Chemopreventive glucosinolate accumulation in various broccoli and collard tissues: Microfluidic-based targeted transcriptomics for by-product valorization.

Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market.

Proposed Method for Estimating Health-Promoting Glucosinolates and Hydrolysis Products in Broccoli (Brassica oleracea var. italica) Using Relative Transcript Abundance.

Due to the importance of glucosinolates and their hydrolysis products in human nutrition and plant defense, optimizing the content of these compounds is a frequent breeding objective for Brassica crops. Toward this goal, we investigated the feasibility of using models built from relative transcript abundance data for the prediction of glucosinolate and hydrolysis product concentrations in broccoli. We report that predictive models explaining at least 50% of the variation for a number of glucosinolates and their hydrolysis products can be built for prediction within the same season, but prediction accuracy decreased when using models built from one season's data for prediction of an opposing season. This method of phytochemical profile prediction could potentially allow for lower phytochemical phenotyping costs and larger breeding populations. This, in turn, could improve selection efficiency for phase II induction potential, a type of chemopreventive bioactivity, by allowing for the quick and relatively cheap content estimation of phytochemicals known to influence the trait.

Transcriptome and Metabolome Analyses of Glucosinolates in Two Broccoli Cultivars Following Jasmonate Treatment for the Induction of Glucosinolate Defense to Trichoplusia ni (Hübner).

Lepidopteran larvae growth is influenced by host plant glucosinolate (GS) concentrations, which are, in turn, influenced by the phytohormone jasmonate (JA). In order to elucidate insect resistance biomarkers to lepidopteran pests, transcriptome and metabolome analyses following JA treatments were conducted with two broccoli cultivars, Green Magic and VI-158, which have differentially induced indole GSs, neoglucobrassicin and glucobrassicin, respectively. To test these two inducible GSs on growth of cabbage looper (Trichoplusia ni), eight neonate cabbage looper larvae were placed onto each of three plants per JA treatments (0, 100, 200, 400 µM) three days after treatment. After five days of feeding, weight of larvae and their survival rate was found to decrease with increasing JA concentrations in both broccoli cultivars. JA-inducible GSs were measured by high performance liquid chromatography. Neoglucobrassicin in Green Magic and glucobrassicin in VI-158 leaves were increased in a dose-dependent manner. One or both of these glucosinolates and/or their hydrolysis products showed significant inverse correlations with larval weight and survival (five days after treatment) while being positively correlated with the number of days to pupation. This implies that these two JA-inducible glucosinolates can influence the growth and survival of cabbage looper larvae. Transcriptome profiling supported the observed changes in glucosinolate and their hydrolysis product concentrations following JA treatments. Several genes related to GS metabolism differentiate the two broccoli cultivars in their pattern of transcriptional response to JA treatments. Indicative of the corresponding change in indole GS concentrations, transcripts of the transcription factor MYB122, core structure biosynthesis genes (CYP79B2, UGT74B1, SUR1, SOT16, SOT17, and SOT18), an indole glucosinolate side chain modification gene (IGMT1), and several glucosinolate hydrolysis genes (TGG1, TGG2, and ESM1) were significantly increased in Green Magic (statistically significant in most cases at 400 µM) while UGT74B1 and MYB122 were significantly increased in VI-158. Therefore, these metabolite and transcript biomarker results indicate that transcriptome profiling can identify genes associated with the formation of two different indole GS and their hydrolysis products. Therefore, these metabolite and transcript biomarkers could be useful in an effective marker-assisted breeding strategy for resistance to generalist lepidopteran pests in broccoli and potentially other Brassica vegetables.

The Role of Glucosinolate Hydrolysis Products from Brassica Vegetable Consumption in Inducing Antioxidant Activity and Reducing Cancer Incidence.

The bioactivity of glucosinolates (GSs), and more specifically their hydrolysis products (GSHPs), has been well documented. These secondary metabolites evolved in the order Brassicales as plant defense compounds with proven ability to deter or impede the growth of several biotic challenges including insect infestation, fungal and bacterial infection, and competition from other plants. However, the bioactivity of GSHPs is not limited to activity that inhibits these kingdoms of life. Many of these compounds have been shown to have bioactivity in mammalian systems as well, with epidemiological links to cancer chemoprevention in humans supported by in vitro, in vivo, and small clinical studies. Although other chemopreventive mechanisms have been identified, the primary mechanism believed to be responsible for the observed chemoprevention from GSHPs is the induction of antioxidant enzymes, such as NAD(P)H quinone reductase (NQO1), heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione S transferases (GSTs), through the Keap1-Nrf2-ARE signaling pathway. Induction of this pathway is generally associated with aliphatic isothiocyanate GSHPs, although some indole-derived GSHPs have also been associated with induction of one or more of these enzymes.

Ether Cleavage Re-Investigated: Elucidating the Mechanism of BBr3-Facilitated Demethylation of Aryl Methyl Ethers.

One of the most well-known, highly utilized reagents for ether cleavage is boron tribromide (BBr3), and this reagent is frequently employed in a 1:1 stoichiometric ratio with ethers. Density functional theory calculations predict a new mechanistic pathway involving charged intermediates for ether cleavage in aryl methyl ethers. Moreover, these calculations predict that one equivalent of BBr3 can cleave up to three equivalents of anisole, producing triphenoxyborane [B(OPh)3] prior to hydrolysis. These predictions were validated by gas chromatography analysis of reactions where the BBr3:anisole ratio was varied. Not only do we confirm that sub-stoichiometric equivalents may be used for ether demethylation, but the findings also support our newly proposed three cycle mechanism for cleavage of aryl methyl ethers.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Blast2GO goes grid: developing a grid-enabled prototype for functional genomics analysis.

The vast amount in complexity of data generated in Genomic Research implies that new dedicated and powerful computational tools need to be developed to meet their analysis requirements. Blast2GO (B2G) is a bioinformatics tool for Gene Ontology-based DNA or protein sequence annotation and function-based data mining. The application has been developed with the aim of affering an easy-to-use tool for functional genomics research. Typical B2G users are middle size genomics labs carrying out sequencing, ETS and microarray projects, handling datasets up to several thousand sequences. In the current version of B2G. The power and analytical potential of both annotation and function data-mining is somehow restricted to the computational power behind each particular installation. In order to be able to offer the possibility of an enhanced computational capacity within this bioinformatics application, a Grid component is being developed. A prototype has been conceived for the particular problem of speeding up the Blast searches to obtain fast results for large datasets. Many efforts have been done in the literature concerning the speeding up of Blast searches, but few of them deal with the use of large heterogeneous production Grid Infrastructures. These are the infrastructures that could reach the largest number of resources and the best load balancing for data access. The Grid Service under development will analyse requests based on the number of sequences, splitting them accordingly to the available resources. Lower-level computation will be performed through MPIBLAST. The software architecture is based on the WSRF standard.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus.

The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A1 (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions.

Pollination Increases Gibberellin Levels in Developing Ovaries of Seeded Varieties of Citrus.

Reproductive and vegetative tissues of the seeded Pineapple cultivars of sweet orange (Citrus sinensis L.) contained the following C-13 hydroxylated gibberellins (GAs): GA53, GA17, GA19, GA20, GA1, GA29, and GA8, as well as GA97, 3-epi-GA1, and several uncharacterized GAs. The inclusion of 3-epi-GA1 as an endogenous substance was based on measurements of the isomerization rates of previously added [2H2]GA1. Pollination enhanced amounts of GA19, GA20, GA29, and GA8 in developing ovaries. Levels of GA1 increased from 5.0 to 9.5 ng/g dry weight during anthesis and were reduced thereafter. The amount of GA in mature pollen was very low. Emasculation reduced GA levels and caused a rapid 100% ovary abscission. This effect was partially counteracted by either pollination or application of GA3. In pollinated ovaries, repeated paclobutrazol applications decreased the amount of GA and increased ovary abscission, although the pattern of continuous decline was different from the sudden abscission induced by emasculation. The above results indicate that, in citrus, pollination increases GA levels and reduces ovary abscission and that the presence of exogenous GA3 in unpollinated ovaries also suppresses abscission. Evidence is also presented that pollination and GAs do not, as is generally assumed, suppress ovary abscission through the reactivation of cell division.

Identification of three C20-gibberellins: GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19).

Three new C20-gibberellins, GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19), have all been isolated from spinach, GA97 also from tomato root cultures and pea pods, and GA98 from maize pollen. The structures of these compounds were established by GC-mass spectrometric comparisons of the trimethylsilylated methyl esters with authentic samples prepared from gibberellic acid (GA3).

Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots.

The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

Phytochrome A overexpression in transgenic tobacco. Correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels.

Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in several plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (GAs) in tobacco (Nicotiana tabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35S promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35S promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ1) promoters. Neither promoter was as effective as CaMV 35S in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome is important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing GA biosynthesis.

Gibberellin A1 is required for stem elongation in spinach.

The effects of the growth retardants 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (AMO-1618) and calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate (BX-112) on stem elongation were investigated in the rosette plant spinach (Spinacia oleracea L.) under long-day (LD) conditions. Stem growth induced by a LD treatment was prevented by both retardants. The inhibition caused by AMO-1618 was reversed by gibberellin A1 (GA1) and GA20, whereas the effects of BX-112 were reversed by GA1 only. Six GAs (GA53, GA44, GA19, GA20, GA1, and GA8) were quantified by gas chromatography-selected ion monitoring using internal standards. Plants treated with BX-112 had reduced levels of GA1 and GA8 and accumulated GA53, GA44, GA19, and GA20. The relative levels of four additional GAs (3-epi-GA1, GA29, GA60, and GA81) were compared by ion intensities only. Relative to GA81, the level of GA29 was decreased by BX-112, whereas the levels of GA60 and 3-epi-GA1 were increased. Transfer of spinach from short-day conditions to LD conditions caused an increase in all identified GAs of the early 13-hydroxylation pathway with GA20, GA1, and GA8 showing the largest increases. These findings support the position that, of the GAs belonging to the early 13-hydroxylation pathway, GA1 is the primary GA active per se for stem elongation in spinach. The increase in endogenous GA1 in plants in LD conditions is most likely the primary factor for stem elongation.

Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins.

Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross-pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA(53), GA(44), GA(17), GA(19), GA(20), GA(29), GA(1), 3-epi-GA(1), GA(8), GA(24), GA(9), and GA(4) have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [(2)H(2)]- and [(13)C]-labeled internal standards, the levels of GA(53), GA(44), GA(19), GA(20), GA(1), GA(8), GA(4), and GA(9) were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13-hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA(53) (10.4x), GA(44) (13.9x), GA(19) (3.0x), GA(20) (11.2x), and GA(1) (2.0x). By contrast, levels of GA(8) were always higher in Clementine, whereas levels of GA(4) did not differ greatly. Levels of GA(9) were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA(1), respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA(3). GA(3) alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits.

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.

Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels.

The endogenous gibberellin (GA) content of spinach (Spinacia oleracea) was reinvestigated by combined gas chromatography-mass spectrometry analysis. The 13-hydroxy GAs: GA(53), GA(44), GA(19), GA(17), GA(20), GA(5), GA(1), GA(29), and GA(8); the non-3, 13-hydroxy GAs: GA(12), GA(15), GA(9), and GA(51); and the 3beta-hydroxy GAs: GA(4), GA(7), and GA(34), were identified in spinach extracts by comparing full-scan mass spectra and Kovats retention indices with those of reference GAs. In addition, spinach plants contained GA(7)-isolactone, 16,17-dihydro-17-hydroxy-GA(53), GA(29)-catabolite, 3-epi-GA(1), and 10 uncharacterized GAs with mass spectra indicative of mono- and dihydroxy-GA(12), monohydroxy-GA(25), dihydroxy-GA(24), and dihydroxy-GA(g). The effect of light-dark conditions on the GA levels of the 13-hydroxylation pathway was studied by using labeled internal standards in selected ion monitoring mode. In short day, the GA levels were higher at the end of the light period than at the end of the dark period. Levels of GAs at the end of each short day were relatively constant. During the first supplementary light period of long day treatment, GA(53) and GA(19) declined dramatically, GA(44) and GA(1) decreased slightly, and GA(20) increased. During the subsequent high-intensity light period, the GA(20) level decreased and the levels of GA(53), GA(44), GA(19), and GA(1) increased slightly. Within 7 days after the beginning of long day treatment, similar patterns for GA(53) and GA(19) occurred. Furthermore, when these plants were transferred to darkness, an increase in the levels of GA(53) and GA(19) was observed. These results are compatible with the idea that in spinach, the flow through the GA biosynthetic pathway is much enhanced during the high-intensity light period, although GA turnover occurs also during the supplementary period of long day, both effects being responsible for the increase of GA(20) and GA(1) in long day.

Cellular changes induced by exogenous and endogenous gibberellins in shoot tips of the long-day plant Silene armeria.

Stem elongation and flowering are two processes induced by long-day (LD) treatment in Silene armeria L. Whereas photoperiodic control of stem growth is mediated by gibberellins (GAs), the flowering response cannot be obtained by GA applications. Microscopic observations on early cellular changes in the shoot meristem following LD induction or GA treatment in short days (SD) were combined with GA analyses of stem sections at various distances below the shoot apex. The earliest effects of both LD and GA induction on the subapical meristem were an increase in the number of cells per cell file and a reduction of cell length in the meristematic tissue approx. 1.0-3.0 mm below the shoot apex. Within 8 d after the beginning of LD induction or after GA application, the cells in the subapical meristem were oriented in long files. In induced tips, cellulose deposition occurred mostly in longitudinal walls, indicating that many transverse cell divisions had taken place which, in turn, increased the length of the stem. In contrast to LD induction, GA treatments did not promote the transition from the vegetative to the floral stage. Endogenous GAs were analyzed by selected ion monitoring (SIM), using labeled internal standards, in extracts from transverse sections of the tip at various distances below the apical meristem. In control plants, the levels of the six 13-hydroxy GAs studied (GA53, GA44, GA19, GA20, GA1, and GA8) decreased as the distance from the apical meristem increased. Except for GA53, GA levels were higher in tips of LD-induced plants, particularly in the meristematic zone approx. 0.5-1.5 mm below the apical meristem. In comparison with SD, the highest increase observed was for GA1, the content of which increased 30-fold in the zone 0.5-3.5 mm below the shoot apex. These data indicate a spatial correlation between the accumulation of GA1 and its precursors, and the enhanced mitotic activity which occurs in the subapical meristem of elongating Silene apices.

Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants.

Twenty gibberellins (GAs) have been identified in extracts from shoots of the Landsberg erecta line of Arabidopsis thaliana by full-scan gas chromatography-mass spectrometry and Kovats retention indices. Eight of them are members of the early-13-hydroxylation pathway (GA53, GA44, GA19, GA17, GA20, GA1, GA29, and GA8), six are members of the early-3-hydroxylation pathway (GA37, GA27, GA36, GA13, GA4, and GA34), and the remaining six are members of the non-3,13-hydroxylation pathway (GA12, GA15, GA24, GA25, GA9, and GA51). Seven of these GAs were quantified in the Landsberg erecta line of Arabidopsis and in the semidwarf ga4 and ga5 mutants by gas chromatography-selected ion monitoring (SIM) using internal standards. The relative levels of the remaining 13 GAs were compared by the use of ion intensities only. In comparison with the Landsberg erecta line, the ga4 mutant had reduced levels of the 3-hydroxy- and 3,13-dihydroxy-GAs, and it accumulated the 13-hydroxy-GAs, except GA53, and the non-3,13-hydroxy-GAs, except GA12. The GA4 gene encodes, therefore, a protein with 3 beta-hydroxylation activity. The ga5 mutant had reduced levels of the C19-GAs, which indicates that the product of the GA5 gene catalyzes the elimination of C-20 at the aldehyde level. The ga5 mutant also had increased levels of certain C20-GAs, which indicates existence of an additional control, possibly hydroxylation of C-20. The growth-response data, as well as the accumulation of GA9 in the ga4 mutant, indicate that GA9 is not active in Arabidopsis, but it must be 3 beta-hydroxylated to GA4 to become bioactive. It is concluded that the reduced levels of the 3 beta-hydroxy-GAs, GA1 and GA4, are the cause of the semidwarf growth habit of both mutants.

Gibberellins and Stem Growth as Related to Photoperiod in Silene armeria L.

Stem growth and flowering in the long-day plant Silene armeria L. are induced by exposure to a minimum of 3 to 6 long days (LD). Stem growth continues in subsequent short days (SD), albeit at a reduced rate. The growth retardant tetcyclacis inhibited stem elongation induced by LD, but had no effect on flowering. This indicates that photoperiodic control of stem growth in Silene is mediated by gibberellins (GA). The objective of this study was to analyze the effects of photoperiod on the levels and distribution of endogenous GAs in Silene and to determine the nature of the photoperiodic after-effect on stem growth in this plant. The GAs identified in extracts from Silene by full-scan combined gas chromatography-mass spectrometry (GC-MS), GA(12), GA(53), GA(44), GA(17), GA(19), GA(20), GA(1), GA(29), and GA(8), are members of the early 13-hydroxylation pathway. All of these GAs were present in plants under SD as well as under LD conditions. The GA(53) level was highest in plants in SD, and decreased in plants transferred to LD conditions. By contrast, GA(19), GA(20), and GA(1) initially increased in plants transferred to LD, and then declined. Likewise, when Silene plants were returned from LD to SD, there was an increase in GA(53), and a decrease in GA(19), GA(20), and GA(1) which ultimately reached levels similar to those found in plants kept in SD. Thus, measurements of GA levels in whole shoots of Silene as well as in individual parts of the plant suggest that the photoperiod modulates GA metabolism mainly through the rate of conversion of GA(53). As a result of LD induction, GA(1) accumulates at its highest level in shoot tips which, in turn, results in stem elongation. In addition, LD also appear to increase the sensitivity of the tissue to GA, and this effect is presumably responsible for the photoperiodic after-effect on stem elongation in Silene.