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Item does not contain fulltext The complexity of hormonal responses and their functional overlap support the presence of an intensive cross-talk between hormone signalling pathways. A detailed analysis of responses induced by ethylene and gibberellin (GA) in a GA-insensitive mutant (gai), an ethylene-resistant mutant (etr1-3), the gai etr1-3 double-mutant, and in wild-type Arabidopsis thaliana plants, revealed multiple interactions between ethylene and GA signal transduction pathways. Ethylene insensitive mutants and wild-type plants treated with 1-methylcyclopropene (1-MCP), an ethylene perception inhibitor, displayed a stronger responsiveness of genes differentially regulated by GA. In addition, microarray-analysis showed that the GA-response in an ethylene-insensitive background is different from that in the wild-type, confirming the importance of ethylene in a plant's response towards GA. In this paper, we present a number of genes with an altered response-pattern as a direct consequence of cross-talk between ethylene and GA.

The availability of short stature apple scions that required minimal applications of chemical growth retardants and could be used with a range of rootstocks would be of considerable benefit to fruit growers. We have suppressed the expression of a gene encoding the gibberellin (GA) biosynthetic enzyme GA 20-oxidase to reduce the levels of bioactive GAs in a scion variety, resulting in significant reductions in stem height. Application of GA3 reversed the effect. The scion remained dwarfed after grafting on to normally invigorating rootstocks, whilst control plants of the same cultivar displayed the expected vigour when grafted on to these rootstocks. This approach could be applicable to any perennial crop variety, allowing dwarf trees to be obtained on any available rootstock or on their own roots without the need for chemical growth retardant application. In effect, seedlings that are well suited to local conditions (drought, salinity) could be employed as tree rootstocks, as could existing rootstocks valued for characters other than vigour control, such as pest and disease resistance.

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.

Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (EbetaF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Menthaxpiperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EbetaF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K(m) for FPP of 1.91+/-0.1 microM and k(cat) of 0.18 s(-1), and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EbetaF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EbetaF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EbetaF synthase activity (K(m) for FPP 0.98+/-0.12 microM, k(cat) 0.1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EbetaF was not present, confirming that this particular mint chemotype had lost EbetaF synthase activity due to the observed mutations.

Background The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification. Results Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a K m of 0.024 μM and a V max of 4.4 pkat/mg toward [1β,2β,3β-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a K m of 0.82 μM and V max of 52,500 pkat/mg toward [1β,2β,3β-3H3]GA20. Conclusion Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.

International audience; The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

The Arabidopsis gene BIG (formerly DOC1/TIR3/UMB1/ASA1) is known to encode a huge calossin-like protein that is required for polar auxin transport (PAT). Mutations at this locus, in addition to reducing PAT, can alter the sensitivity of plants to several hormones and light. The tir3-1 allele of BIG reduces the response of plants to application of the gibberellin (GA) precursors ent-kaurenoic acid and GA12 and its semidwarf phenotype is partially reversed by C19-GAs. The effects of auxin transport inhibitors (ATIs) on GA 20-oxidation was examined in wild-type and tir3-1 seedlings. 1-N-naphthylphthalamic acid (NPA) and triiodobenzoic acid lead to overexpression of the GA-biosynthetic gene AtGA20ox1 comparable in magnitude to the overexpression observed in seedlings treated with paclobutrazol, a GA biosynthesis inhibitor. In contrast to that of AtGA20ox1, overexpression of AtGA20ox2 is pronounced only in paclobutrazol-treated Col and Ler, and is less in tir3-1 and in all NPA-treated seedlings. Thus the effects of BIG and ATIs on the expression of genes encoding GA 20-oxidases are complex, and suggest that at least in some tissues ATIs, directly or indirectly, may reduce the level of bioactive GA and/or alter GA signal transduction.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC–MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, α-humulene, and β-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC–MS analysis of products from of 3H- and 2H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

A gibberellin 20-oxidase gene from pumpkin and the rolC gene from Agrobacterium rhizogenes were introduced into Solanum dulcamara using A. tumefaciens-mediated delivery. Southern hybridisation confirmed the presence of transgenes in kanamycin-resistant regenerants. The phenotypes of transgenic plants indicated significant alterations in development, especially in relation to stature. Dwarfism in transgenic S. dulcamara is discussed in relation to GA metabolism.

A major catabolic pathway for the gibberellins (GAs) is initiated by 2β-hydroxylation, a reaction catalyzed by 2-oxoglutarate-dependent dioxygenases. To isolate a GA 2β-hydroxylase cDNA clone we used functional screening of a cDNA library from developing cotyledons of runner bean ( Phaseolus coccineus L.) with a highly sensitive tritium-release assay for enzyme activity. The encoded protein, obtained by heterologous expression in Escherichia coli , converted GA 9 to GA 51 (2β-hydroxyGA 9 ) and GA 51 -catabolite, the latter produced from GA 51 by further oxidation at C-2. The enzyme thus is multifunctional and is best described as a GA 2-oxidase. The recombinant enzyme also 2β-hydroxylated other C 19 -GAs, although only GA 9 and GA 4 were converted to the corresponding catabolites. Three related cDNAs, corresponding to gene sequences present in Arabidopsis thaliana databases, also encoded functional GA 2-oxidases. Transcripts for two of the Arabidopsis genes were abundant in upper stems, flowers, and siliques, but the third transcript was not detected by Northern analysis. Transcript abundance for the two most highly expressed genes was lower in apices of the GA-deficient ga1–2 mutant of Arabidopsis than in wild-type plants and increased after treatment of the mutant with GA 3 . This up-regulation of GA 2-oxidase gene expression by GA contrasts GA-induced down-regulation of genes encoding the biosynthetic enzymes GA 20-oxidase and GA 3β-hydroxylase. These mechanisms would serve to maintain the concentrations of biologically active GAs in plant tissues.

Er-doped SiO2 powders were prepared by a simple and straightforward sol–gel method using tetraethoxysilane and erbium triflate as sources of SiO2 and Er2O3. Excitation with near-infrared 800 and 980 nm light initiated an energy up-conversion process in these materials, which resulted in the emission in the predominantly red and green parts of the spectrum. Laser power dependence of the intensity of the up-converted emission was studied both experimentally and by using a numerical model to understand the up-conversion mechanism. Excited state absorption (ESA) and five unique ion–ion energy transfer up-conversion (ETU) processes between the relevant levels are identified as possible mechanisms for the observed luminescence. The efficient NIR-visible upconversion was observed even at comparatively low powers of 200 mW cm−2.

The optical efficiency of GaN-based multiple quantum well (MQW) and light emitting diode (LED) structures grown on Si(111) substrates by metal-organic vapor phase epitaxy was measured and compared with equivalent structures on sapphire. The crystalline quality of the LED structures was comprehensively characterized using x-ray diffraction, atomic force microscopy, and plan-view transmission electron microscopy. A room temperature photoluminescence (PL) internal quantum efficiency (IQE) as high as 58% has been achieved in an InGaN/GaN MQW on Si, emitting at 460 nm. This is the highest reported PL-IQE of a c-plane GaN-based MQW on Si, and the radiative efficiency of this sample compares well with similar structures grown on sapphire. Processed LED devices on Si also show good electroluminescence (EL) performance, including a forward bias voltage of ∼3.5 V at 20 mA and a light output power of 1 mW at 45 mA from a 500×500 μm2 planar device without the use of any additional techniques to enhance the output cou...

A functional analysis of the promoter from the wheat alpha-amylase gene alpha-Amy2/54 is described. Mutant alpha-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene beta-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and alpha-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the alpha-Amy2/54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal (-55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid.

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K m for ribulose-1,5-bisphosphate from 82 to 190 μM but its replacement with glutamine, which has polar properties, had no appreciable effect.

(20R)- and (20S)-[20- 2 H 1 ]-gibberellins A 15 and A 44 have been used to determine the stereochemistry of the conversion to the 20-aldehyde catalysed by gibberellin 20-oxidases.

The structural genes for three forms of Rubisco have been isolated from bacteria and introduced into various plasmids. Apart from details of the sequences which have been obtained from these constructs, they are now being exploited for mutagenesis to determine the identity and specific function of the individual amino acid residues that compose the active site. These methods have been applied to a plasmid that contains the structural gene for the simplest form of Rubisco from Rhodospirillum rubrum to obtain mutant enzymes with altered activity. The construct pRR2119 is also expressed to very high levels in Escherichia coli and enough recombinant protein of both the wild-type and m utant enzymes can be obtained for detailed physico-chemical studies. Other vectors have now been constructed, containing the genes of prokaryotic Rubisco that assemble into an active form I enzyme. The levels of expression are acceptable and the product is similar to the authentic enzyme. These constructs are now being used for mutagenesis in vitro to attempt to alter the relative rates of the oxygenase and carboxylase activities.

The genes for the ribosomal RNAs and for 14 chloroplast proteins have been identified and located on tomato plastid DNA by hybridisation of probes from pea, wheat or spinach chloroplast DNA. These include the genes for the large subunit of RuBP carboxylase, ATP synthase subunits alpha, beta, epsilon, I and III, photosystem I reaction centre P700, photosystem II subunits 44 kDa, 51 kDa, D2 and 32 kDa and cytochromeb-f complex subunits cytochromeb-563, cytochromef and 15 kDa polypeptide. The identity of eight of these genes was confirmed by coupled transcription-translation of plasmids and immunoprecipitation by specific antibodies. The locations of the genes are similar to their positions on spinach and tobacco plastid DNA.

Pea chloroplast DNA and cloned restriction fragments of pea chloroplast DNA have been used as templates in a cell-free coupled transcription-translation system from E. coli and have been shown to direct the synthesis of a 39 kD polypeptide related to cytochrome f (37.3 kD). The product was shown to be similar to cytochrome f by immunoprecipitation with specific antibodies and by peptide mapping of the products of limited chymotrypsin digestion. The structural gene for this higher molecular weight form of cytochrome f has been located within a 3.3 kbp BglII fragment situated 18 kbp from the single 16S rRNA gene.

Tomato plastid DNA has been isolated from leaf chloroplasts andPst1 fragments cloned in the plasmid vector pUC8. Recombinant plasmids containing all but the largestPst1 fragment were identified by colony hybridisation. A restriction map of the plastid DNA for four restriction endonucleases was generated by digestion of the clonedPst1 fragments and by hybridisation to Southern blots. The plastid DNA is a circular molecule of 156.6–159.4 kbp with a large inverted repeat, and shows high conservation of restriction sites with plastid DNA from tobacco andPetunia.

The gene for the 15.2 kDa polypeptide of the pea cytochrome b-f complex has been located in pea chloroplast DNA by coupled transcription-translation of fragments of chloroplast DNA in a cell-free system from Escherichia coli. The gene is located 36 kbp away from the gene for cytochrome f and is transcribed from the opposite strand. Nucleotide sequence analysis of an 850 bp MspI fragment containing the gene reveals an open reading frame coding for a hydrophobic protein of 139 amino acid residues (M, 15266). A sequence of 50 amino acid residues in the middle of the 15.2 kDa polypeptide shows homology with a sequence near the C-terminus of mitochondrial cytochrome b.

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

A cytochrome b–f complex has been extracted from pea chloroplast from pea chloroplast membranes with octyl glucoside and cholate and has been purified by ammonium sulphate fractionation and polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium deoxycholate. The complex contains cytochromes f and b-563 in a ratio of 1:2, but shows very low levels of the Rieske iron sulphur centre and plastoquinol-plastocyanin oxidoreductase activity. The complex consists of four polypeptides of molecular weights 37300, 34000, 19500 and 15200. The complex is dissociated by electrophoresis in the presence of 2M urea to give pure preparations of cytochrome f and cytochrome b-563, which on sodium dodecylsulphate-polyacrylamide gel electrophoresis give single bands of apparent molecular weights 37300 and 19500 respectively.

Three components of the cytochrome b-f complex, cytochrome f, cytochrome b-563 and a 15.2-kDa polypeptide, were labelled with radioactive amino acids in isolated pea chloroplasts incubated in the light with added Mg-ATP. The assembly of cytochrome b-563 (19.5 kDa) into the cytochrome complex required the presence of added Mg-ATP whereas cytochrome f (37.3 kDa) and the 15.2-kDa polypeptide were assembled in its absence. Incorporation of [35S]methionine into the polypeptide chain of cytochrome b-563 and the 15.2-kDa component was confirmed by peptide mapping of the products of partial digestion with papain.

DNA from pea and tobacco chloroplasts hybridizes specifically with probes derived from the gene for a membrane component, the a subunit of ATP synthase of the cyanobacterium, Synechococcus 6301. DNA sequence of the hybridizing region of the pea plastid DNA has revealed that it encodes a protein of 247 amino acids related in sequence to the a subunits of ATP synthase of Escherichia coli, Synechococcus and mitochondria. This is the sixth component of chloroplast ATP synthase that is plastid coded. The gene is located upstream from the genes for three other ATP synthase subunits and a transcript of 6 kb contains coding sequences from each of these genes. Thus the subunit a gene is part of a co-transcribed cluster of four ATP synthase genes arranged in the order a:c(or III):b(or I):alpha. Two other ATP synthase genes, those for beta and epsilon subunits, are known to form a separate cluster. These gene arrangements are most closely related to those found in the cyanobacterium, Synechococcus 6301. Hence, this finding provides strong evidence for a common origin for cyanobacteria and plant chloroplasts.

Purified Fab fragments of digoxin-specific antibodies obtained from sheep were used to treat 26 patients with advanced, life-threatening digoxin (23 cases) or digitoxin (3 cases) toxicity. These patients had advanced cardiac arrhythmias, and in some cases hyperkalemia, which were resistant to conventional treatment. All patients had an initial favorable response to doses of Fab fragments calculated (in most cases) to be equivalent, on a molar basis, to the amount of cardiac glycoside in the patient's body. In four patients treated after prolonged hypotension and low cardiac output, death ensued from cerebral or myocardial hypoperfusion. In one case the available Fab fragment supply was inadequate to reverse a massive suicidal ingestion of digoxin, and the patient died after recurrent ventricular arrhythmias. In the remaining 21 patients, cardiac rhythm disturbances and hyperkalemia were rapidly reversed, and full recovery ensued. There were no adverse reactions to the treatment. We conclude that the use of purified digoxin-specific Fab fragments is a safe and effective means to reverse advanced, life-threatening digitalis intoxication.

Ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39, Rubisco) catalyses both the carboxylation and oxygenation of ribulose 1,5-bisphosphate (RuBP) with CO2 and O2 acting as competitive alternative substrates. The oxygenation of the RuBP results in the formation of phosphoglycollate and initiates a metabolic pathway that releases CO2 during a step in which glycine is converted to serine. The exchange of CO2 and O2 between plants and the atmosphere resulting from this pathway is, by one definition, photorespiration. In C4 plants, and in certain algae and cyanobacteria, photorespiration is controlled by the ability of the organisms to concentrate CO2 in the cell compartment containing the Rubisco and thereby overcoming the inhibitory effect of oxygen and decreasing the extent of the competitive reaction of the RuBP with oxygen. The concentrating mechanisms require energy to drive them and, in C4 plants, a more elaborate leaf structure. Possibly these added requirements prevent such mechanisms from having a selective advantage in most land plants adapted to cooler climates.

The cytochrome b-f complex of higher plant chloroplasts is a multi-subunit protein complex located in the thylakoid membrane and forms part of the electron transport chain between the photosystems. The complex isolated from spinach chloroplasts has five polypeptides of 34, 33, 23.5, 20 and 17.5 kDa (Hurt, Hauska 1981). The 34 and 33 kDa polypeptides are reported as being two forms of cytochrome f, the 23.5 and 20 kDa components as cytochrome b-563 and the Rieske FeS centre respectively, and the 17.5 kDa component has no known redox function (Hurt, Hauska 1982). All chloroplast multisubunit complexes hitherto examined have at least one component which is plastid-synthesised, the remainder being products of cytoplasmic protein synthesis (Ellis 1981). It is known that cytochrome f is synthesised in isolated pea chloroplasts (Doherty, Gray 1979), but the site of synthesis of the other components has not been established. We have purified the cytochrome complex from pea (Pisum sativum cv. Feltham First) thylakoids in order to examine the sites of synthesis of each of the components, and to investigate the location of genes for components.

The potential for exploitation of the gibberellin (GA) plant growth hormones in agriculture was a major factor driving research on the physiological function of these substances following their introduction to the West from Japan in the 1950s. As a consequence of its vital role in promoting organ growth as well as in stimulating seed germination and reproductive development, this class of hormone has assumed considerable importance as a plant growth regulator (PGR) and a wide range of applications of GAs are known. However, in terms of treated area and sales, the commercial utilisation of GAs is dwarfed by that of the growth retardants, which, as inhibitors of GA biosynthesis, function by reducing the bioactive GA content of treated plants, thereby reducing shoot elongation. Several inhibitors of GA biosynthesis are of practical relevance with important uses. The annual global PGR market accounts for approximately €700 million, of which some 50% can be assumed to be represented by growth retardants. Area- and value-wise, stem shortening in small grains and in rice production to reduce the risk of lodging is the main application of PGRs worldwide. It is estimated that some 30% of global PGR sales (equalling approximately €210 million) are represented by stem stabilisers. In addition to inhibitors of GA biosynthesis, ethylene-releasing ethephon is also used for this purpose, particularly in barley. The usage of such products is general practice in countries with intense production of wheat, barley, rye, triticale, and oats such as France, Germany and Great Britain. For instance, 92% of the winter wheat, 81% of the winter barley, 67% of the oats, and 100% of the rye acreage were treated with anti-lodging products in Great Britain in 2008. Despite this, it has been estimated that losses due to lodging cost the British wheat industry about €50 million per year and these costs are likely to be even higher to date. The application of a PGR allows for an active regulation of developmental processes. For example, control of stem elongation in cereals may be “fine-tuned” according to need, both in terms of affecting the right growth stage (by timing of treatment) and the intensity of growth reduction (by dosage). Also, but within limits, distinct parts of a plant may be targeted. It can be counter-productive in cereal production if, in addition to shoot growth, root growth is also reduced. This can be avoided by spray-applying non-systemic compounds such as prohexadione-Ca. Thus, in terms of flexibility, immediate growth control and potential specificity, PGRs can offer advantages over conventional breeding and genetic engineering approaches. On the other hand, there is wide-spread concern about the use of chemicals in crop production. In order to comply with the principles of integrated crop production better, it would be preferable if problems solved with presently existing PGRs could be overcome by introducing improved genotypes and/or by making available new and more specific PGRs with an even further reduced risk to the grower, environment and consumer. The introduction of semi-dwarfing genes into wheat and rice in the 1960s played an important role in the Green Revolution that resulted in dramatic increases in crop yield. Shorter, more stable stems were necessary to avoid lodging of the crop under high levels of nitrogenous fertiliser, but semidwarfism also improved yields by increasing grain numbers, probably by allowing more assimilate to partition into the developing spike. The dwarfing genes in both rice and wheat act on the GA-signalling system: semi-dwarf rice contains mutations in a GA-biosynthesis gene that result in reduced GA content while the reduced height (Rht) mutation in wheat compromises the ability of GA to stimulate growth. In view of the importance of the GA signalling pathway in regulating physiological processes of relevance to agriculture, it is a prime target in crop improvement programmes. To date, most interest has been focussed on the GA-biosynthetic and inactivation pathways that determine GA concentration since they have been more clearly understood than the mechanisms for GA perception and signal transduction. A number of studies describing the genetic modification of GA metabolism, either to enhance or restrict plant growth, have been published. For example, increasing GA content by ectopic expression of GA 20-oxidase genes, which encode a rate-limiting enzyme in GA biosynthesis, has been shown to result in higher biomass in tobacco and in longer fibres in aspen, resulting in improved paper-making quality. Conversely, ectopic expression of GA 2-oxidase genes, which encode GA-inactivating enzymes, provides a particularly efficient method for introducing dwarfism. These approaches are clearly effective and even though breeding by genetic engineering is not publicly acceptable in a number of countries, they highlight potential gene targets for breeding to improve plant architecture or yield. Recently, the range of targets has been potentially broadened by impressive advances in our understanding of GA signal transduction.

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C(19)-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C(19)-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA(1) content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA(1) content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.

The generation of new ideas and scientific hypotheses is often the result of extensive literature and database searches, but, with the growing wealth of public and private knowledge, the process of searching diverse and interconnected data to generate new insights into genes, gene networks, traits and diseases is becoming both more complex and more time‐consuming. To guide this technically challenging data integration task and to make gene discovery and hypotheses generation easier for researchers, we have developed a comprehensive software package called KnetMiner which is open‐source and containerized for easy use. KnetMiner is an integrated, intelligent, interactive gene and gene network discovery platform that supports scientists explore and understand the biological stories of complex traits and diseases across species. It features fast algorithms for generating rich interactive gene networks and prioritizing candidate genes based on knowledge mining approaches. KnetMiner is used in many plant science institutions and has been adopted by several plant breeding organizations to accelerate gene discovery. The software is generic and customizable and can therefore be readily applied to new species and data types; for example, it has been applied to pest insects and fungal pathogens; and most recently repurposed to support COVID‐19 research. Here, we give an overview of the main approaches behind KnetMiner and we report plant‐centric case studies for identifying genes, gene networks and trait relationships in Triticum aestivum (bread wheat), as well as, an evidence‐based approach to rank candidate genes under a large Arabidopsis thaliana QTL. KnetMiner is available at: https://knetminer.org.

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1–2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1–2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

The identification of most of the genes involved in the metabolic pathways for gibberellin hormones has helped us to understand these pathways and their regulation. Many of these enzymes are multifunctional and therefore fewer enzymes than might be expected are required to synthesize the various gibberellin structures. However, several of the enzymes are encoded by multiple genes that are regulated differently, adding unexpected genetic complexity. Several endogenous and environmental factors modify the expression of gibberellin biosynthesis genes, including developmental stage, hormonal status and light. A future challenge will be to dissect the complex, interacting pathways that mediate the regulation of gibberellin metabolism.&nbsp

Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.

Highlights • How wheat responds to physical and nutritional stresses was investigated. • An evident interaction between mechanical impedance and phosphorus supply were observed. • Mechanical impedance restricted wheat growth under sufficient phosphorus supply • Wheat responses to mechanical impedance were reduced under low phosphorus supply • Tall and dwarf wheat genotypes performed similarly under impedance and P stresses., Increased mechanical impedance induced by soil drying or compaction causes reduction in plant growth and crop yield. However, how mechanical impedance interacts with nutrient stress has been largely unknown. Here, we investigated the effect of mechanical impedance on the growth of wheat seedlings under contrasting phosphorus (P) supply in a sand culture system which allows the mechanical impedance to be independent of water and nutrient availability. Two wheat genotypes containing the Rht-B1a (tall) or Rht-B1c (gibberellin-insensitive dwarf) alleles in the Cadenza background were used and their shoot and root traits were determined. Mechanical impedance caused a significant reduction in plant growth under sufficient P supply, including reduced shoot and root biomass, leaf area and total root length. By contrast, under low P supply, mechanical impedance did not affect biomass, tiller number, leaf length, and nodal root number in both wheat genotypes, indicating that the magnitude of the growth restriction imposed by mechanical impedance was dependent on P supply. The interaction effect between mechanical impedance and P level was significant on most plant traits except for axial and lateral root length, suggesting an evident physical and nutritional interaction. Our findings provide valuable insights into the integrated effects of plants in response to both soil physical and nutritional stresses. Understanding the response patterns is critical for optimizing soil tillage and nutrient management in the field.

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C(19)-GAs, including the bioactive GAs GA(1 )and GA(4), by 2beta-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA(1) and GA(4). Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds.