Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.     

Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation

The gibberellins (GAs) are endogenous regulators of plant growth. Experiments are described here that test the hypothesis that GA regulates hypocotyl growth by altering the extent of hypocotyl cell elongation. These experiments use GA-deficient and altered GA-response mutants of Arabidopsis thaliana (L.) Heyhn. It is shown that GA regulates elongation, in both light- and dark-grown hypocotyls, by influencing the rate and final extent of cellular elongation. However, light- and dark-grown hypocotyls exhibit markedly different GA dose-response relationships. The length of dark-grown hypocotyls is relatively unaffected by exogenous GA, whilst light-grown hypocotyl length is significantly increased by exogenous GA. Further analysis suggests that GA control of hypocotyl length is close to saturation in dark-grown hypocotyls, but not in light grown hypocotyls. The results show that a large range of possible hypocotyl lengths is achieved via dose-dependent GA-regulated alterations in the degree of elongation of individual hypocotyl cells.Key words: Arabidopsis, cell elongation, gibberellin (GA), GA mutants, hypocotyl.      

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

Cell Elongation and Microtubule Behavior in the Arabidopsis Hypocotyl: Responses to Ethylene and Auxin

During elongation of the Arabidopsis hypocotyl, each cell reacts to light and hormones in a time- and position-dependent manner. Growth in darkness results in the maximal length a wild-type cell can reach. Elongation starts at the base and proceeds in the acropetal direction. Cells in the upper half of the hypocotyl can become the longest of the whole organ. Light strongly inhibits cell elongation all along the hypocotyl, but proportionally more in the upper half. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is known to stimulate hypocotyl elongation in the light. Here we show that this stimulation only occurs in cells of the apical half of the hypocotyl. Moreover, ACC application can partially overcome light inhibition, whereas indole-3-acetic acid (IAA) cannot. On low-nutrient medium (LNM) in the light, elongation is severely reduced as compared to growth on rich medium, and both ACC and IAA can stimulate elongation to the levels reached on a nutrient-rich medium. Furthermore, microtubule orientation was studied in vivo. During elongation in darkness, transverse and longitudinal patterns are clearly related with rates of elongation. In other conditions, except for the association of longitudinally orientated microtubules with growth arrest, microtubule orientation is merely an indicator of developmental age, not of elongation activity. A hypothesis on the relation between microtubules and elongation rate is discussed.     

Light‐induced stabilization of ACS contributes to hypocotyl elongation during the dark‐to‐light transition in Arabidopsis seedlings

Hypocotyl growth during seedling emergence is a crucial developmental transition influenced by light and phytohormones such as ethylene. Ethylene and light antagonistically control hypocotyl growth in either continuous light or darkness. However, how ethylene and light regulate hypocotyl growth, including seedling emergence, during the dark‐to‐light transition remains elusive. Here, we show that ethylene and light cooperatively stimulate a transient increase in hypocotyl growth during the dark‐to‐light transition via the light‐mediated stabilization of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthases (ACSs), the rate‐limiting enzymes in ethylene biosynthesis. We found that, in contrast to the known inhibitory role of light in hypocotyl growth, light treatment transiently increases hypocotyl growth in wild‐type etiolated seedlings. Moreover, ACC, the direct precursor of ethylene, accentuates the effects of light on hypocotyl elongation during the dark‐to‐light transition. We determined that light leads to the transient elongation of hypocotyls by stabilizing the ACS5 protein during the dark‐to‐light transition. Furthermore, biochemical analysis of an ACS5 mutant protein bearing an alteration in the C‐terminus indicated that light stabilizes ACS5 by inhibiting the degradation mechanism that acts through the C‐terminus of ACS5. Our study reveals that plants regulate hypocotyl elongation during seedling establishment by coordinating light‐induced ethylene biosynthesis at the post‐translational level. Moreover, the stimulatory role of light on hypocotyl growth during the dark‐to‐light transition provides additional insights into the known inhibitory role of light in hypocotyl development.     

The distribution of hydroxyproline-rich glycoprotein mRNAs in shoots of cucumber and pea

The distribution of hydroxyproline-rich glycoprotein (HRGP) mRNAs in the shoots of dark-grown and irradiated cucumber ( Cucumis sativus L. cv. Burpee pickler) and pea ( Pisum sativum L. cv. Alaska) was studied. A cloned genomic DNA fragment encoding carrot ( Daucus carota ) root extensin (pDC5A1) was used to measure HRGP mRNAs from cucumber and pea along the length of dark-grown and irradiated shoots. There was a marked difference in the levels of HRGP mRNAs isolated from apical and basal regions of cucumber. Whereas apical, elongating regions had low levels of HRGP mRNAs, basal regions of the shoot had high levels. Levels of HRGP mRNAs were also compared in shoots of dark-grown and irradiated cucumber. Although light inhibits hypocotyl growth, it had no effect on levels of HRGP mRNAs. There was no gradient in the distribution of HRGP mRNAs along the epicotyl of dark-grown pea. As was the case with cucumber, light did not affect the accumulation of HRGP mRNAs in pea shoots. We conclude that light does not affect elongation by regulating the accumulation of HRGP mRNAs. The gradient of accumulation of HRGP mRNAs along the hypocotyl of cucumber probably reflects differences in cellular differentiation along the shoot.     

Genetic and developmental control of nuclear accumulation of COP1, a repressor of photomorphogenesis in Arabidopsis.

Using a beta-glucuronidase (GUS) reporter-COP1 fusion transgene, it was shown previously that Arabidopsis COP1 acts within the nucleus as a repressor of seedling photomorphogenic development and that high inactivation of COP1 was accompanied by a reduction of COP1 nuclear abundance (A.G. von Arnim, X.-W. Deng [1994] Cell 79: 1035-1045). Here we report that the GUS-COP1 fusion transgene can completely rescue the defect of cop1 mutations and thus is fully functional during seedling development. The kinetics of GUS-COP1 relocalization in a cop1 null mutant background during dark/light transitions imply that the regulation of the functional nuclear COP1 level plays a role in stably maintaining a committed seedling's developmental fate rather than in causing such a commitment. Analysis of GUS-COP1 cellular localization in mutant hypocotyls of all pleiotropic COP/DET/FUS loci revealed that nuclear localization of GUS-COP1 was diminished under both dark and light conditions in all mutants tested, whereas nuclear localization was not affected in the less pleiotropic cop4 mutant. Using both the brassinosteroid-deficient mutant det2 and brassinosteroid treatment of wild-type seedlings, we have demonstrated that brassinosteroid does not control the hypocotyl cell elongation through regulation nuclear localization of COP1. The growth regulator cytokinin, which also dramatically reduced hypocotyl cell elongation in the absence of light, did not prevent GUS-COP1 nuclear localization in dark-grown seedlings. Our results suggest that all of the previously characterized pleiotropic COP/DET/FUS loci are required for the proper nuclear localization of the COP1 protein in the dark, whereas the less pleiotropic COP/DET loci or plant regulators tested are likely to act either downstream of COP1 or by independent pathways.     

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.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

An Arabidopsis cell cycle -dependent kinase-related gene, CDC2b, plays a role in regulating seedling growth in darkness.

The Arabidopsis CDC2b gene has been defined as a plant-specific cell cycle-dependent kinase-related gene, although it lacks the conserved cyclin binding motif, and its exact function is not known. Here, we report that in etiolated seedlings, the expression of the CDC2b gene is correlated with elongation rate of the hypocotyl. Inhibition of CDC2b gene expression by using an inducible antisense construct resulted in short-hypocotyl and open-cotyledon phenotypes when transgenic seedlings were grown in the dark. The severity of these phenotypes in dark-grown seedlings could be correlated with the level of the antisense gene expression. The short hypocotyl of seedlings underexpressing CDC2b was a result of inhibition of cell elongation rather than a reduction in cell number, whereas in cotyledons, inhibition of CDC2b expression resulted in large, open cotyledons with amyloplasts rather than etioplasts. Although the nuclear DNA was less compact in the antisense hypocotyl cells, DNA content and endoreduplication were not affected. Cell division of the shoot apical meristem also was not affected by antisense expression. The short-hypocotyl phenotype of these transgenic plants was partially rescued by the addition of brassinolide. Brassinolide can only induce CDC2b expression in darkness. These results suggest a role for the CDC2b gene in seedling growth via regulation of hypocotyl cell elongation and cotyledon cell development.     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

Structure–Function Relationships of Four Stereoisomers of a Brassinolide Mimetic on Hypocotyl and Root Elongation of the Brassinosteroid-Deficient <Emphasis Type="Italic">det2-1</Emphasis> Mutant of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Brassinosteroids (BRs) are a group of plant hormones and the bioactive BR, brassinolide (BL), is causally implicated in promoting cell elongation and cell proliferation. In Arabidopsis, the biosynthesis of BL is essential for hypocotyl etiolation in the dark, and application of bioactive BRs can promote both hypocotyl and root elongation, although high concentrations of applied BRs result in inhibition of root elongation. A non-steroidal structure consisting of four stereoisomers was designed to contain subunits bearing key functional groups mimicking those of BL. The bioactivity of each of these individual stereoisomers was tested using the Arabidopsis thaliana det2-1 mutant line, which is deficient in BL, and thus does not etiolate in the dark. Application of BL at each of 0.1, 1.0, and 10.0 µM promotes hypocotyl elongation in dark-grown det2-1 plants while simultaneously inhibiting elongation of their primary root. In contrast, the mimetic structures, when applied to dark-grown det2-1 plants, promote hypocotyl elongation without negatively affecting primary root elongation. In fact, two of the mimetic structures, applied at a 10 µM concentration, significantly promoted both hypocotyl and root elongation. Correlation of this contrasting behavior with the configurations of the hydroxylated stereocenters of the mimetics is described. This is the first example of a non-steroidal BL mimetic where the biological activities of individual stereoisomers were tested and compared.     

Photoregulation of asymmetric cell division followed by rhizoid development in the fern Ceratopteris prothalli

Strap-shaped prothalli of CERATOPTERIS: richardii grown in the dark have an apical meristem, a subapical elongation zone and a basal growth cessation zone [Murata et al. (1997) Plant Cell Physiol. 38: 201]. When the dark-grown prothalli were irradiated with continuous white light, marginal cells of the elongation zone divided asymmetrically, and the resulting smaller cells developed into rhizoids. The asymmetric division was also induced by brief irradiation of red light. The effect of red light was cancelled by subsequent irradiation of far-red light, indicating that the asymmetric division was regulated by phytochrome. Since the response to red light was not observed at 10(1) J m(-2) and saturated at 10(2) J m(-2) and the response is photoreversible by far-red light, the photoresponse was classified as a low-fluence response of phytochrome. Although the asymmetric division was induced by brief irradiation of red light, continuous irradiation of white, blue or red light was necessary to induce rhizoid growth. These results indicate that asymmetric division and subsequent cell growth are independently regulated by light in CERATOPTERIS: prothalli.     

Hormonal influence on photocontrol of the protandry in the genus Helianthus

Under natural photoperiodic conditions protandry in hermaphrodite disc flowers of sunflower (Helianthus annuus L.) is determined by the different elongation rates of the style and filaments. The elongation of the filament and style starts simultaneously after the daily dark period, but the style growth rate is slower. When plants close to anthesis are exposed to continuous white light (WL) a loss of protandry occurs: the filaments do not grow far enough to extrude the anthers from the corolla. The histological analyses show that the number of filament epidermal cells remains unaltered after organ elongation and that cells respond to photoperiod only by cell expansion. Emasculation does not substantially inhibit filament cell expansion, whereas isolation of the filament or stamen from the corolla suggests that this organ could be the perception site of the filament growth stimulus. In vitro treatments with auxin (indole-3-acetic acid, IAA or alpha-naphthaleneacetic acid, NAA) reverses the inhibition of cell expansion caused by continuous WL, whereas gibberellic acid (GA(3)) at high concentrations reproduces the effect of continuous WL. Experiments carried out on various Helianthus spp. show that all these plants have evolved the same photo- and hormonal-control of the protandry. In experiments in which the light treatments were continued for 24 h, the auxins drastically reduced the inhibiting effect of red light (R) and dichromatic treatments FR (far red)+R, whereas GA(3) repressed filament extension regardless of light quality. As far as auxins are concerned, the response of sunflower filaments does not appear to be connected with the polar transport of the hormone. Moreover, the promoting effect of darkness is not mediated by an increase of endogenous free IAA in disc flowers. However, sunflower filaments manifested a similar temporal pattern of response to the light/dark cycle and to auxin.     

Effect of light on ethylene production and hypocotyl growth of soybean seedlings

The apical 1-cm hypocotyl of dark-grown `Clark' soybean (Glycine max [L.] Merr.) seedlings produced ethylene at rates of 7 to 11 nanoliters per hour per gram when attached to the cotyledons. Such physiologically active rates occurred prior to the deceleration of hypocotyl elongation caused by the temperature of 25 C.

Daily exposure of the etiolated seedlings to red light promoted hypocotyl elongation and prevented its lateral swelling. Red light treatment also caused a 45% decrease in ethylene production. Far red irradiation following the red treatment reversed the red effects, suggesting that the ethylene intervenes as a regulator in the phytochrome control of `Clark' soybean hypocotyl growth at 25 C.

     

Transient gibberellin application promotes Arabidopsis thaliana hypocotyl cell elongation without maintaining transverse orientation of microtubules on the outer tangential wall of epidermal cells

The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth.