(Δ ² ‐isopentenyl) adenine (iP)‐type cytokinins (CKs). IPT3 expression is localized in the phloem, and iP‐type CKs, which are synthesized by IPT3, are phloem‐mobile. As CKs are a class of phytohormones that control growth and genome‐wide gene expression, shoot‐expressed IPT3 may mediate the systemic regulation of growth and transcriptomic responses to shoot nitrate status. To examine this, we developed a novel system to manipulate nitrate levels and IPT3 expression in a shoot‐specific manner and performed growth analysis, CK determination, and RNA‐sequencing. Our results demonstrated that shoot nitrate accumulation significantly promoted shoot growth and elevated shoot concentrations of iP ribotides, iP‐7‐ N ‐glucoside, and iP‐9‐ N ‐glucoside through the action of shoot IPT3. Transcriptomic responses to shoot nitrate accumulation were largely tuned by shoot IPT3, with opposite effects in shoots and roots. Shoot IPT3 amplified shoot responses of nitrate‐inducible genes and immune response genes to shoot nitrate accumulation, while it dampened root responses of nitrate transport/assimilation genes. This transcriptomic modulation via shoot IPT3 was accompanied by coherent transcriptional changes in genes encoding mobile peptides and transcriptional repressors. Here we present a novel scheme integrating shoot nitrate status responses with CK signaling.

Root-type ferredoxin:NADP(H) oxidoreductase (RFNR) is believed to reduce ferredoxin using NADPH in nonphotosynthetic tissues, facilitating ferredoxin-dependent biological processes. However, the physiological functions of RFNR remain unclear due to the difficulty in obtaining mutants lacking redundant RFNR isoproteins. The present study successfully generated Arabidopsis homozygous rnfr1 ; 2 double mutants by traditional crossing and selection. However, they displayed severely stunted roots, challenging subsequent growth and abundant seed recovery. Notably, grafted plants combining mutant scions with wild-type rootstocks exhibited normal growth and produced abundant mutant seeds. Growth analysis employing reciprocal grafts with the wild-type and mutant plants showed that primary root growth was inhibited only when the rootstock was derived from the mutants. Meanwhile, the absence of RFNR1 and 2 in the scion had no apparent impact on shoot and root growth. Root transcriptome analysis revealed that RFNR1 and 2 deficiency upregulated genes encoding ferredoxin-dependent enzymes and root-type ferredoxin, leading to genome-wide reprogramming associated with cell walls, lipids, photosynthesis, secondary metabolism, and biotic/abiotic stress responses. Thus, Arabidopsis RFNR1 and 2 are crucial for root growth and various ferredoxin-dependent biological processes.

Plants systemically regulate growth and gene expression according to their internal nitrate status. Our previous study reported that shoot nitrate accumulation increases shoot expression of adenosine phosphate- ISOPENTENYLTRANSFERASE 3 ( IPT3 ) and shoot levels of N ⁶ -(Δ ² -isopentenyl) adenine (iP)-type cytokinins (CKs). IPT3 expression is localized in the phloem, and iP-type CKs, which are synthesized by IPT3, are phloem-mobile. As CKs are a class of phytohormones that control growth and genome-wide gene expression, shoot-expressed IPT3 may mediate the systemic regulation of growth and transcriptomic responses to shoot nitrate status. To examine this, we developed a novel system to manipulate nitrate levels and IPT3 expression in a shoot-specific manner and performed growth analysis, CK determination, and RNA-sequencing. Our results demonstrated that shoot nitrate accumulation significantly promoted shoot growth and elevated shoot concentrations of iP ribotides, iP-7- N -glucoside, and iP-9- N -glucoside through the action of shoot IPT3. Transcriptomic responses to shoot nitrate accumulation were largely tuned by shoot IPT3, with opposite effects in shoots and roots. Shoot IPT3 amplified shoot responses of nitrate-inducible genes and immune response genes to shoot nitrate accumulation, while it dampened root responses of nitrate transport/assimilation genes. This transcriptomic modulation via shoot IPT3 was accompanied by coherent transcriptional changes in genes encoding mobile peptides and transcriptional repressors. Here we present a novel scheme integrating shoot nitrate status responses with CK signaling.

Helicases are involved in almost every nucleic acid metabolism process. Within this family, RecQ helicase proteins protect genome integrity across all organisms through DNA recombination, repair, and replication. This study focused on five Arabidopsis thaliana RecQ-like (AtRecQl) genes with diverse functionalities. Analysis of ProAtRecQl:GUS expression during vegetative and reproductive development stages revealed organ- and tissue-specific patterns. Changes in AtRecQls transcript levels in response to abiotic stressors suggest their involvement in diverse stimuli responses. Notably, germination and growth rates were lower in atrecql2 and atrecql3 mutants under various salt concentrations and cold conditions. These findings indicate that AtRecQl2 and AtRecQl3 act as positive regulators of abiotic stress tolerance during the germinative and postgerminative phases.

The conversion of nitrate to ammonium, i.e., nitrate reduction, is a major consumer of reductants in plants. Previous studies have reported that the mitochondrial alternative oxidase (AOX) is upregulated under limited nitrate reduction conditions, including no/low nitrate or when ammonium is the sole nitrogen (N) source. Electron transfer from ubiquinone to AOX bypasses the proton-pumping complexes III and IV, thereby consuming reductants efficiently. Thus, upregulated AOX under limited nitrate reduction may dissipate excessive reductants and thereby attenuate oxidative stress. Nevertheless, so far there is no firm evidence for this hypothesis due to the lack of experimental systems to analyze the direct relationship between nitrate reduction and AOX. We therefore developed a novel culturing system for A. thaliana that manipulates shoot activities of nitrate reduction and AOX separately without causing N starvation, ammonium toxicity, or lack of nitrate signal. Using shoots processed with this system, we examined genome-wide gene expression and growth to better understand the relationship between AOX and nitrate reduction. The results showed that, only when nitrate reduction was limited, AOX deficiency significantly upregulated genes involved in mitochondrial oxidative stress, reductant shuttles, and non-phosphorylating bypasses of the respiratory chain, and inhibited growth. Thus, we conclude that AOX alleviates mitochondrial oxidative stress and sustains plant growth under limited nitrate reduction.