Phototropic bending of non-elongating and radially growing woody stems results from asymmetrical xylem formation

Active phototropic bending of non-elongating and radially growing portion of stems (woody stems) has not been previously documented, whereas negative gravitropic bending is well known. We found phototropic bending in woody stems and searched for the underlying mechanism. We inclined 1-year-old Quercus crispula Blume seedlings and unilaterally illuminated them from a horizontal direction perpendicular to ('normal' illumination) or parallel to ('parallel' illumination) the inclination azimuth. With normal illumination, active phototropic bending and xylem formation could be evaluated separately from the negative gravitropic response and vertical deflection resulting from the weight of the seedlings. One-year-old stems with normal illumination bent significantly, with asymmetrical xylem formation towards the illuminated upper surface and side of the stem, whereas those with parallel illumination showed non-significant lateral bending, with asymmetrical xylem formation only on the upper side. A mechanical model was built on the assumption that a bending moment resulted from the asymmetrical xylem formation during phototropic bending of the woody stems. The model fitted the relationship between the observed spatial distributions of the xylem and the observed lateral bending, and thus supported the hypothesis that phototropic bending of woody stems results from asymmetrical xylem formation, as such occurs during gravitropism.     

Proton Efflux from the Outer Layer of the Peduncle of Tulip in Gravitropism and Circumnutation*

Epidermal plus hypodermal peels from tulip peduncles produced bands of acidity on agar containing bromocresol purple. Peels from horizontally oriented peduncles gave rise to an acidity band which corresponded to the lower side of the peduncle. The band began 3–6 cm beneath the flower and extended basipetally within the region of gravitropic bending. No corresponding band appeared in an agar layer laid on the cortical surface exposed by peeling. Peduncles growing in the normal vertical position showed circumnutations with a period in the range of 4 h. The peels from these stalks produced one or two bands more acid than the remaining part of the peel. Since the acidity band in horizontally positioned stalks corresponds to the zone of faster growth causing gravitropic bending, we infer that the band(s) produced by vertical stalks also correspond to zones of differential growth involved in circumnutation. On the basis of a previous finding that tulip leaves give rise to an oscillating acidity pattern, we infer that vertical stalks also show such a pattern. This inference fits the model proposing the involvement of an internal oscillator in circumnutation. However, the ratio of the circumnutation period to the gravitropic lag phase in tulip peduncles is such as predicted by the gravitropic-feedback model of circumnutation.     

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.     

Quantification of curvature production in cylindrical organs, such as roots and hypocotyls

Differential growth curvature rate (DGCR), defined as the spatial derivative of the tropic speed, was derived as a measure of curvature production in cylindrical organs. Its relation to usual concepts, such as curvature (kappa), rate of curvature (dkappa/dt) and differential growth profiles, was determined. A root gravitropism model, testing the hypothesis of one and two motors, exemplified its capabilities.DGCR was derived using cylindrical geometry and its meaning was obtained through a curvature conservation equation. The root gravitropism model was solved using a discrete difference method on a computer.DGCR described curvature production independently of growth, and was superior to dkappa/dt, which underestimated production. Moreover, DGCR profiles were able to differ between one and two motors, while profiles of kappa and dkappa/dt were not.The choice of the measure of curvature production has a large impact on experimental results, in particular when spatial and temporal patterns of differential growth need to be determined. DGCR was shown to fulfill the accuracy needed in the quantification of curvature production and should thus serve as a helpful tool for measurements.     

The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 ± 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (di-nitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N,N-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the upper and lower sides of gravistimulated maize root tips which is necessary for gravitropism.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - FC fusicoccin - Mes 2-(N-morpholino)ethanesulfonic acid The authors thank Phyllis Woolwine for drawing Fig. 1, Dr. Sarbjit Virk for assistance with total calcium measurements, Dr. Paul Sampson for statistical advice, and Michael Newton for developing the EM algorithm to analyze the time-series data. This work was supported by NASA grant NAGW-1394 and by a NASA Research Associateship to T.B. through NASA grant NAGW-70.     

Effects of norflurazon, an inhibitor of carotenogenesis, on abscisic acid and xanthoxin in the caps of gravistimulated maize roots

Maize seeds were germinated in the dark in the presence of the carotenoid synthesis inhibitor norflurazon and the teveis of abscisic acid, xanthoxin and total carotenoids were measured in the root cap and in the adjacent 1.5 mm segment. In norflurazon-treated roots abscisic acid levels were markedly reduced, but an increase occurred in the levels of xanthoxin, a compound structurally and physiologically similar to abscisic acid. In the cultivar of maize ( Zea mays L. cv. Merit) used for this work, brief illumination of the root is required for gravitropic curving. Following illumination both control and norflurazon-treated roots showed normal gravitropic curvature, however, the rate of curvature was delayed in norflurazon-treated roots. Our data from norflurazon-treated roots are consistent with a role for xanthoxin in maize root gravitropism. The increase in xanthoxin in the presence of an inhibitor of carotenoid synthesis suggests that xanthoxin and abscisic acid originate, at least in part, via different metabolic pathways.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.     

How do plant shoots bend up? — The initial step to elucidate the molecular mechanisms of shoot gravitropism usingArabidopsis thaliana

In higher plants, shoots show a negative gravitropic response. To elucidate the molecular mechanisms of this phenomenon, mutational analyses usingArabidopsis thaliana are in progress. This minireview aims to present recent developments in the genetic analysis of shoot gravitropism in this organism. We focus mainly on our studies on the novelshootgravitropic (sgr) mutants inArabidopsis thaliana that have dramatic defects in shoot gravitropism.     

Chromium in a series of Portuguese plants used in the herbal treatment of diabetes

Chromium (Cr³⁺) is an essential micronutrient for humans. Its main action is thought to be the regulation of blood sugar, because chromium deficiency is associated with diabetic-like symptoms, and chromium supplementation is correlated with increased glucose tolerance and insulin sensivity. Some Portuguese aromatic plants are utilized as tisanes by diabetic people as medicinal plants. Their active principle is not yet known, and the importance of their chromium content in the claimed therapeutic properties should not be discarded. Therefore, determination of chromium in some Portuguese medicinal plants was performed by flameless atomic absorption. All the analyzed plants contain chromium at the normal level for this element, but the plants used to prepare tisanes to help diabetic conditions contain higher levels (2.2 μg/g dry wt ±0.88;n=11) than the others (0.88 μg/g dry wt±0.18;n=17).     

Chromium in plants

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1–4 μg/g compared to 0.5–1.5 μg/g).     

Changes in IAA responsiveness in the elongation region of graviresponding mung bean roots

IAA responsiveness of sections of root tissue taken from the top and bottom of mung bean roots was assessed prior to and at varying times following gravistimulation. Prior to gravistimulation, root tissue sections from the sides of the elongation zone responded similarly to IAA. After gravistimulation (within 5 min), root sections from the bottom of the elongation zone became more responsive to IAA than sections collected from the upper side of the elongation zone. The change in IAA responsiveness of these tissue sections was transient with root sections from both the top and bottom of the elongation zone again exhibiting similar responsiveness to IAA following 15 minutes of gravistimulation.These studies also examined if the root tip is required for the gravity-induced shift in IAA responsiveness in the tissues of the elongation zone. The IAA responsiveness of top and bottom sections of the elongation zone from decapped mung bean roots was assessed at varying times following gravistimulation. The responsiveness to IAA of top and bottom sections changed rapidly in decapped roots, just as had been previously found for intact roots. Although the alteration in responsiveness was transient in decapped roots (just as intact roots), the time it took for the sections to recover previous responsiveness to IAA was extended.These results suggest that the initial growth response of graviresponding roots may be due to a change in the IAA responsiveness of tissues in the elongation zone and not an asymmetric accumulation of IAA on the lower side of the elongation zone. The results also indicate that the gravity-induced shift in IAA responsiveness in the elongation zone occurs independently of the root cap, suggesting that the cells in the elongation region can perceive and respond to gravity independently of the root cap during the intial phases of the gravity response.     

Dispersive and non-dispersive waves through plants: implications for arthropod vibratory communication

Vibratory communication in arthropods is a widespread phenomenon. Arthropods living on plants have been reported to use only dispersive bending waves in the context of prey-predator, competition, social and sexual interactions. Differences in signal structure have also been postulated to work as species recognition mechanisms and speciation agents. Using two identical laser Doppler vibrometers and a wavelet analysis, we quantified the wave propagation modes in rush stems (Juncus effusus) over the whole range of frequencies used by arthropods. A non-dimensionalized analysis shows that mechanical waves propagate not only as dispersive bending waves, but also as non-dispersive waves. Our analysis implies that an arthropod can communicate through non-dispersive bending waves by either producing signals of high frequencies or by choosing large stems, two widely different options tapping into the physiological and the behavioural repertoires, respectively. Non-dispersive waves, unreported so far in insect vibratory communication in plants, present serious advantages over dispersive bending waves in terms of signal integrity and may well be much more widely used than anticipated, in particular for species recognition.     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

Stem phototropism of trees: a possible significant factor in determining stem inclination on forest slopes

BACKGROUND AND AIMS: The main stems of trees on forest slopes incline down the slope to various extents that are characteristic of the species. The inclination has been explained as an active response to a horizontally asymmetrical light environment, but the contributing physiological mechanisms are unknown. The present study tested the hypothesis that stem phototropism, gravitropism, or a combination of the two determines the inclination of tree stems on forest slopes. METHODS: Cryptomeria japonica, Pinus densiflora, Quercus myrsinaefolia and Q. serrata were studied. Measurements were made of stem inclination of mature trees on forest slopes in uniform plantations of each species, and changes in stem inclination of potted seedlings in response to illumination treatments (unilateral or overhead) and inclination treatments (artificially inclined or erect). Indices of phototropic and gravitropic responsiveness were evaluated for each species, calculated from the change in stem inclination in response to artificial inclination with unilateral or overhead illumination. KEY RESULTS: Stem inclination on forest slopes varied significantly among species: Q. serrata inclined most in the down-slope direction, C. japonica inclined the least, and P. densiflora and Q. myrsinaefolia were intermediate. The change in stem inclination of seedlings in each treatment varied significantly among species. One-year-old stems of Q. serrata and 2-year-old stems of Q. myrsinaefolia bent toward the light source. Interspecific variation in the change in stem inclination in response to the unilateral illumination or that in the index of phototropic responsiveness was strongly correlated with the variation in stem inclination on forest slopes. CONCLUSIONS: The orientation of woody stems that have finished elongation can be actively controlled by phototropism. Interspecific variation in phototropic responsiveness of trees is a possible significant determinant of interspecific variation in stem inclination on forest slopes.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.