NPA Treatment Research Articles

Genome mining of WOX-ARF gene linkage in Machilus pauhoi underpinned cambial activity associated with IAA induction.

As an upright tree with multifunctional economic application, Machilus pauhoi is an excellent choice in modern forestry from Lauraceae. The growth characteristics is of great significance for its molecular breeding and improvement. However, there still lack the information of WUSCHEL-related homeobox (WOX) and Auxin response factor (ARF) gene family, which were reported as specific transcription factors in plant growth as well as auxin signaling. Here, a total of sixteen MpWOX and twenty-one MpARF genes were identified from the genome of M. pauhoi. Though member of WOX conserved in the Lauraceae, MpWOX and MpARF genes were unevenly distributed on 12 chromosomes as a result of region duplication. These genes presented 45 and 142 miRNA editing sites, respectively, reflecting a potential post-transcriptional restrain. Overall, MpWOX4, MpWOX13a, MpWOX13b, MpARF6b, MpARF6c, and MpARF19a were highly co-expressed in the vascular cambium, forming a working mode as WOX-ARF complex. MpWOXs contains typical AuxRR-core and TGA-element cis-acting regulatory elements in this auxin signaling linkage. In addition, under IAA and NPA treatments, MpARF2a and MpWOX1a was highly sensitive to IAA response, showing significant changes after 6 hours of treatment. And MpWOX1a was significantly inhibited by NPA treatment. Through all these solid analysis, our findings provide a genetic foundation to growth mechanism analysis and further molecular designing breeding in Machilus pauhoi.

Endogenous IAA affected fluoranthene accumulation by regulating H+-ATPase and SOD activity in ryegrass

This study explores the role of endogenous indole-3-acetic acid (IAA) in modulating plant responses to pollution stress and its effect on pollutant accumulation, with a focus on fluoranthene (Flu) in ryegrass. To elucidate the mechanism, we employed an IAA promoter (α-aminobutyric acid [α-AB]) and an IAA inhibitor (naphthylphthalamic acid [NPA]) to regulate IAA levels and analyze Flu uptake characteristics. The experimental setup included a Flu treatment group (ryegrass with Flu addition) and a control group (ryegrass without Flu). Our findings demonstrate that Flu treatment enhanced IAA content and plant growth in ryegrass compared to the control. The Flu+AB treatment further enhanced these effects, while the Flu+NPA treatment exhibited a contrasting trend. Moreover, Flu+AB treatment led to increased Flu accumulation, in contrast to the inhibitory effect observed with Flu+NPA treatment. Flu treatment also enhanced the activities of key antioxidant enzymes (SOD, POD, CAT) and increased soluble sugar and protein levels, indicative of enzymatic and nonenzymatic defense responses, respectively. The Flu+AB treatment amplified these responses, whereas the Flu+NPA treatment attenuated them. Significantly, Flu treatment raised H+-ATPase activity compared to the control, an effect further elevated by Flu+AB treatment and diminished by Flu+NPA treatment. A random forest analysis suggested that Flu accumulation dependency varied under different treatments: it relied more on H+-ATPase activity under Flu+AB treatment and more on SOD activity under Flu+NPA treatment. Additionally, Flu+AB treatment boosted the transpiration rate in ryegrass, thereby increasing the Flu translocation factor, a trend reversed by Flu+NPA treatment. This research highlights crucial factors influencing Flu accumulation in ryegrass, offering potential new avenues for controlling the gathering of contaminants within plant systems.

Effects of Exogenous Auxin on Mesocotyl Elongation of Sorghum.

The length of sorghum mesocotyl plays a vital role in seed emergence from the soil, which is the foundation of healthy growth. In this study, we aimed to understand how exogenous auxin (IAA) promoted mesocotyl elongation of sorghum and its physiology mechanism. The results presented that exogenous IAA significantly promoted mesocotyl elongation in MS24B (short mesocotyl inbred line) by increasing the cell length, while with extra exogenous NPA (IAA inhibitor) application, the mesocotyl length presented a significant short phenotype. In Z210 (long mesocotyl inbred line), exogenous IAA had a slight effect on mesocotyl length elongation, while the NPA treatment decreased the mesocotyl length considerably. In MS24B, IAA treatment increased the activity of amylase to degrade starch to soluble sugar, and the activity of hexokinase was improved to consume the increased soluble sugar to offer more energy. The energy will help to increase the activity of PM H+-ATPase and the expression of expansin-related genes, which ultimately will promote the acidification of the plasma membrane in MS24B for cell elongation. Overall, the exogenous IAA functioned on the activation of energy metabolism, which in turn, inducted the acidification of the plasma membrane for mesocotyl elongation.

Auxin efflux carrier ZmPIN1a modulates auxin reallocation involved in nitrate-mediated root formation

BackgroundAuxin plays a crucial role in nitrate (NO3–)-mediated root architecture, and it is still unclear that if NO3– supply modulates auxin reallocation for regulating root formation in maize (Zea mays L.). This study was conducted to investigate the role of auxin efflux carrier ZmPIN1a in the root formation in response to NO3– supply.ResultsLow NO3– (LN) promoted primary root (PR) elongation, while repressed the development of lateral root primordia (LRP) and total root length. LN modulated auxin levels and polar transport and regulated the expression of auxin-responsive and -signaling genes in roots. Moreover, LN up-regulated the expression level of ZmPIN1a, and overexpression of ZmPIN1a enhanced IAA efflux and accumulation in PR tip, while repressed IAA accumulation in LRP initiation zone, which consequently induced LN-mediated PR elongation and LR inhibition. The inhibition rate of PR length, LRP density and number of ZmPIN1a-OE plants was higher than that of wild-type plants after auxin transport inhibitor NPA treatment under NN and LN conditions, and the degree of inhibition of root growth in ZmPIN1a-OE plants was more obvious under LN condition.ConclusionThese findings suggest that ZmPIN1a was involved in modulating auxin levels and transport to alter NO3–-mediated root formation in maize.

Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism.

The distribution and content of auxin within plant tissues affect a variety of important growth and developmental processes. Polar auxin transport (PAT), mainly mediated by auxin influx and efflux transporters, plays a vital role in determining auxin maxima and gradients in plants. The auxin efflux carrier PIN-FORMED (PIN) family is one of the major protein families involved in PAT. Rice (Oryza sativa L.) genome possesses 12 OsPIN genes. However, the detailed functions of OsPIN genes involved in regulating the rice architecture and gravity response are less well understood. In the present study, OsPIN1b was disrupted by CRISPR/Cas9 technology, and its roles in modulating rice architecture and root gravitropism were investigated. Tissue-specific analysis showed that OsPIN1b was mainly expressed in roots, stems and sheaths at the seedling stage, and the transcript abundance was progressively decreased during the seedling stages. Expression of OsPIN1b could be quickly and greatly induced by NAA, indicating that OsPIN1b played a vital role in PAT. IAA homeostasis was disturbed in ospin1b mutants, as evidenced by the changed sensitivity of shoot and root to NAA and NPA treatment, respectively. Mutation of OsPIN1b resulted in pleiotropic phenotypes, including decreased growth of shoots and primary roots, reduced adventitious root number in rice seedlings, as well as shorter and narrower leaves, increased leaf angle, more tiller number and decreased plant height and panicle length at the late developmental stage. Moreover, ospin1b mutants displayed a curly root phenotype cultured with tap water regardless of lighting conditions, while nutrient solution culture could partially rescue the curly root phenotype in light and almost completely abolish this phenotype in darkness, indicating the involvement of the integration of light and nutrient signals in root gravitropism regulation. Additionally, amyloplast sedimentation was impaired in the peripheral tiers of the ospin1b root cap columella cell, while it was not the main contributor to the abnormal root gravitropism. These data suggest that OsPIN1b not only plays a vital role in regulating rice architecture but also functions in regulating root gravitropism by the integration of light and nutrient signals.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock

Adventitious root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

Auxin regulated metabolic changes underlying sepal retention and development after pollination in spinach

BackgroundPollination accelerate sepal development that enhances plant fitness by protecting seeds in female spinach. This response requires pollination signals that result in the remodeling within the sepal cells for retention and development, but the regulatory mechanism for this response is still unclear. To investigate the early pollination-induced metabolic changes in sepal, we utilize the high-throughput RNA-seq approach.ResultsSpinach variety ‘Cornel 9’ was used for differentially expressed gene analysis followed by experiments of auxin analog and auxin inhibitor treatments. We first compared the candidate transcripts expressed differentially at different time points (12H, 48H, and 96H) after pollination and detected significant difference in Trp-dependent auxin biosynthesis and auxin modulation and transduction process. Furthermore, several auxin regulatory pathways i.e. cell division, cell wall expansion, and biogenesis were activated from pollination to early developmental symptoms in sepals following pollination. To further confirm the role auxin genes play in the sepal development, auxin analog (2, 4-D; IAA) and auxin transport inhibitor (NPA) with different concentrations gradient were sprayed to the spinach unpollinated and pollinated flowers, respectively. NPA treatment resulted in auxin transport weakening that led to inhibition of sepal development at concentration 0.1 and 1 mM after pollination. 2, 4-D and IAA treatment to unpollinated flowers resulted in sepal development at lower concentration but wilting at higher concentration.ConclusionWe hypothesized that sepal retention and development might have associated with auxin homeostasis that regulates the sepal size by modulating associated pathways. These findings advanced the understanding of this unusual phenomenon of sepal growth instead of abscission after pollination in spinach.

The mechanism of auxin transport in lateral root spacing

The mechanism of auxin transport in lateral root spacing

Auxin regulation involved in gynoecium morphogenesis of papaya flowers

The morphogenesis of gynoecium is crucial for propagation and productivity of fruit crops. For trioecious papaya (Carica papaya), highly differentiated morphology of gynoecium in flowers of different sex types is controlled by gene networks and influenced by environmental factors, but the regulatory mechanism in gynoecium morphogenesis is unclear. Gynodioecious and dioecious papaya varieties were used for analysis of differentially expressed genes followed by experiments using auxin and an auxin transporter inhibitor. We first compared differential gene expression in functional and rudimentary gynoecium at early stage of their development and detected significant difference in phytohormone modulating and transduction processes, particularly auxin. Enhanced auxin signal transduction in rudimentary gynoecium was observed. To determine the role auxin plays in the papaya gynoecium, auxin transport inhibitor (N-1-Naphthylphthalamic acid, NPA) and synthetic auxin analogs with different concentrations gradient were sprayed to the trunk apex of male and female plants of dioecious papaya. Weakening of auxin transport by 10 mg/L NPA treatment resulted in female fertility restoration in male flowers, while female flowers did not show changes. NPA treatment with higher concentration (30 and 50 mg/L) caused deformed flowers in both male and female plants. We hypothesize that the occurrence of rudimentary gynoecium patterning might associate with auxin homeostasis alteration. Proper auxin concentration and auxin homeostasis might be crucial for functional gynoecium morphogenesis in papaya flowers. These results will lead to further investigation on the auxin homeostasis and gynoecium morphogenesis in papaya.

Auxin signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis.

Auxin signaling mediated by various auxin/indole-3-acetic acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of the SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin-inducible gene. The precise developmental role and molecular regulation of LRP1 in root development remain to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides the primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin-dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs-ARFs modules during LR development. We showed that auxin-mediated induction of LRP1 is lost inemerging LRs of slr-1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric divisionduring LR development. Overexpression of LRP1 (LRP1 OE) showed an increased number of LR primordia (LRP) at stages I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1-induced expression of YUC4, which is involved in auxin biosynthesis, contributes to the increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3, SRS6 and SRS7 proteins of the SHI/STY family, indicating their possible redundant role during root development. Our results suggested that auxin and histone deacetylation affect LRP1 expression andit acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.

Bacterial communities related to 3-nitro-1-propionic acid degradation in the rumen of grazing ruminants in the Qinghai-Tibetan Plateau

The objectives of this current study were to characterize the overall rumen bacterial community in grazing yak and two sheep species (Tibetan and Small Tail Han sheep) reared in the unique environmental conditions of the Qinghai-Tibetan Plateau, as well as the bacterial community associated with the detoxification of a phytotoxin, 3-nitro-1-propionic acid (NPA), during in vitro culture with 4.2 mM NPA. Using 16S rRNA gene high-throughput sequencing, it was found that the yak rumen harbored populations showing a higher bacterial diversity compared to Tibetan sheep. The rumen bacterial community in the three ruminant species differed from each other. PICRUSt analysis identified that the pathway involved in nitrogen metabolism was enriched in Tibetan sheep while that related to fatty acid biosynthesis was over-represented in the yak. The methane metabolism pathway was dominant in bacterial populations from the Small Tail Han sheep. Comparisons between freshly collected rumen fluid and populations subjected to consecutive 72 h batch cultures revealed substantial decreases in alpha diversity in populations cultured with NPA. Moreover, the relative abundances of some bacterial taxa changed significantly, with increased abundance of Proteobacteria and Actinobacteria. In addition, the overall community structure of the bacterial population in the freshly collected ruminal fluid was clearly different than that within populations observed in the 72 h batch cultures likely due to the impact of NPA treatments and the more restrictive growth conditions of the culture medium. In regard to PICRUSt analysis, the methane metabolism pathway became scarce in Tibetan and Small Tail Han sheep, whereas the energy and carbohydrate metabolic pathways such as nitrogen metabolism, ABC transporters and glycolysis/gluconeogenesis were found to be maintained across all populations. Results from the present study provide new information on the bacterial and functional composition within ruminal populations adapted to three economically important grazing ruminant species prominent on the Qinghai-Tibetan Plateau. The results further reveal that effects of NPA treatment on community structure can have an impact not only the metabolism of NPA but on other digestive functions as well.

AtRBOH I confers submergence tolerance and is involved in auxin-mediated signaling pathways under hypoxic stress

Plants suffer from oxygen deficiency (hypoxia) and energy starvation under flooding conditions. Higher plants have evolved complex adaptive mechanisms to flooding that are induced by changes in the cellular redox state and phytohormones. Previously, we showed that the transcript levels of respiratory burst oxidase homolog I (AtRBOH I) in Arabidopsis increase under hypoxic stress. In this study, we used two independent Atrboh I-knockout lines to assess the molecular function of AtRBOH I in hypoxic signaling pathways. Under submergence conditions, the Atrboh I-knockout lines had a reduced survival rate and lower chlorophyll contents than those of wild type. The patterns of AtRBOH I expression were analyzed by fusing its promoter to the GUS reporter. These expression analyses indicated that AtRBOH I expression was activated by hypoxia, but this induction was reduced by the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Quantitative RT-PCR analyses showed that the transcript levels of hypoxia-inducible genes (AtHRE1, AtADH1, AtLDH, and AtSUS1) were reduced in AtRBOH I-knockout lines under hypoxic conditions. The transcript levels of AtSUS1 were lower in AtRBOH I-knockout lines than in wild type in the hypoxia combined with NPA treatment. Hypoxic conditions increased the transcript levels of the auxin-responsive genes At1g19840, At3g23030, and At5g19140, and hypoxia combined with NPA resulted in increased transcript levels of the ethylene biosynthetic genes AtACS7 and AtACS8. Together, these results show that AtRBOH I regulates the expression of genes involved in ethylene biosynthesis and down-stream of hypoxia signaling, and that there is some interplay between hypoxia signaling and auxin-mediated signaling pathways under hypoxic stress.

A Novel Functional Variant of nNOS Mediates Anoxic Injury in Brain Microvascular Endothelial Cells via Mitochondrial Superoxide Generation

Deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms in mice have been shown to elicit opposite effects on ischemic brain injury following experimental stroke. Recently, we have demonstrated the expression of nNOS in human, rat, and mouse primary brain microvascular endothelial cells (MECs). The constitutively active endothelial nNOS (enNOS) was found to be functionally distinct from the nNOS expressed in neurons and the eNOS but sensitive to inhibition by pharmacological nNOS inhibitors. Under physiological conditions, enNOS was found to produce superoxide instead of nitric oxide (NO), unlike all known NOS isoforms. The objective of the present study was to examine the pathophysiological role of enNOS in endothelial injury. We hypothesized that enNOS mediates the anoxic injury underlying the blood‐brain barrier (BBB) dysfunction and cell death by instigating mitochondrial oxidative stress. We utilized primary human brain MECs (passage# 7–9) and employed inhibitors of nNOS (N‐ω‐Propyl‐L‐arginine or NPA and ARL‐17477; 1 μmol/L each) and eNOS (L‐N5‐(1‐Iminoethyl)ornithine or NIO, 1 μmol/L). Electron spin resonance spectrometry measurements with mitochondria‐targeted mito‐TEMPO‐H (50 μmol/L) showed that cells treated with enNOS inhibitor (ARL‐17477, 165±16 a.u, n=7; p < 0.05) reduced the mitochondrial superoxide in compared to the untreated MECs (108±10 a.u, n=7). Oxygen consumption rate (OCR; pmol/minute/pg protein) measured in MECs by Seahorse Analyzer showed increase in maximal respiration and decreased non‐mitochondrial OCR with NPA treatment (355 ± 60 and 7 ± 4, respectively; n=11; p < 0.05) compared to untreated cells (189 ± 49 and 19 ± 4, respectively; n=11). Transendothelial electrical resistance (TEER) measurements were made to quantitate the BBB integrity in confluent monolayers of hMECs grown in transwells and subjected to oxygen‐glucose deprivation (OGD; 6 h). Inhibition of enNOS with ARL‐17477 (50 ± 3 vs. 39 ± 2 untreated, n=6–8; p < 0.05) and NPA (46 ± 2 vs. 36 ± 2 untreated, n=7–9; p < 0.05) increased TEER (Ω.cm2) in MECs. In addition, ARL‐17477 and NPA (119 ± 15 and 116 ± 9, n=9 each; p<0.05) sustained recovery of TEER (% change) at 30 hours following OGD compared to the eNOS inhibited (89 ± 5, n=9; p=NS) and untreated cells (75 ± 8, n=9). Western blot studies have shown that treatment with enNOS inhibitors increases the levels of the tight junction protein, Zona Occludens‐1, in MECs compared to untreated cells. In contrast, viability measurements using CCK‐8 assay (% viable cells) observed that MECs treated with each NOS inhibitor (NPA: 42 ± 3; ARL‐17477: 52 ± 3; NIO: 70 ± 5; n = 16, p < 0.05) and exposed to OGD increased cell survival compared to untreated cells (26 ± 2; n = 16). Thus, enNOS mediates anoxic‐injury leading to the impairment of BBB integrity as well as cell death.Support or Funding InformationAHA Predoctoral (VNS: 16PRE31450006) and postdoctoral (IR: 15POST23040005) Fellowship Grant, AHA Scientist Development Grant (PVK: 14SDG20490359), and NIH grants (DWB: HL093554 and PVK: NS094834).

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

3-Nitropropionic acid-induced ischemia tolerance in the rat brain is mediated by reduced metabolic activity and cerebral blood flow.

Tissue tolerance to ischemia can be achieved by noxious stimuli that are below a threshold to cause irreversible damage ('preconditioning'). Understanding the mechanisms underlying preconditioning may lead to the identification of novel therapeutic targets for diseases such as stroke. We here used the oxidative chain inhibitor 3-nitropropionic acid (NPA) to induce ischemia tolerance in a rat middle cerebral artery occlusion (MCAO) stroke model. Cerebral blood flow (CBF) and structural integrity were characterized by longitudinal magnetic resonance imaging (MRI) in combination with behavioral, histologic, and biochemical assessment of NPA-preconditioned animals and controls. Using this approach we show that the ischemia-tolerant state is characterized by a lower energy charge potential and lower CBF, indicating a reduced baseline metabolic demand, and therefore a cellular mechanism of neural protection. Blood vessel density and structural integrity were not altered by NPA treatment. When subjected to MCAO, preconditioned animals had a characteristic MRI signature consisting of enhanced CBF maintenance within the ischemic territory and intraischemic reversal of the initial cytotoxic edema, resulting in reduced infarct volumes. Thus, our data show that tissue protection through preconditioning occurs early during ischemia and indicate that a reduced cellular metabolism is associated with tissue tolerance to ischemia.

Cytokinin treatments affect the apical-basal patterning of the Arabidopsis gynoecium and resemble the effects of polar auxin transport inhibition.

The apical-basal axis of the Arabidopsis gynoecium is established early during development and is divided into four elements from the bottom to the top: the gynophore, the ovary, the style, and the stigma. Currently, it is proposed that the hormone auxin plays a critical role in the correct apical-basal patterning through a concentration gradient from the apical to the basal part of the gynoecium, as chemical inhibition of polar auxin transport through 1-N-naphtylphtalamic acid (NPA) application, severely affects the apical-basal patterning of the gynoecium. In this work, we show that the apical-basal patterning of gynoecia is also sensitive to exogenous cytokinin (benzyl amino purine, BAP) application in a similar way as to NPA. BAP and NPA treatments were performed in different mutant backgrounds where either cytokinin perception or auxin transport and perception were affected. We observed that cytokinin and auxin signaling mutants are hypersensitive to NPA treatment, and auxin transport and signaling mutants are hypersensitive to BAP treatment. BAP effects in apical-basal gynoecium patterning are very similar to the effects of NPA, therefore, it is possible that BAP affects auxin transport in the gynoecium. Indeed, not only the cytokinin-response TCS::GFP marker, but also the auxin efflux carrier PIN1 (PIN1::PIN1:GFP) were both affected in BAP-induced valveless gynoecia, suggesting that the BAP treatment producing the morphological changes has an impact on both in the response pattern to cytokinin and on auxin transport. In summary, we show that cytokinin affects proper apical-basal gynoecium patterning in Arabidopsis in a similar way to the inhibition of polar auxin transport, and that auxin and cytokinin mutants and markers suggest a relation between both hormones in this process.

Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos

Polar auxin transport provides a developmental signal for cell fate specification during somatic embryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somatic embryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somatic embryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somatic embryogenesis.

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t-GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1-N-naphthylphthalamic acid (NPA) treatment. The tissue-specific expression patterns of OsPIN3t were also investigated using a β-glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.

Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis.

Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

Differential regulation of Knotted1-like genes during establishment of the shoot apical meristem in Norway spruce (Picea abies)

Establishment of the shoot apical meristem (SAM) in Arabidopsis embryos requires the KNOXI transcription factor SHOOT MERISTEMLESS. In Norway spruce (Picea abies), four KNOXI family members (HBK1, HBK2, HBK3 and HBK4) have been identified, but a corresponding role in SAM development has not been demonstrated. As a first step to differentiate between the functions of the four Norway spruce HBK genes, we have here analyzed their expression profiles during the process of somatic embryo development. This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA. Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment. In contrast, HBK1 and HBK3 were up-regulated prior to SAM formation, and their temporal expression was not affected by NPA. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants further supported similar functions of HBK2 and HBK4, distinct from those of HBK1 and HBK3. Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-011-1224-6) contains supplementary material, which is available to authorized users.