Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Gibberellins and photoperiodic control of shoot elongation in Salix

Effects of exogenous gibberellins GA₅₃, GA₄₄, GA₁₉, GA₂₀ and GA₁ on photoperiodically controlled shoot elongation in seedlings of Salix pentandra L. were studied. Gibberellins GA₂₀ and GA₁ induced shoot elongation under short days (SD) and could substitute for a transfer to long day (LD), while gibberellins A₅₃, A₄₄ and A₁₉ were inactive. In seedlings exposed to a prolonged SD-treatment (30 days) there was a significant positive interaction between a transfer to LD and a treatment with GA₂₀ and GA₁ on shoot elongation. In addition, GA₁₉ enhanced the growth promotive effect of LD in these seedlings. The results are compatible with the suggestion that conversion of GA₁₉ to GA₂₀ is blocked under SD. This effect is supposed to be an early process leading to the cessation of shoot elongation under SD. Responsiveness of the seedlings to LD and to a GA-treatment gradually decreased with an increasing length of exposure to SD.     

Effects of prohexadione (BX-112) and gibberellins on shoot growth in seedlings of Salix pentandra

Effects of gibberellins A₁, A_4/7, A₉, A₁₉ and A₂₀ and growth retardants were studied on shoot elongation in seedlings of Salix pentandra L. The growth-retarding effects of CCC and ancymidol were antagonized by all the gibberellins tested. The novel plant growth regulator prohexadione (free acid of BX-112), which is suggested to block 3β-hydroxylation of gibberellins, effectively prevented shoot elongation in seedlings grown under long photoperiod. Initiation of new leaves was only slightly reduced. GA₁, but not GA₁₉ and GA₂₀, was active in overcoming the inhibition of stem elongation of seedlings, treated with prohexadione, GA₁₉, GA₂₀ and GA₁ are native in S. pentandra , and the results are compatible with the hypothesis that GA₁ is active per se in shoot elongation, and that the effect of GA₁₉ and GA₂₀ is dependent on their conversion to GA₁.
A mixture of GA₄ and GA₇ was as active as GA₁ in promoting shoot elongation in seedlings treated with prohexadione, while GA₉ showed slight activity only when applied at high doses.     

Prolonged salt stress alters the ratios of protochlorophyllide spectral forms in dark-grown wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and influences chlorophyll <Emphasis Type="Italic">a</Emphasis> accumulation following irradiation

To examine the effects of salt stress on dark-grown wheat (Triticum aestivum), seedlings of the salt-tolerant cultivar Sids 1 and the susceptible cultivar Giza 168 were grown in darkness for 14 days in nutrient solution with and without 200 mM of supplementary salt (100 mM of NaCl and 100 mM of KCl). During this time, we monitored their protochlorophyllide (Pchlide) contents, ratios of photoactive to non-photoactive forms of Pchlide (from 655/633-nm emission ratios in their 77 K fluorescence emission spectra) and (following flash irradiation) ratios of newly formed chlorophyllide (Chlide) to non-photoactive Pchlide. In addition, the accumulation of chlorophyll a in leaf sections was monitored during prolonged (24 h) irradiation. The results depended on the developmental state of the seedlings. However, the salt stress treatment caused marked increases in both Pchlide contents in dark-grown leaves and in Chlide contents following irradiation of leaf sections of both cultivars. The ratio of phototransformable to non-phototransformable Pchlide and the abundance of newly formed Chlide were also increased by the salt stress. Further, leaves of salt-stressed seedlings consistently accumulated more chlorophyll a than leaves of unstressed seedlings when floating on the nutrient solution (with or without supplementary salt) in continuous white light. The findings are consistent with the hypothesis that increased levels of the long-wavelength form of Pchlide contribute to protective mechanisms against salt stress.     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

A localised decrease of GA1 in shoot tips of Salix pentandra seedings precedes cessation of shoot elongation under short photoperiod

By application of a recently developed method allowing analysis of gibberellins (GAs) in mg amounts of tissue, the effect of photoperiod on levels of GAs in shoot tips of individual seedlings of the woody species Salix pentandra was studied. In elongating long day-grown seedlings, maximum levels of GA₁ were found 5–20 mm below the apex, approximately twice the levels in other segments. After exposure of plants to 5 or 15 short days, the levels of GA₁ were about 50% lower within this specific region of the stem, as compared with seedlings grown under long days. Short day-induced cessation of shoot elongation also correlated with overall declines in the levels of GA₅₃, GA₁₉, GA₂₀ and GA₈, Within each photoperiodic treatment the levels of these GAs were generally relatively similar throughout the upper 35 mm of stems. No differences in internode lengths or in lengths of pith or epidermal cells were found in plants grown under long days compared with those exposed to 5 short days. In both cases, cells in mitosis were observed in the subapical stem tissues of shoot tips. After 15 short days, stem elongation was completed, and dividing cells were generally not found in the subapical part of the stem. However, short day exposure did not prevent elongation of internodes and cells differentiated before the treatment was started. Thus, the localised decrease in level of GA₁ in shoot tips under short days precedes the morphological and anatomical changes connected with the short day-induced cessation of elongation growth. This supports the hypothesised role for GA₁ in photoperiodic control of shoot elongation in S. pentandra .     

Photoperiodic control of endogenous gibberellins in seedlings of Salix pentandra

In the temperate-zone woody species Salix pentandra elongation growth is regulated by the photoperiod. Long days sustain active growth, whereas short days induce cessation of apical growth, which is a prerequisite for winter hardening. It is shown that this is correlated to quantitative changes in levels of endogenous GA₁₉ GA₂₀, and GA₁. Within two short days the amount of the active GA₁ and its immediate precursor GA₂₀, decreased markedly in young leaves us well as in stem tissue. Also, the amount of GA¹⁹, declined, but the decrease was delayed relative to that of GA₁ and GA₂₀. The ability of S. pentandra seedlings to respond to exogenous GA₁₉, decreased with increasing numbers of short days. Observations that support the hypothesis that the level of GA₁ in S. pentandra is regulated by the photoperiod in a quantitative mode with conversion of GA₁₉, to GA₂₀, being one target for control.
Different distribution of GAs in various plant parts was observed. The level of GA was higher in young leaves than in other plant parts, and the amount of GA₁₉ was 5–10 times higher in stem tissue than in leaves and roots. The ratios of GA₈ to GA¹ and GA₂₀, were higher in roots as compared with other parts, as rods contained very low levels of GA₁ and GA₂₀, but amounts of GA₂₀ comparable with other parts.     

The plant-growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins

The plant-growth-promoting rhizobacteria (PGPR), Bacillus pumilus and Bacillus licheniformis, isolated from the rhizosphere of alder ( Alnus glutinosa [L.] Gaertn.) have a strong growth-promoting activity. Bioassay data showed that the dwarf phenotype induced in alder seedlings by paclobutrazol (an inhibitor of gibberellin [GA] biosynthesis) was effectively reversed by applications of extracts from media incubated with both bacteria and also by exogenous GA₃. Full-scan gas chromatography-mass spectrometry analyses on extracts of these media showed the presence of GA₁, GA₃, GA₄and GA₂₀, in addition to the isomers 3- epi -GA₁ and iso -GA₃. Isotope dilution analysis indicated that epi -GA₁ was an artefact. Likewise, iso -GA₃ is also probably an artifact spontaneously formed during extraction and/or analysis. In both culture media, GA₁ was present in higher concentrations (130–150 ng ml⁻¹) than GA₃ (50–60 ng ml⁻¹), GA₄ (8–12 ng ml⁻¹) and GA₂₀ (2–3 ng ml⁻¹). The data indicated that culture of both bacteria accumulate bioactive C19-gibberellins in relative high amounts and that these GAs appear to be physiologically active in the host plant. The evidence suggests that the promotion of stem elongation induced by the PGPR could be mediated by bacterial GAs.     

Growth-promoting activity of gibberellins on shoot elongation in Salix pentandra is reduced by 16,17-dihydro derivatisation

The metabolism of GA₁₀ is thought to be under photoperiodic control in the woody plant Salix pentandra . However, in a recent study using 16,17-[³H₂]GA₁₉ as a mimic of Ga₁₀, no effect of photoperiod was found on its metabolism to 16,17-dihydro-GA₂₀ and 16,17-dihydro-GA₁. To investigate if this was due to differential action of exogenous 16,17-dihydro-GAs and GAs, the effects of the 16,17-dihydro-derivatives of the gibberellins GA₁₉, GA₁, and GA₁ as compared with their parent GAs, on shoot elongation in seedlings of S. pentandra were studied. 16,17-Dihydro-GA₁₉, and -GA₂₀ were both almost inactive, while 16,17-dihydro-GA₁ induced some shoot elongation in seedlings treated with ancymidol as well as under short days. GA₁₉, GA₂₀ and GA₁ were all able to counteract the inhibitory effect of ancymidol under continuous light, while inhibition induced by a 12-h photoperiod was antagonised only by GA₂₀ and GA₁. Thus, the growth-stimulating activity of the tested GAs is significantly reduced by 16,17-dihydro derivatisation, but the derivatives do not inhibit stem elongation in S, pentandra , as has been found in monocotyledons.     

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid (GA₃) applied at different times during the growth of wild carrot ( Daucus carota ssp. Carota ) cell suspension cultures inhibited anthocyanin accumulation. Application of 3 × 10^–6 M GA₃ to cultures on day 0 or day 4 gave, respectively, 10 or 35% of anthocyanin accumulation relative to levels occurring when GA₃ was applied at the end of the growth period. Endogenous GAs were separated by high pressure liquid chromatography, and identified and quantified by gas chromatography-selected ion monitoring. Gibberellins GA₁, GA₃ and traces of GA₈. GA₁₉ and GA₂₀ were identified in carrot cell suspension cultures of both high and low anthocyanin-accumulating clones. The concentrations of GA₁. GA₃ and GA₈ in the two clones were similar and were not significantly different after the application of uniconazole which promoted anthocyanin accumulation. This suggests that these endogenous GAs are not the sole factors controlling the accumulation of anthocyanin in these different clones. Exogenous GA₃ and uniconazole had no effect on 3'-nucleotidase and 5'-nucleotidase activity in the carrot cell suspension cultures. Thus 3'-nucleotidase does not appear to play a role in the inhibition of anthocyanin accumulation by exogenous GA₃.     

Metabolism of C19- and C20-gibberellins by cell-free preparations from immature Phaseolus coccineus seed

Gibberellin biosynthesis pathways were investigated using isotopically-labelled C₁₉- and C₂₀-gibberellins and cell-free preparations from immature seed of Phaseous coccineus cv. Prizewinner. The initial steps in an early 13-hydroxylation pathway involved the conversion gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to GA₁₂ which was 13-hydroxylated to yield GA₅₃, Metabolism of GA₅₃ yielded GA₄₄. In contrast to other cell-free systems, GA₄₄ was not further converted, either as a δ-lactone or an open-lactone structure, to the C-20 aldehyde GA₁₉. GA₁₉ was, however, metabolised to GA₂₀, GA₅ and GA₁. GA₂₀ represented a branch point in the pathway as it was converted both to GA₁, which was an end product, and GA₅ which was further converted to GA₆. Like GA₁, GA₆ was also an end-product of the early 13-hydroxylation pathway.
A non-13-hydroxylation pathway involving GA₄, GA₁₅, GA₂₄ GA₃₇ and GA₃₆ also originated from GA₁₂. The terminal product of this pathway was the 3β-hydroxy C₁₉-gibberellin, GA₄.     

Gibberellin status and responsiveness in shoots of tall and dwarf genotypes of diploid rye (Secale cereale)

The recessive dwarfing alleles of rye ( Secale cereale L.), ct1 and ct2 , caused a 35–55% reduction in the length of leaf 2 compared with corresponding tall lines grown at both 10°C and 20°C. The dwarf lines were 45–50% as responsive to applied GA₃ as the tall lines at 20°C but the absolute GA-responsiveness of the dwarfs was greater at 10°C than at 20°C. There was no significant difference in the contents of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₃ and GA₈ in the leaf extension zone of tall and dwarf seedlings grown at 20°C. It was concluded that the mechanism whereby GA homeostasis is maintained is functional in both tall and dwarf lines despite marked differences in leaf extension rate. The recessive rye mutations may cause loss of function late in the GA-cell elongation pathway or, alternatively, indirectly affect GA-responsiveness in vegetative tissues. The genetic and physiological evidence indicates that ct1 and ct2 are unrelated to the GA-insensitive Rht genes in hexaploid bread wheat.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Effects of different gibberellins on elongation growth under short day conditions in seedlings of Salix pentandra

Fifteen different gibberellins (GA's) were tested for their ability to induce elongation growth under short day conditions in seedlings of Salix pentandra L. GA's were applied either to the apex or they were injected into a mature leaf. GA₃ was highly active and also GA₄₊₇ and GA₄ showed high activity. GA₁, GA₂, GA₅, GA₉, GA₁₃, GA₂₀, GA₃₆ and GA₄₇ showed moderate activity. GA₁₆, GA₁₇, GA₂₇ and GA₄₁ exhibited low or no activity in doses up to 10 μg per plant. In general, a better growth response was obtained with an application to the apex than with an injection into the leaf.     

Interaction of potassium ions and gibberellin in the control of hypocotyl growth in Amaranthus caudatus

Potassium promotes growth in several plant tissues. Elongation growth of the hypocotyls of Amaranthus caudatus L. ev. Lalsag is mainly controlled by gibberellins, but K⁺ also promotes growth. In the present study the interaction of K⁺ with gibberellin (GA₃) and chlorocholine chloride (CCC) has been investigated. When K⁺ was applied externally in the dark, hypocotyl growth was promoted in the seedlings. External application of GA₃ did not promote growth in the dark. GA₃ was effective in the light and K⁺ was synergistic with GA₃ in promoting elongation. Application of CCC in the dark makes the seedlings sensitive to GA₃. The inhibition of growth by CCC was also reversed by K⁺. The results indicate a possible role of K⁺ in GA₃ induced elongation of hypocotyls.