Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes.

Regulated secretory vesicle delivery, vesicle fusion and rapid membrane recycling are all contentious issues with respect to tip growth in plant, fungal and animal cells. To examine the organisation and dynamics of membrane movements at the growing pollen tube apex and address the question of their relationship to growth, we have used the membrane stain FM4-64 both as a structural marker and as a quantitative assay. Labelling of living Lilium Longiflorum pollen tubes by FM4-64 resulted in a distinct staining pattern in the tube apex, which corresponds spatially to the previously identified cone-shaped 'apical clear zone' containing secretory vesicles. Dye uptake could be inhibited by sodium azide and followed a strict temporal sequence from the plasma membrane to a population of small (1-2 microm diameter) discrete internal structures, with subsequent appearance of dye in the apical region and ultimately in vacuolar membranes. Washout of the dye rapidly removed the plasma membrane staining, which was followed by a gradual decline in the apical fluorescence over more than an hour. Injected aqueous FM4-64 solution showed a relatively even distribution within the pollen tube. Association of FM4-64 with apical secretory vesicles was supported by the effects of the inhibitors Brefeldin-A and Cytochalasin-D, which are known to affect the localisation and number of such vesicles, on the FM4-64 staining pattern. Examination of the dynamics of FM4-64 labelling in the pollen tube tip by time-lapse observation, supported by fluorescence-recovery-after-photobleaching (FRAP) analysis, suggested the possibility of distinct pathways of bulk membrane movement both towards and, significantly, away from the apex. Quantitative analysis of FM4-64 distribution in the apex revealed that fluctuations in fluorescence 5 to 10 microm subapically, and to a lesser extent the apical 3 microm, could be related to the periodic oscillation in pollen tube growth rate. This data reveals a quantitative relationship between FM4-64 staining and growth rate within an individual tube.

An agravitropic mutant of Arabidopsis, endodermal-amyloplast less 1, that lacks amyloplasts in hypocotyl endodermal cell layer.

We have isolated a new recessive mutant of Arabidopsis thaliana for gravitropism, endodermal-amyloplast less 1 (eal1). eal1 shows reduced gravitropism in hypocotyl, and completely lacks gravitropism in inflorescence stems; root gravitropism is not affected. Starch staining with I-KI solution reveals almost no amyloplasts in eal1 hypocotyls when grown on a sucrose-free medium, though the root columella cells contain as many amyloplasts as wild type. On a medium containing 1% sucrose, eal1 hypocotyls contain as many starch granules as those of wild type, suggesting that starch synthesis is not affected in eal1. The endodermal cell layer which is thought to function as statocytes in hypocotyls is present in eal1. These results suggest that differentiation or development of gravity-responsive amyloplasts are affected in eal1 hypocotyls.

The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue.

Organ bending through differential growth represents a major mechanism by which plants are able to adaptively alter their morphology in response to local changes in the environment. Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses; however, the mechanisms by which they elicit their effects remain largely unknown. Here, we describe isolation of the NPH4 gene of Arabidopsis, which is conditionally required for differential growth responses of aerial tissues, and we report that NPH4 encodes the auxin-regulated transcriptional activator ARF7. The phenotypes of nph4 mutants, which include multiple differential growth defects associated with reduced auxin responsiveness, including impaired auxin-induced gene expression, are consistent with the predicted loss of function of a transcriptional activator, and these phenotypes indicate that auxin-dependent changes in gene transcription are prerequisite for proper organ bending responses. Although NPH4/ARF7 appears to be a major regulator of differential growth, it is not the sole regulator because phenotypes of nph4 null mutants were suppressed by application of ethylene. This latter finding illustrates the intimate connection between auxin and ethylene in the control of growth in higher plants.

The MSG1 and AXR1 genes of Arabidopsis are likely to act independently in growth-curvature responses of hypocotyls.

Growth-curvature responses of hypocotyls of Arabidopsis thaliana (L.) Heynh. were measured in double mutants between msg1 and axr1, both of which are auxin-resistant and defective in hypocotyl growth curvature induced upon unilateral application of auxin. The msg1 axr1 double mutants showed no auxin-induced growth curvature, that is, they exhibited the msg1 phenotype, though the axr1 defects were partial. Hypocotyls of both the msg1 and axr1 mutants were partially defective in second-positive phototropism, whereas the double mutants lost the response completely. When grown on vertically held agar plates, the axr1 mutant showed normal hypocotyl gravitropism and the mutation did not affect the reduced hypocotyl gravitropism of msg1. Hypocotyls of msg1 and axr1 mutants grew upward like wild-type ones when grown along an agar surface, while they grew more randomly when grown without an agar support, suggesting that axr1 hypocotyls are not completely normal in gravitropism. The extent of defects in growth orientation increased in the order: msg1 axr1 double mutants > msg1 > axr1 > wild type. The hypocotyls of these mutants showed auxin resistance in the order: msg1 axr1 > axr1 > msg1 > wild type. The msg1 mutant had epinastic leaves and axr1 had wrinkled leaves; leaves of the msg1 axr1 double mutants were epinastic and wrinkled. These results suggest that MSG1 and AXR1 act independently in separate pathways of the reactions tested in the present study. In contrast, the phenotype of the msg1 aux1 double mutants shows that AUX1 is not significantly involved in these phenomena.

The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf.

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 microM were studied, with maximum IAA-induced curvature at 100 microM. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg 1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which it effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.