Hiromi Suzuki

Plant hormones play critical roles in many aspects of plant life cycles including development, growth, reproduction and responses to environmental stimuli. These processes are often associated with changes in endogenous plant hormone levels and locations. Therefore, to understand the modes of action of plant hormones, it is important to accurately quantify these chemical compounds in a high-definition tissue map. In this study, we developed a system to quantify indole-3-acetic acid (IAA), the major endogenous auxin, from small tissue samples using laser microdissection (LMD) coupled with nano-flow liquid chromatography (nano-LC)-mass spectrometry (MS), which improved detection limits, allowing quantification of IAA from a single 10 μm cryosection of maize coleoptile. Our results reveal that IAA is actively synthesized in the apical 400 μm region of the coleoptiles and is preferentially accumulated in vascular tissues. This technique can provide a precise view of the spatiotemporal distribution of plant hormones and their significance in regulating physiological responses at tissue or cellular levels.

The use of genes derived from halophytes represents a promising strategy for enhancing salt tolerance. Among salt tolerance mechanisms, vacuolar Na⁺/H⁺ exchangers (NHXs) play a central role in cellular Na⁺ sequestration in both halophytes and glycophytes. In this study, we identified the SvNHX2 gene from the halophyte Sporobolus virginicus and characterized its role in salinity tolerance. SvNHX2 expression was strongly induced by salt stress, particularly in shoots in S. virginicus. Arabidopsis thaliana plants overexpressing SvNHX2 exhibited significantly improved growth at the vegetative growth stage under 100 and 125 mM NaCl on agar plates and at the reproductive growth stage under 100 mM NaCl in hydroponic systems, although no clear correlation was observed between transgene expression levels and tolerance. Ion analysis showed that SvNHX2 overexpression increased Na⁺ accumulation in roots under NaCl stress, suggesting enhanced vacuolar Na⁺ sequestration, while K⁺ levels remained comparable to the wild type. These findings indicated that SvNHX2 contributes to salt tolerance primarily by promoting Na⁺ compartmentalization, and that its capacity is comparable to those of NHX proteins in glycophytes. In this study, we provide additional insights into the function of halophytic NHX and demonstrated that SvNHX2 is a valuable gene for engineering salt-tolerant crops.

The plant hormone abscisic acid (ABA) is actively synthesized in vascular tissues and transported to guard cells to promote stomatal closure. Although several transmembrane ABA transporters have been identified, how the movement of ABA within plants is regulated is not fully understood. In this study, we determined that Arabidopsis NPF4.6, previously identified as an ABA transporter expressed in vascular tissues, is also present in guard cells and positively regulates stomatal closure in leaves. We also found that mutants defective in NPF5.1 had a higher leaf surface temperature compared to the wild type. Additionally, NPF5.1 mediated cellular ABA uptake when expressed in a heterologous yeast system. Promoter activities of NPF5.1 were detected in several leaf cell types. Taken together, these observations indicate that NPF5.1 negatively regulates stomatal closure by regulating the amount of ABA that can be transported from vascular tissues to guard cells.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues. Significance Plant roots grow toward gravity. The phenomenon is known as “root gravitropism” as early described by Charles Darwin, and it has been well established that the plant hormone auxin plays a central role in this response. The major endogenous auxin, indole-3-acetic acid (IAA), is mainly synthesized from tryptophan via indole-3-pyruvic acid, whereas indole-3-butyric acid (IBA) is a minor precursor of IAA. Previous studies have shown that IAA derived from IBA plays a specific role in regulating lateral root development. However, in the present study we demonstrate through the identification and characterization of IBA transporter NPF7.3/NRT1.5 that IAA synthesized from IBA regulates root gravitropism.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

Abscisic acid (ABA) is an important hormone for seed development and germination whose physiological action is modulated by its endogenous levels. Cleavage of carotenoid precursors by 9-cis epoxycarotenoid dioxygenase (NCED) and inactivation of ABA by ABA 8′-hydroxylase (CYP707A) are key regulatory metabolic steps. In Arabidopsis (Arabidopsis thaliana), both enzymes are encoded by multigene families, having distinctive expression patterns. To evaluate the genome-wide impact of ABA deficiency in developing seeds at the maturation stage when dormancy is induced, we used a nced2569 quadruple mutant in which ABA deficiency is mostly restricted to seeds, thus limiting the impact of maternal defects on seed physiology. ABA content was very low in nced2569 seeds, similar to the severe mutant aba2; unexpectedly, ABA Glc ester was detected in aba2 seeds, suggesting the existence of an alternative metabolic route. Hormone content in nced2569 seeds compared with nced259 and wild type strongly suggested that specific expression of NCED6 in the endosperm is mainly responsible for ABA production. In accordance, transcriptome analyses revealed broad similarities in gene expression between nced2569 and either wild-type or nced259 developing seeds. Gene ontology enrichments revealed a large spectrum of ABA activation targets involved in reserve storage and desiccation tolerance, and repression of photosynthesis and cell cycle. Proteome and metabolome profiles in dry nced2569 seeds, compared with wild-type and cyp707a1a2 seeds, also highlighted an inhibitory role of ABA on remobilization of reserves, reactive oxygen species production, and protein oxidation. Down-regulation of these oxidative processes by ABA may have an essential role in dormancy control.

Plant roots play important roles in absorbing water and nutrients, and in tolerance against environmental stresses. Previously, we identified a rice root‐specific pathogenesis‐related protein (RSOsPR10) induced by drought, salt, and wounding. RSOsPR10 expression is strongly induced by jasmonate (JA)/ethylene (ET), but suppressed by salicylic acid (SA). Here, we analyzed the promoter activity of RSOsPR10. Analyses of transgenic rice lines harboring different‐length promoter::β‐glucuronidase (GUS) constructs showed that the 3‐kb promoter region is indispensable for JA/ET induction, SA repression, and root‐specific expression. In the JA‐treated 3K‐promoter::GUS line, GUS activity was mainly observed at lateral root primordia. Transient expression in roots using a dual luciferase (LUC) assay with different‐length promoter::LUC constructs demonstrated that the novel transcription factor OsERF87 induced 3K‐promoter::LUC expression through binding to GCC‐cis elements. In contrast, the SA‐inducible OsWRKY76 transcription factor strongly repressed the JA‐inducible and OsERF87‐dependent expression of RSOsPR10. RSOsPR10 was expressed at lower levels in OsWRKY76‐overexpressing rice, but at higher levels in OsWRKY76‐knockout rice, compared with wild type. These results show that two transcription factors, OsERF87 and OsWRKY76, antagonistically regulate RSOsPR10 expression through binding to the same promoter. This mechanism represents a fine‐tuning system to sense the balance between JA/ET and SA signaling in plants under environmental stress.

Gravitropism refers to the growth or movement of plants that is influenced by gravity. Roots exhibit positive gravitropism, and the root cap is thought to be the gravity-sensing site. In some plants, the root cap requires light irradiation for positive gravitropic responses. However, the mechanisms regulating this phenomenon are unknown. We herein report that maize roots exposed to white light continuously for ≥1–2h show increased indole-3-acetic acid (IAA) levels in the root tips, especially in the transition zone (1–3mm from the tip). Treatment with IAA biosynthesis inhibitors yucasin and l-kynurenine prevented any increases in IAA content and root curvature under light conditions. Analyses of the incorporation of a stable isotope label from tryptophan into IAA revealed that some of the IAA in roots was synthesized in the root apex. Furthermore, Zmvt2 and Zmyuc gene transcripts were detected in the root apex. One of the Zmyuc genes (ZM2G141383) was up-regulated by light irradiation in the 0–1mm tip region. Our findings suggest that IAA accumulation in the transition zone is due to light-induced activation of Zmyuc gene expression in the 0–1mm root apex region. Light-induced changes in IAA levels and distributions mediate the maize root gravitropic U-turn.

ZmPHOT1 and ZmPHOT2 are expressed differentially in maize coleoptiles and leaves, with Zmphot1 possibly involved in first-positive phototropic curvature of red-light-adapted maize coleoptiles exposed to pulsed low-fluence blue light. Unilateral blue-light perception by phototropin(s) is the first event of phototropism, with the subsequent signal causing lateral transport of auxin at the coleoptile tip region of monocots. In this study, we analyzed the behavior of two maize phototropin genes: ZmPHOT1 and ZmPHOT2, the latter identified from the maize genome database and newly characterized. Quantitative real-time PCR analysis demonstrated that ZmPHOT1 was abundantly expressed in etiolated coleoptiles, while lower expressions of both ZmPHOT1 and ZmPHOT2 were observed in young leaves. Interestingly, these genes were not specifically expressed in the coleoptile tip region, a key position for photoperception in phototropism. Exposure to pulsed low-fluence blue light (LBL) (0.33 µmol m⁻² s⁻¹ × 8 s) and continuous high-fluence blue light (HBL) (10 µmol m⁻² s⁻¹) rapidly decreased ZmPHOT1 gene expression in coleoptiles, with levels of ZmPHOT2 not significantly altered in that tissue. In young leaves, no drastic expression changes were induced in either ZmPHOT1 or ZmPHOT2 by LBL or HBL irradiation. The Zmphot1 protein was investigated by Western blot analysis with anti-Osphot1 antibodies. Zmphot1 was detected in microsomal fractions, with higher levels in coleoptiles than in leaves. HBL caused rapid phosphorylation of the protein, whereas no phot1 phosphorylation was induced by LBL. The involvement of Zmphot1 in LBL-induced phototropic curvature of maize coleoptiles is discussed.