Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings

Modern corn ( Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection of the modern hybrid, 3394, for increased crop yield includes changes in responsiveness to auxin and light. Etiolated seedlings in the modern line are shorter than in an older hybrid, 307, since they produce shorter coleoptile, mesocotyl, and leaves (blade as well as sheath). Etiolated 3394 seedlings, as well as isolated mesocotyl and sheath segments, were less responsive to auxin and an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA). Reduced response of 3394 to auxin was associated with less reduction of elongation growth by light (white, red, far-red, blue) than in 307, whereas the activity of polar auxin transport (PAT) and its reduction by red or far-red light was similar in both genotypes. NPA reduced PAT in etiolated 3394 seedlings much less than in 307. A characteristic feature of 3394 plants is more erect leaves. In both hybrids, light (white, red, blue) increases leaf declination from the vertical, whereas NPA reduces leaf declination in 307, but not in 3394. Our results support findings that auxin and PAT are involved in elongation growth of corn seedlings, and we show that light interacts with auxin or PAT in regulation of leaf declination. We hypothesize that, relative to 307, more erect leaves in the modern hybrid may be primarily a consequence of a reduced amount of auxin receptor(s) and reduced responsiveness to light in etiolated 3394 plants. The more erect leaves in 3394 may contribute to the tolerance of the modern corn hybrid to dense planting.     

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3–5 pmol/mg protein with an apparent dissociation constant (K _d) between 3 and 5 μM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22–24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31–33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-³H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Comparison of Site I auxin binding and a 22-kilodalton auxin-binding protein in maize

Several properties of a 43-kilodalton (kDa) auxin-binding protein (ABP) having 22-kDa subunits are shared by a class of auxin binding designated Site I. The spatial distribution of the ABP in the maize (Zea mays L.) mesocotyl corresponds with the distribution of growth induced by naphthalene-1-acetic acid and with the distribution of Site I binding as previously shown by J.D. Walton and P.M. Ray (1981, Plant Physiol. 68, 1334–1338). The greatest abundance of both ABP and Site I activity is at the apical region of the mesocotyl. The ABP and Site I activity co-migrate in isopycnic centrifugation with the endoplasmic-reticulum marker, cytochrome-c reductase. Red light, at low and high fluence, far-red and white light were used to alter the elongation rate of apical 1-cm sections of etiolated maize mesocotyls, the amount of auxin binding, and the abundance of the ABP. Relative changes in auxin binding and the ABP were correlated, but the growth rate was not always correlated with the abundance of the ABP.Abbreviations ABP auxin-binding protein - ER endoplasmic reticulum - FR far-red light - kDa kilodalton - NAA naphthalene-1-acetic acid - PM plasma membrane - R red light - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Auxin receptors: recent developments

Rapid advances have been made in the study of auxin binding proteins (ABPs) in the last five years. In particular, an ABP in maize membranes has been cloned, sequenced and both monoclonal and polyclonal antibodies to this ABP have been developed. Structural and functional analysis has begun and there is good electrophysiological evidence that ABP in the plasma membrane functions as a receptor, probably involved in auxin-induced cell expansion. The role of the large amount of ABP in the endoplasmic reticulum is less clear, as is the relationship to soluble ABPs. At present there is only some circumstantial evidence relating any ABP to cell division. Receptors for synthetic inhibitors of auxin transport (phytotropins) are also of interest in relation to auxin action, but are less well characterised. Identification of new naturally-occurring phytotropins could lead to novel plant growth regulators.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

Demonstration of auxin binding to strawberry fruit membranes

Presence of specific auxin-binding sites in strawberry fruit (Fragaria ananassa Duch. cv. Ozark Beauty) membranes has been demonstrated. These 1-naphthaleneacetic acid (NAA)-binding sites in the 80,000g to 120,000g fraction of the strawberry fruit membrane were pronase sensitive with an estimated equilibrium dissociation constant for NAA of 1.1 × 10⁻⁶ molar. The minimum concentration of NAA required to stimulate strawberry fruit growth was at least one order of magnitude higher than the minimum concentration of NAA required to stimulate corn coleoptile elongation. This was consistent with the higher equilibrium dissociation constant (lower affinity) for auxin binding to strawberry fruit membranes than to corn coleoptiles. Twelve auxin analogs, ranging from very strong to weak auxins, were tested for abilities to stimulate in situ strawberry fruit growth and to bind (displace or compete with NAA) to strawberry fruit membranes. The observed positive correlation (r = 0.74) between the in vitro binding to the 80,000 to 120,000 membrane fraction and the in situ biological activity of these analogs indicated that the NAA-binding sites in strawberry fruit membranes may represent physiologically relevant auxin receptors.     

转异源abp基因烟草的叶外植体不同培养中的形态发生

以转正义和反义abp基因[生长素结合蛋白（ABP）的基因]烟草及对照的叶外植体为材料,在附加2mg／L或1mg／L的6－BA和不同浓度的NAA的MS培养基上进行不定芽分化试验。在较低NAA浓度条件下,转正义abp基因烟草（SE2）的叶外植体分化的不定芽数高于对照（SR1）和转反abp基因烟草（AS3）,说明SE2对生长素的敏感性比SR1和AS3高;而在较高NAA浓度条件下,AS3分化的不定芽数大于SE2和SR1,说明AS3对生长素的敏感性比SE2和SR1低。在培养基中加入生长素极性运输抑制剂HFCA后,不定芽分化受到抑制,分化不定芽中部分叶的形态由两侧对称性生长转变为形成不对称的喇叭状叶。在HFCA浓度为0－7．5mg／L条件下,SE2的喇叭叶发生频率明显高于SR1和AS3,其中SE2和SR1的喇叭叶发生频率分别在HFCA浓度为6．5和7．5mg／L时达到最高以后保持平稳,而AS3的喇叭叶发生频率在HFCA处理浓度直到15mg／L时仍保持上升趋势。这些结果说明ABP具有结合外源生长素,从而促进芽器官分化的功能,同时可能作为生长素受体参与了生长素的极性运输。     

Plasmalemma hyperpolarity and culture of sense and antisenseabp transgenic tobacco protoplasts

The relationship among transfer and expression of auxin binding protein gene (abp), auxin (NAA)-induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisenseabp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of senseabp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisenseabp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there.     

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

Interactions of auxin and gibberellin in the control of basal growth of <Emphasis Type="Italic">Arabidopsis</Emphasis> rosette leaves

Arabidopsis is a species that naturally displays the rosette form. Therefore, elucidation of the factors, which control basal leaf development, is of particular interest. Most evidence points that auxins and gibberellins are important in the control of rosette leaf development. In this paper, we report on a regimen that disrupts the normal rosette growth in Arabidopsis and induces internodal growth, which we have termed unbasal. The growth conditions are: (1) seed germination in the presence of 2,3,5-triiodobenzoic acid (TIBA); (2) transfer of the seedlings to a medium containing exogenous auxin (NAA) and GA₃; (3) transfer of the seedlings to a GA₃-only medium for all subsequent growth. Under these conditions, auxin and GA interact to induce internode elongation. Polar auxin transport appears to have a temporal effect on this synergistic interaction. In this regimen, GA increases auxin activity in the basal portions of the stem. Cross sectional morphology of the elongated internodes between two rosette leaves in an un-basal plant was similar to that seen for the pin1 Arabidopsis mutation.     

Role of basipetal auxin transport and lateral auxin movement in rooting and growth of etiolated lupin hypocotyls

The involvement of polar auxin transport (PAT) on the growth of light-grown seedlings and rooting is generally accepted, while the role of auxin and PAT on the growth of dark-grown seedlings is subject to controversy. To further investigate this question, we have firstly studied the influence of NPA, a known inhibitor of PAT, on the rooting and growth of etiolated Lupinus albus hypocotyls. Rooting was inhibited when the basal ends of de-rooted seedlings were immersed in 100 micro m NPA but was partially restored after immersion in NPA + auxin. However, NPA applied to de-rooted seedlings or the roots of intact seedlings did not inhibit hypocotyl growth. It was taken up and distributed along the organ, and actually inhibited the basipetal transport of ((3)H)-IAA applied to isolated hypocotyl sections. Since the apex is the presumed auxin source for hypocotyl growth and rooting, and the epidermis is considered the limiting factor in auxin-induced growth, the basipetal and lateral auxin movement (LAM) after application of ((3)H)-IAA to decapitated seedlings were studied, in an attempt to evaluate the role of PAT and LAM in the provision of auxin to competent cells for growth and rooting. Local application of ((3)H)-IAA to the stele led to the basipetal transport of auxin in this tissue, but the process was drastically reduced when roots were immersed in NPA since no radioactivity was detected below the apical elongation region of the hypocotyl. LAM from the stele to the cortex and the epidermis occurred during basipetal transport, since radioactivity in these tissues increased as transport time progressed. Radioactivity on a per FW basis in the epidermis was 2-4 times higher than in the cortex, which suggests that epidermal cells acted as a sink for LAM. NPA did not inhibit LAM along the elongation region. These results suggest that while PAT was essential for rooting, LAM from the PAT pathway to the auxin-sensitive epidermal cells could play a key role in supplying auxin for hypocotyl elongation in etiolated lupin seedlings.     

The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Auxin Receptors and Auxin Binding Proteins

In the last few years, a large number of auxin-binding proteins (ABPs) have been reported. Implicitly or explicitly, interest in such proteins resides in their possible role as auxin receptors. Many of these proteins are characterized as ABPs solely by their susceptibility to covalent photolabeling by tritiated azido-indole-3-acetic acid. In most cases where the labeled polypeptides have been identified, they turn out to have roles unconnected with primary auxin perception. It seems likely that auxin is binding to sites of catholic specificity in these cases and the influence of experimental protocols on the data is discussed. Because the term ABP implies that auxin binding affects the function of that protein, the importance of establishing further criteria before photolabeled peptides can be termed ABPs is emphasized. Applying such criteria, only a very few ABPs are currently of interest and only one of these, maize ABP1, has been characterized in detail. This protein is located primarily within the lumen of the endoplasmic reticulum, although an important fraction appears to function on the outside of the plasma membrane. The protein has a wide species distribution and it now seems highly probable that it is a genuine auxin receptor, the only protein for which such a function has yet been established. This conclusion is based on three independent lines of electrophysiological evidence, together with confocal imaging of cytoplasmic pH changes.     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.