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     

Heat Shock–Induced Fluctuations in Clock and Light Signaling Enhance Phytochrome B–Mediated Arabidopsis Deetiolation

Moderately warm constant ambient temperatures tend to oppose light signals in the control of plant architecture. By contrast, here we show that brief heat shocks enhance the inhibition of hypocotyl growth induced by light perceived by phytochrome B in deetiolating Arabidopsis thaliana seedlings. In darkness, daily heat shocks transiently increased the expression of PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and markedly enhanced the amplitude of the rhythms of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. In turn, these rhythms gated the hypocotyl response to red light, in part by changing the expression of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5. After light exposure, heat shocks also reduced the nuclear abundance of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and increased the abundance of its target ELONGATED HYPOCOTYL5 (HY5). The synergism between light and heat shocks was deficient in the prr7 prr9, lhy cca1, pif4 pif5, cop1, and hy5 mutants. The evening element (binding site of LHY and CCA1) and G-box promoter motifs (binding site of PIFs and HY5) were overrepresented among genes with expression controlled by both heat shock and red light. The heat shocks experienced by buried seedlings approaching the surface of the soil prepare the seedlings for the impending exposure to light by rhythmically lowering LHY, CCA1, PIF4, and PIF5 expression and by enhancing HY5 stability.     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998     

Response of seedlings of a dwarf and a normal strain of watermelon to gibberellins

Hypocotyl and root elongation in a dwarf and a normal strain of watermelon (Citrullus lanatus [Thunb.] Matsu.) in the absence or presence of different gibberellins was investigated in seedlings grown under gold fluorescent light or in darkness. The normal strain, “Sugar Baby,” responded only slightly to the gibberellic acids employed. At appropriate concentrations all of the gibberellic acids were capable of normalizing growth in the monorecessive dwarf strain, WB-2, in darkness or in light. Gibberellins A₄₊₇ and A₇ were effective in stimulating hypocotyl elongation at concentrations 10 to 15 times lower than that needed for a response to GA₁ or GA₃. Dark-grown dwarfs responded to about a 3-fold lower concentration of GA₄₊₇ than those grown in light.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

     

Gibberellin response in lettuce hypocotyl sections

Excised lettuce (Lactuca sativa L.) hypocotyl sections retain the ability to elongate in response to gibberellic acid (GA₃) addition. In 48 hr at 30 C a GA₃-treated segment more than doubles while a control segment elongates less than 50%. Auxin has no detectable effect on this system. Sensitivity to GA₃ is not decreased by apex or root removal. Of the experimental variables tested, temperature, sucrose, and preincubation in water affect growth both with and without GA₃. Blue and far red light inhibit growth without GA₃; this inhibition is reversed by GA₃. Potassium chloride stimulates growth of illuminated sections treated with GA₃ but has no effect on control growth. When sections are incubated in the dark, KCl has a promotive effect on elongation.     

Interaction of Brassinosteroids with Light Quality and Plant Hormones in Regulating Shoot Growth of Young Sunflower and <Emphasis Type="Italic">Arabidopsis</Emphasis> Seedlings

Sunflower hypocotyls elongate as light quality changes from the normal red to far-red (R/FR) ratio of sunlight to a lower R/FR ratio. This low R/FR ratio-induced elongation significantly increases endogenous concentrations of indole-3-acetic acid (IAA) and also of three gibberellins (GAs): GA₂₀, GA₁, and GA₈. Of these, it is likely GA₁ that drives low R/FR-induced growth. Brassinosteroids are also involved in shoot growth. Here we tested three R/FR ratios: high, normal, and low. Significant hypocotyl elongation occurred with this stepwise reduction in R/FR ratio, but endogenous castasterone concentrations in the hypocotyls remained unchanged. Brassinolide was also applied to the seedlings and significantly increased hypocotyl growth, though one that was uniform across all three R/FR ratios. Applied brassinolide increased hypocotyl elongation while significantly reducing (usually) levels of IAA, GA₂₀, and GA₈, but not that of GA₁, which remained constant. Given the above, we conclude that endogenous castasterone does not mediate the hypocotyl growth that is induced by enriching FR light, relative to R light. Similarly, we conclude that the hypocotyl growth that is induced by applied brassinolide does not result from an interaction of brassinolide with changes in light quality. The ability of applied brassinolide to influence IAA, GA₂₀, and GA₈ content, yet have no significant effect on GA₁, is hard to explain. One speculative hypothesis, though, could involve the brassinolide-induced reductions that occurred for endogenous IAA, given IAA’s known ability to differentially influence the expression levels of GA20ox, GA3ox, and GA2ox, key genes in GA biosynthesis.     

Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis

Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.     

Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria

The effects of differential photoperiodic treatments applied to shoot tips and mature leaves of the long-day (LD) plant Silene armeria L. on growth and flowering responses, and on the levels of endogenous gibberellins (GAs), were investigated. Gibberellins were analyzed by gaschromatography-mass spectrometry and the use of internal standards. Exposure of mature leaves to LD, regardless of the photoperiodic conditions of the shoot tips, short days (SD), LD, or darkness, promoted elongation of the stems and of the immature leaves. Long-day treatment of the mature leaves modified the levels of endogenous GAs in shoot tips kept under LD, SD, or darkness. In shoot tips kept in LD or darkness the levels of GA₅₃ were reduced, whereas the levels of GA₁₉ and GA₂₀ were increased. The contents of GA₁ were increased in all three types of shoots: SD twofold, LD fivefold, and darkness eightfold. Dark treatment of the shoot tips on plants of which the mature leaves were grown in SD promoted elongation of the immature etiolated leaves and increased the GA₁ content of the shoot tips threefold. However, this treatment did not cause stem elongation. The different photoperiodic treatments applied to the shoot tips did not change the levels of GAs in mature leaves. These results indicate that both LD and dark treatments result in an increase in GA₁ in shoot tips. In addition, it is proposed that LD treatment induces the formation of a signal that is transmitted from mature leaves to shoot tips where it enhances the effect of GA on stem elongation.Abbreviations GA_n gibberellin A_n - LD long day(s) - SD short day(s) We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]-gibberellins and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility, East Lansing, for advice with mass spectrometry. This work was supported, in part, by a fellowship from the Spanish Ministry of Agriculture (Instituto Nacional de Investigaciones Agrarias) to M.T., by the U.S. Department of Energy grant No. DE-FG02-91ER20021, and by the U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

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.     

Promotive and inhibitory effects of uniconazole and prohexadione on the sprouting of bulbils of Chinese yam,Dioscorea opposita

Gibberellin A₁ (GA₁), GA₃ and GA₄ inhibited the sprouting of nondormant bulbils of Chinese yam, Dioscorea opposita, where the effectiveness of the GAs was as follows: GA₄>GA₁+GA₃. Uniconazole and prohexadione, plant growth retardants, promoted the sprouting of half-dormant bulbils. By contrast, these retardants inhibited the sprouting of nondormant bulbils. Gibberellin A₃ (GA₃) and A₄ (GA₄) which were applied to the stems of the sprouted bulbils, promoted stem elongation, but GAs applied to the bulbous parts inhibited this process. The effectiveness of the GAs on stem elongation was as follows: GA₃+GA₄ for the promotion and GA₄ > GA₃ for the inhibition. Uniconazole applied to the stem inhibited the stem elongation of the sprouted bulbils. These results suggest the possible involvement of endogenous GAs in the induction and maintenance of bulbil dormancy of D. opposita, as well as in the bulbil sprouting and subsequent stem elongation.     

Dormancy and germination in Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee seeds: Effects of short-time moist chilling and plant growth regulators

We investigated the germination requirements of the species Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee (Lamiaceae). We studied the effects of scarification, short-time moist chilling (+4 °C) for 15 and 30 days, and various doses of gibberellic acid (GA₃; 0, 100, 150 and 250 ppm), Kinetin (KIN; 50 ppm) and a combination of 250 ppm GA₃ and 50 ppm KIN. The hormone and moist chilling treatments were carried out under both continuous darkness (20 °C) and photoperiodic (20/10 °C; 12/12 h, respectively) conditions. Seeds failed to germinate in response to short-time moist chilling treatments with distilled water under both continuous darkness and photoperiodic conditions. Seeds were found to have dormancy. Treatments with GA₃ or a combination of GA₃ and KIN were successful at breaking seed dormancy. A maximum of 37% of the seeds germinated after GA₃ application in all series. When only KIN was applied at a 50 ppm concentration, germination (12%) was found only with moist chilling for 30 days under continuous darkness. The highest germination rates were found in seeds treated with combination of 250 ppm GA₃ and 50 ppm KIN. In the combination treatments, while the moist chilling treatments for 15 days resulted in 68 and 73% germination, respectively, these rates were up to 95% in the moist chilling treatments for 30 days under continuous darkness and photoperiodic conditions. Mean germination time (MGT) in GA₃ and KIN combinations was lower than in other treatments. Scarification with 80% sulphuric acid did not promote germination. The characteristics of physiological dormancy of S. germanica ssp. bithynica seeds are consistent with conditions of existence in the in alpine habitat of this species.