Maize Coleoptiles Research Articles

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.

Maize acetylcholinesterase is a positive regulator of heat tolerance in plants

We previously reported that native tropical zone plants showed high acetylcholinesterase (AChE) activity during heat stress, and that AChE activity in endodermal cells of maize seedlings was increased by heat treatment. However, the physiological role of AChE in heat stressed plants is still unclear. Here we report (1) tissue-specific expression and subcellular localization of maize AChE, (2) elevation of AChE activity and possible post-translational modifications of this enzyme under heat stress, and (3) involvement of AChE in plant heat stress tolerance. Maize AChE was mainly expressed in coleoptile nodes and seeds. Maize AChE fused with green fluorescent protein (GFP) was localized in extracellular spaces of transgenic rice plants. Therefore, in maize coleoptile nodes and seeds AChE mainly functions in the cell wall matrix. After heat treatment, enhanced maize AChE activity was observed by in vitro activity measurement and by in situ cytochemical staining; transcript and protein levels, however, were not changed. Protein gel blot analysis revealed two AChE isoforms (upper and lower); the upper-form gradually disappeared after heat treatment. Thus, maize AChE activity might be enhanced through a post-translational modification response to heat stress. Finally, we found that overexpression of maize AChE in transgenic tobacco plants enhanced heat tolerance relative to that of non-transgenic plants, suggesting AChE plays a positive role in maize heat tolerance.

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m−2 s−1). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0–3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0–3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0–2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.

Fusicoccin Counteracts the Toxic Effect of Cadmium on the Growth of Maize Coleoptile Segments

The effects of cadmium (Cd; 0.1–1000 μM) and fusicoccin (FC) on growth, Cd2+ content, and membrane potential (E m) in maize coleoptile segments were studied. In addition, the E m changes and accumulation of Cd and calcium (Ca) in coleoptile segments treated with Cd2+ combined with 1 μM FC or 30 mM tetraethylammonium (TEA) chloride (K+-channel blocker) were also determined. In this study, the effects of Ca2+-channel blockers [lanthanum (La) and verapamil (Ver)] on growth and content of Cd2+ and Ca2+ in coleoptile segments were also investigated. It was found that Cd at high concentrations (100 and 1000 μM) significantly inhibited endogenous growth of coleoptile segments and simultaneously measured proton extrusion. FC combined with Cd2+ counteracted the toxic effect of Cd2+ on endogenous growth and significantly decreased Cd2+ content (not the case for Cd2+ at the highest concentration) in coleoptile segments. Addition of Cd to the control medium caused depolarization of E m, the extent of which was dependent on Cd concentration and time of treatment with Cd2+. Hyperpolarization of E m induced by FC was suppressed in the presence of Cd2+ at 1000 μM but not Cd2+ at 100 μM. It was also found that treatment of maize coleoptile segments with 30 mM TEA chloride caused hyperpolarization of E m and decreased Cd2+ content in coleoptile segments, suggesting that, in the same way as for FC, accumulation of Cd2+ was dependent on plasma membrane (PM) hyperpolarization. Similar to FC, TEA chloride also decreased Ca2+ content in coleoptile segments. La and Ver combined with Cd2+ (100 μM) significantly decreased Cd content in maize coleoptile segments, but only La completely abolished the toxic effect of Cd2+ on endogenous growth and growth in the presence of FC. Taken together, these results suggest that the mechanism by which FC counteracts the toxic effect of Cd2+ (except at 1000 μM Cd2+) on the growth of maize coleoptile segments involves both stimulation of PM H+-ATPase activity by FC as well as Cd2+-permeable, voltage-dependent Ca channels, which are blocked by FC and TEA chloride-induced PM hyperpolarization.

Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

Preparation, characterization, and biological properties of β-glucans

β-Glucans are soluble fibers with physiological functions, such as, interference with absorption of sugars and reduction of serum lipid levels. β-glucans are found in different species, such as, Rhynchelytrum repens, Lentinus edodes, Grifola frondosa, Tremella mesenterica, Tremella aurantia, Zea may, Agaricus blazei, Phellinus baummi, Saccharomyces cerevisae (yeast), and Agaricus blazei murell (mushroom). Analysis of the fractions reveals the presence of arabinose, glucose, xylose, and traces of rhamnose and galactose. The presence of β-glucan in these fractions is confirmed by hydrolyzing the polymers with endo-β-glucanase from Bacillus subtilis, followed by high-performance liquid chromatography (HPLC) analysis of the characteristic oligosaccharides produced. The 4 M KOH fractions from different tissues are subjected to gel permeation chromatography on Sepharose 4B, with separation of polysaccharides, with different degrees of polymerization, the highest molecular mass (above 2000 kDa) being found in young leaves. The molecular mass of the leaf blade polymers is similar (250 kDa) to that of the maize coleoptiles β-glucan used for comparison. The 4 M KOH fraction injected into rats with streptozotocin-induced diabetes has shown hypoglycemic activity, reducing blood sugar to normal levels for approximately 24 hours. This performance is better than that obtained with pure β-glucan from barley, which decreases blood sugar levels for about four hours. These results suggest that the activity of β-glucans is responsible for the use of this plant extract as a hypoglycemic drug in folk medicine.

The Maize Mixed-Linkage (1→3),(1→4)-β-d-Glucan Polysaccharide Is Synthesized at the Golgi Membrane

With the exception of cellulose and callose, the cell wall polysaccharides are synthesized in Golgi membranes, packaged into vesicles, and exported to the plasma membrane where they are integrated into the microfibrillar structure. Consistent with this paradigm, several published reports have shown that the maize (Zea mays) mixed-linkage (1-->3),(1-->4)-beta-D-glucan, a polysaccharide that among angiosperms is unique to the grasses and related Poales species, is synthesized in vitro with isolated maize coleoptile Golgi membranes and the nucleotide-sugar substrate, UDP-glucose. However, a recent study reported the inability to detect the beta-glucan immunocytochemically at the Golgi, resulting in a hypothesis that the mixed-linkage beta-glucan oligomers may be initiated at the Golgi but are polymerized at the plasma membrane surface. Here, we demonstrate that (1-->3),(1-->4)-beta-D-glucans are detected immunocytochemically at the Golgi of the developing maize coleoptiles. Further, when maize seedlings at the third-leaf stage were pulse labeled with [(14)C]O(2) and Golgi membranes were isolated from elongating cells at the base of the developing leaves, (1-->3),(1-->4)-beta-D-glucans of an average molecular mass of 250 kD and higher were detected in isolated Golgi membranes. When the pulse was followed by a chase period, the labeled polysaccharides of the Golgi membrane diminished with subsequent transfer to the cell wall. (1-->3),(1-->4)-beta-D-Glucans of at least 250 kD were isolated from cell walls, but much larger aggregates were also detected, indicating a potential for intermolecular interactions with glucuronoarabinoxylans or intermolecular grafting in muro.

Auxin biosynthesis site and polar transport in maize coleoptiles

Since Darwins’ pioneering experiments, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production and polar transport. In a recent study, using maize coleoptiles, we first showed that the asymmetric IAA flow from the tip in response to gravistimulus directly affects the TIR/AFBs-mediated auxin signaling pathway, which results in tropic curvature. In this work, we also showed that IAA is synthesized from tryptophan (Trp) in the apical 1 mm region, and from there the synthesized IAA moves to the basal part via polar transport by ZmPIN1(s). These results clearly show the importance of the tip region in perception of gravistimulus and in transmitting the perceived information to the lower region using IAA as a messenger signal. Thus, it is concluded that IAA production and transport from the tip are key factors controlling the cell elongation rate in the lower part of the coleoptiles, by making a regulated and dynamic IAA flow net work in the coleoptiles.

Effect of Ca2+ and cAMP on protein phosphorylation in mitochondria of maize seedlings

The effect of common intracellular signals (Ca2+ and cAMP) on the activity of protein phosphorylation in mitochondria was investigated in coleoptiles of maize (Zea mays L.). Treatment of isolated mitochondria with 2 mM CaCl2 brought about an increase in the level of phosphorylation of proteins with mol ws of 74, 60, and 33 kD but considerably reduced phosphorylation of the protein with a mol wt of 51.5 kD. In the presence of Ca2+, phosphorylation of polypeptides with mol wts of 59 and 66 kD was also detected. cAMP considerably reduced phosphorylation of essentially all the investigated proteins in isolated mitochondria, which could be explained by activation of their dephosphorylation. Phosphorylation of mitochondrial proteins involves a polypeptide of about 94 kD showing kinase activity, which may be proper protein kinase or one of the subunits of a compound structure. In maize mitochondria, PP1A phosphatases were found. A hypothesis was advanced that redox-dependent phosphorylation/dephosphorylation of mitochondrial proteins plays an important role in mitochondrial signaling in higher plants.

1. NPH3- and PGP-like genes exclusively expressed at tip specific region where apical photo-sensing followed by lateral transport of IAA takes place in phototropic response of maize coleoptiles

1. NPH3- and PGP-like genes exclusively expressed at tip specific region where apical photo-sensing followed by lateral transport of IAA takes place in phototropic response of maize coleoptiles

Differential Downward Stream of Auxin Synthesized at the Tip Has a Key Role in Gravitropic Curvature via TIR1/AFBs-Mediated Auxin Signaling Pathways

Since the early days of Darwin, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production, polar transport and tropisms. Here, using maize coleoptiles, we first showed that polar transport of IAA synthesized at the tip region is regulated by ZmPIN(s). Then, the TIR/AFBs-mediated auxin signaling pathway corresponds to the asymmetric IAA flow after gravi-stimulus, which results in tropic curvature. When [(13)C(11)(15)N(2)]Trp was applied to coleoptile tips, substantial amounts of the stable isotope were incorporated into IAA at the tip region, and the labeled IAA was transported in a polar manner at approximately 7 mm h(-1). Immunohistochemical analyses revealed that ZmPIN1(s) was present in almost all cells. ZmPIN1(s) showed a relatively non-polar distribution at the tip, but a basal cellular localization at lower regions. Application of the IAA transport inhibitors 1-N-naphthylphthalamic acid (NPA) and brefeldin A (BFA) at the very tip region almost completely inhibited IAA movement from the tip. These inhibitors also severely suppressed gravitropic bending. PEO-IAA, an auxin antagonist that binds to TIR1/AFBs, suppressed not only the expression of an auxin-responsive ZmSAUR2 gene, but also gravitropic curvature. Expression of ZmSAUR2 was up-regulated on the lower side and down-regulated on the upper side of the coleoptile elongation zone, corresponding to the asymmetric IAA distribution. These results indicate that the asymmetric downward streams of IAA control the differential growth rate of the cells by attenuating TIR1/AFBs-mediated auxin response genes, including ZmSAUR2, and therefore result in tropic curvature.

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.

Alteration of plasmalemma H+-ATPase activity in maize coleoptile cells at different age of seedlings

An irregular alteration of the activity and character of the distribution of plasmalemma H + -ATPase has been shown in parenchymal cells of the coleoptile subapical zone within the period from the third to fifth day of ethiolated maize seedling development. The study was carried out by the cytochemical method. The highest enzyme activity was determined in the cells of four-day-old coleoptiles. The revealed change in H + -ATPase activity does not correspond to the dynamics of the growth intensity of the elongation of coleoptile cells within the studied period of seedling development.

10. Is the tissue/cell structure of coleoptile tip necessary for IAA biosynthesis in maize coleoptiles?

10. Is the tissue/cell structure of coleoptile tip necessary for IAA biosynthesis in maize coleoptiles?

14. Screening of novel chemicals that affect auxin biosynthesis activity by observing the degree of gravitropic response of maize coleoptiles

14. Screening of novel chemicals that affect auxin biosynthesis activity by observing the degree of gravitropic response of maize coleoptiles

11. Establishment of experimental system to analyze phototropic curvature of excised maize coleoptiles

11. Establishment of experimental system to analyze phototropic curvature of excised maize coleoptiles

13. Screening of novel chemicals that affect IAA biosynthetic activity in maize coleoptiles tip

13. Screening of novel chemicals that affect IAA biosynthetic activity in maize coleoptiles tip

77. Indole-3-acetic acid synthesized at the tip and its polar transport by ZmPIN1 is a key factor for gravity bending of maize coleoptiles

77. Indole-3-acetic acid synthesized at the tip and its polar transport by ZmPIN1 is a key factor for gravity bending of maize coleoptiles

Direct Involvement of Benzoxazinoids in the Growth Suppression Induced by Phototropic Stimulation in Maize Coleoptiles

The process of growth suppression observed in the illuminated sides in response to phototropic stimulation was investigated in maize coleoptiles. In our previous studies, the increased level of benzoxazinoids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 6-methoxybenzoxazolinone (MBOA), and the up-regulation of β-glucosidase (DIMBOA-glucosidase) activity have been observed in the illuminated halves. In this report, significant accumulation of hydrogen peroxide (H 2 O 2 ) was observed in the phototropically stimulated sides. Moreover, exogenously applied DIMBOA and MBOA induced a significant up-regulation of H 2 O 2 in the applied sides, respectively. In addition, lignin level was also slightly increased in the illuminated sides within 60 min. These results suggest that phototropic stimulation induced the production of benzoxazinoids, resulting to the up-regulation of H 2 O 2 in the illuminated halves. Consequently, the up-regulated H 2 O 2 promoted the cell-wall stiffness, leading to the growth suppression in the illuminated sides. This is the first indication that blue-light induced benzoxazinoids play the role of directional effecter in the growth suppression in the illuminated sides in maize coleoptiles.

7. Changes of IAA level and direction in IAA transport upon gravi-stimulation in maize coleoptiles

7. Changes of IAA level and direction in IAA transport upon gravi-stimulation in maize coleoptiles