Indoleacetic Acid and Molecular Differentiation Evidence for Interaction

Proteins of hypocotyls of bean were studied by electrophoresis. Proteins were extracted from hypocotyl segments of various stages of development starting with the relatively undifferentiated hook regions and proceeding by 2 cm segments down the hypocotyl. The proteins were the soluble (pH 7.4), the basic nuclear (histones), acidic ribonuclear and acidic chromosomal. Soluble proteins reflected differentiation of the hypocotyl in that lower hypocotyl segments had more different protein types than did the hook region. Indoleacetic acid (IAA) at 10^?6M when applied to the lower hypocotyl appeared to induce still more different proteins. However, at 10^?3M, IAA appeared to induce molecular dedifferentiation in that hypocotyl protein patterns began to resemble those of the hook. Histones also reflected differentiation, the hook having more histone types than the lower hypocotyl. IAA had no effect on histones. The hook region had two types of acidic chromosomal proteins, the lower hypocotyl one. When lower hypocotyl segments were incubated in 10^?3M IAA, the protein pattern resembled that of the hook in that the second protein normally present in the hook and not in the hypocotyl was in fact induced in the hypocotyl. The hook had two acidic ribonuclear proteins, the lower hypocotyl one. IAA did not affect this protein. These experiments suggest that IAA in some manner regulates molecular (protein) differentiation. It is further suggested that IAA accomplishes this control through the acidic nuclear proteins which are closely associated with genetic material and which reflect differentiation and are also affected by IAA.     

Growth patterns and gravitropic curvature of radish hypocotyls

The rapid growth rate of radish cells makes the hypocotyl particularly valuable in research on growth phenomena and gravitropism. An examination of mean data regarding the growth of aetiolated radish seedlings not subjected to gravitropic stimulation showed that there was an increase in the growth rate from day 3 to day 5. When the hypocotyl was placed horizontally all zones showed an increase in growth of the lower surface of the hypocotyl and generally a decrease in the growth rate in the upper surface. The upper apical part of the gravistimulated hypocotyl grew almost the same as controls. In some cases, in both 4- and 5-day-old seedlings, in the upper median part there was a lower growth rate than in controls. The zones of growth during the development of the hypocotyl were different. Analysis of curvature showed that the growth rate of the different zones was a function of their location in the hypocotyl and that the rate of curvature was different in various parts of the hypocotyl.     

Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.     

Transcriptome analysis of age-related gain of callus-forming capacity in Arabidopsis hypocotyls

Callus-forming capacity is enhanced with hypocotyl maturity in Arabidopsis. However, the genetic regulation of age-related gain in capacity for callus formation is unclear. We used a gene expression microarray assay to characterize the underlying mechanisms during callus formation in young and mature hypocotyl explants of Arabidopsis. As expected, genes involved in photosynthesis and cell wall thickening showed altered expression during hypocotyl maturation. In addition, genes involved in cytokinin perception were enriched in mature hypocotyl tissues. Phytohormone-induced callus formation in hypocotyl explants was accompanied by increased expression of genes mainly related to the cell cycle, histones and epigenetics. The induction level of these genes was higher in mature hypocotyl explants than young explants during callus formation. We identified a number of genes, including those with unknown function, potentially involved in age-related gain in callus formation. Our results provide insight into the effect of hypocotyl age on callus formation. Altered cytokinin signaling components, cell cycle regulation and epigenetics may work in concert to lead to gain of callus-forming capacity in hypocotyls with age.     

The Effects of Cytokinin and Light on Hypocotyl Elongation in Arabidopsis Seedlings Are Independent and Additive

Cytokinin has been reported to mimic some of the effects of light on de-etiolation responses in dark-grown Arabidopsis seedlings. The interaction between cytokinin and light was examined by analyzing cytokinin dose and light fluence effects on hypocotyl elongation in wild-type and mutant Arabidopsis seedlings with defects in light or hormone responses. It was found that (a) cytokinin and light-response systems have independent and additive effects on the inhibition of hypocotyl elongation and (b) either cytokinin or light can saturate the morphogenic responses. As a consequence, cytokinin has no effect on hypocotyl elongation under normal growth conditions because light levels saturate the hypocotyl inhibition response. To determine whether a functional light-response pathway is required for cytokinin responses, light-insensitive long hypocotyl (hy) mutants were tested for cytokinin responses. The hy mutants (hy1 to hy6) had normal cytokinin responses, except phyB-1 (hy3-1), in which hypocotyl elongation was insensitive to cytokinin. Cytokinin insensitivity in phyB-1 was attributed to an indirect effect of the mutation on cytokinin responses. The effects of cytokinin on the inhibition of hypocotyl elongation are largely mediated by ethylene, and blocking the ethylene-response pathway through the action of a cytokinin-resistant, ethylene-insensitive mutant (ckr1/ein2) had no effect on the light inhibition of hypocotyl elongation. These results do not support the idea that cytokinin mediates the action of light on hypocotyl elongation.     

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.      

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in osmotic potential and cell wall composition

The changes in osmotic potential and cell wall composition of hypocotyl cell walls from different hypocotyl regions were investigated during growth of etiolated seedlings of Pinus pinaster Aiton. The osmotic potential in the subapical 5 mm part was minimum when hypocotyl growth rate was low, and increased when the fast growth phase began. The main non-cellulosic sugars of the cell wall from pine hypocotyl were arabinose, galactose, xylose, glucose and uronic acids, although their relative proportions were different from those found for angiosperm cell walls. Non-cellulosic glucose was the sugar showing the most important changes during hypocotyl growth as well as along the hypocotyl, suggesting that a glucose-rich polysaccharide is involved in a very active turnover during growth. A partial degradation of a xyloglucan during growth is suggested.     

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.     

In vitro morphogenetic response and distribution of endogenous plant hormones in hypocotyl segments of snapdragon (Antirrhinum majus L.)

Snapdragon seedlings, 20 mm in length, were cut into 5 segments from the cotyledon to the root, which were cultured in vitro on hormone-free MS medium. Adventitious shoot formation was highest in the basal hypocotyl segments with stimulation by the addition of BA. Endogenous cytokinins were higher in the basal hypocotyl segments than in the two upper hypocotyl segments, whereas auxin content was higher in the two upper than in the basal hypocotyl segments. Ratios of cytokinins to auxin were also the highest in the basal hypocotyl segment. A general principle in in vitro culture that a high concentration of cytokinin and a low concentration of auxin promotes the induction of shoot morphogenesis was confirmed from measurements of endogenous growth regulator concentrations.     

MDP25, a novel calcium regulatory protein, mediates hypocotyl cell elongation by destabilizing cortical microtubules in Arabidopsis

The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.     

Growth and stomata development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins

The plant hormones gibberellin (GA), ethylene and auxin can promote hypocotyl elongation of Arabidopsis seedlings grown in the light on a low nutrient medium (LNM). In this study, we used hypocotyl elongation as a system to investigate interactions between GA and ethylene or auxin and analysed their influence on the development of stomata in the hypocotyl. When applied together, GA and ethylene or auxin exerted a synergistic effect on hypocotyl elongation. Stimulated cell elongation is the main cause of hypocotyl elongation. Furthermore, hypocotyls treated with GA plus either ethylene or auxin show an increased endoreduplication. In addition, a small but significant increase in cell number was observed in the cortical cell files of hypocotyls treated with ethylene and GA together. However, studies with transgenic seedlings expressing CycB1::uidA genes revealed that cell division in the hypocotyl occurs only in the epidermis and mainly to form stomata, a process strictly regulated by hormones. Stomata formation in the hypocotyl is induced by the treatment with either GA or ethylene. The effect of GA could be strongly enhanced by the simultaneous addition of ethylene or auxin to the growth medium. Gibberellin is the main signal inducing stomata formation in the hypocotyl. In addition, this signal regulates hypocotyl elongation and is modulated by ethylene and auxin. The implication of these three hormones in relation to cell division and stomata formation 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 EFFECTS OF TEN PHENOLIC COMPOUNDS ON HYPOCOTYL GROWTH AND MITOCHONDRIAL METABOLISM OF MUNG BEAN

Ten phenolic compounds were examined for their effect on mung bean (Phaseolus aureus L.) hypocotyl growth and on respiration and coupling parameters of isolated mung bean hypocotyl mitochondria. Three compounds—tannic, gentisic, and p-coumaric acids—inhibited hypocotyl growth and when incubated with isolated hypocotyl mitochondria released respiratory control, inhibited respiration, and prevented substrate-supported Ca²⁺ and PO₄ transport. Vanillic acid also inhibited hypocotyl growth and reduced mitochondrial Ca²⁺ uptake but did not affect respiration or respiratory control of isolated mitochondria. This is the first compound reported to selectively inhibit Ca²⁺ uptake in plant mitochondria. Two other phenolic compounds—α, 3,5-resorcylic and protocatechuic acids—showed no significant effect on hypocotyl growth and did not affect mitochondrial oxidative phosphorylation either separately or in various combinations. Four phenolic compounds—ferulic, caffeic, p-hydroxybenzoic, and syringic acids—showed a significant reduction in mung bean hypocotyl growth but did not inhibit any of the mitochondrial processes examined. The results show that phenolic compounds which alter respiration or coupling responses in isolated mitochondria also inhibit hypocotyl growth and may reflect a mechanism of action for these natural growth inhibitors.     

Seed Coat Retention and Hypocotyl Hook Development in Mutants of Arabidopsis thaliana L.

Germination and growth of wild-type and two mutant strains (aux-1and Dwf) of Arabidopsis thaliana L. have been examined. Seedlingsof aux-1 exhibit agravitropic roots whereas Dwf display bothagravitropic roots and shoots. Wild-type seedlings retained the seed coat at the root-hypocotyltransition zone and developed hypocotyl hooks. In contrast,aux-I and Dwf seedlings did not retain their seed coats andlacked hypocotyl hooks. A positive gravitropic response of theroots was essential for the retention of the seed coat at theroot—hypocotyl transition zone by the attachment of roothairs to the seed coat. The development of the hypocotyl hookwas aided by the retention of the seed coat. The apical regionof the hypocotyl apparently remained agravitropic during formationand maintenance of the hypocotyl hook. Arabidopsis thaliana L., auxins, gravitropism, hypocotyl hook, mutants, peg formation, germination     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana , two dominant photomorphogenic mutants, shy1-1D and shy2-1D , for two genetic loci designated as SHY1 and SHY2 ( s uppressor of hy 2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion, and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red/far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis . This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.     

Comparative Enzymology of the Adenosine Triphosphate Sulfurylases from Leaf and Swollen Hypocotyl Tissue of Beta vulgaris: Multiple Enzyme Forms in Hypocotyl Tissue

ATP sulfurylase activity was partially purified from the swollen hypocotyl of beetroot (Beta vulgaris); activity was measured by sulfate-dependent PPi-ATP exchange. The ATP sulfurylase activity was separated from pyrophosphatase and ATPase activities which interfere with the assay of ATP sulfurylase activity. The ATP sulfurylase activity from hypocotyl tissue was invariably resolved into two approximately equal activities (hypocotyls I and II) by ion exchange chromatography and polyacrylamide gradient gel electrophoresis. Both enzymes catalyzed selenate- and sulfate-dependent PPi-ATP exchange; the affinity of hypocotyl II for these substrates was greater than for hypocotyl I. It is unlikely that the two activities arise by allelic variation or as an artifact of purification; they are most probably isoenzymes. Studies of the subcellular localization of the two hypocotyl enzymes were inconclusive.     

UVB诱导的绿豆下胚轴原生质体收缩效应

ＵＶ－Ｂ处理外起绿豆幼苗下胚轴原生质体的收缩;绿豆幼苗的下胚轴的伸长亦受ＵＶ－Ｂ处理的显著抑制。统计分析证实两者呈显著正相关（ｒ＾２＝０．８０６６）。这一结果表明,ＵＶ－Ｂ对绿豆下胚轴生长的抑制作用与不胚轴细胞伸长受到抑制相关。     

Characterization of post-flooding recovery-responsive enzymes in soybean root and hypocotyl

Soybean exhibits markedly reduced growth and yields under flooding stress. To determine the functional roles of four soybean proteins in post-flooding recovery, the organ/stress specificity and time-dependency of their enzymatic activities were analyzed. Peroxidase activity decreased in root and hypocotyl exposed to flooding and cold stresses, but increased during the post-stress recovery period. In contrast, its activity increased in both root and hypocotyl under drought stress. Acid phosphatase activity was suppressed in root treated with flooding and cold stresses, and slightly increased during the recovery period; however, the opposite profile was observed in hypocotyl. In response to drought stress, it did not change in root, but was decreased in hypocotyl. Beta-ketoacyl reductase activity did not change in root under flooding conditions, but was decreased in hypocotyl, although the activity increased slightly during the recovery period. In addition, it was decreased in both organs under drought and cold stresses, but again increased during the recovery period. Nucleotidylyl transferase activity was increased in root under flooding and drought stresses, but was decreased in hypocotyl. It was decreased in response to cold stress, but exhibited a slight increase during the recovery period. Furthermore, the treatment with jasmonate and salicylate suppressed the activities of peroxidase and acid phosphatase in root and hypocotyl under flooding stress; however, the activity of acid phosphatase increased during the recovery period. Nucleotidylyl transferase activity in root was also elevated by treatment with jasmonate, but gradually decreased during the recovery period. These results suggest that jasmonate-induced changes in nucleotidylyl transferase activity may facilitate soybean root recovery after flooding stress.