RESUMO
The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a 'signalling-precursor' concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function.
RESUMO
Transgenic maize (Zea mays) that expresses rice (Oryza sativa) TREHALOSE PHOSPHATE PHOSPHATASE1 (TPP1) from the rice MADS6 promoter, which is active over the flowering period, produces higher yields than wild type. This yield increase occurs with or without drought conditions during flowering. To understand the mechanistic basis of the increased yield, we characterized gene expression and metabolite profiles in leaves and developing female reproductive tissue, comprising florets, node, pith, and shank, over the flowering period with and without drought. The MADS6 promoter was most active in the vasculature, particularly phloem companion cells in florets and pith, consistent with the largest decreases in trehalose 6-phosphate (T6P) levels (2- to 3-fold) being found in pith and florets. Low T6P led to decreased gene expression for primary metabolism and increased gene expression for secondary metabolism, particularly lipid-related pathways. Despite similar changes in gene expression, the pith and floret displayed opposing assimilate profiles: sugars, sugar phosphates, amino acids, and lipids increased in florets, but decreased in pith. Possibly explaining this assimilate distribution, seven SWEET genes were found to be up-regulated in the transgenic plants. SnRK1 activity and the expression of the gene for the SnRK1 beta subunit, expression of SnRK1 marker genes, and endogenous trehalose pathway genes were also altered. Furthermore, leaves of the transgenic maize maintained a higher photosynthetic rate for a longer period compared to wild type. In conclusion, we found that decreasing T6P in reproductive tissues down-regulates primary metabolism and up-regulates secondary metabolism, resulting in different metabolite profiles in component tissues. Our data implicate T6P/ SnRK1 as a major regulator of whole-plant resource allocation for crop yield improvement.
Assuntos
Flores/metabolismo , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/genética , Floema/genética , Floema/crescimento & desenvolvimento , Floema/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transgenes/genética , Trealose/metabolismo , Zea mays/enzimologia , Zea mays/genéticaRESUMO
Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g(-1) fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metabolismo dos Carboidratos , Carboidratos , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Nitrogênio , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Plântula , Sacarose/metabolismo , Sacarose/farmacologia , Trealose/metabolismoRESUMO
Trehalose 6-phosphate (T6P) is an important regulator of plant metabolism and development. T6P content increases when carbon availability is high, and in young growing tissue, T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Here, strong accumulation of T6P was found in senescing leaves of Arabidopsis (Arabidopsis thaliana), in parallel with a rise in sugar contents. To determine the role of T6P in senescence, T6P content was altered by expressing the bacterial T6P synthase gene, otsA (to increase T6P), or the T6P phosphatase gene, otsB (to decrease T6P). In otsB-expressing plants, T6P accumulated less strongly during senescence than in wild-type plants, while otsA-expressing plants contained more T6P throughout. Mature otsB-expressing plants showed a similar phenotype as described for plants overexpressing the SnRK1 gene, KIN10, including reduced anthocyanin accumulation and delayed senescence. This was confirmed by quantitative reverse transcription-polymerase chain reaction analysis of senescence-associated genes and genes involved in anthocyanin synthesis. To analyze if the senescence phenotype was due to decreased sugar sensitivity, the response to sugars was determined. In combination with low nitrogen supply, metabolizable sugars (glucose, fructose, or sucrose) induced senescence in wild-type and otsA-expressing plants but to a smaller extent in otsB-expressing plants. The sugar analog 3-O-methyl glucose, on the other hand, did not induce senescence in any of the lines. Transfer of plants to and from glucose-containing medium suggested that glucose determines senescence during late development but that the effects of T6P on senescence are established by the sugar response of young plants.
Assuntos
Arabidopsis/fisiologia , Metabolismo dos Carboidratos , Folhas de Planta/fisiologia , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Antocianinas/genética , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Meios de Cultura/metabolismo , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Flores/fisiologia , Glucose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trealose/metabolismoRESUMO
Trehalose metabolism and signaling is an area of emerging significance. In less than a decade our views on the importance of trehalose metabolism and its role in plants have gone through something of a revolution. An obscure curiosity has become an indispensable regulatory system. Mutant and transgenic plants of trehalose synthesis display wide-ranging and unprecedented phenotypes for the perturbation of a metabolic pathway. Molecular physiology and genomics have provided a glimpse of trehalose biology that had not been possible with conventional techniques, largely because the products of the synthetic pathway, trehalose 6-phosphate (T6P) and trehalose, are in trace abundance and difficult to measure in most plants. A consensus is emerging that T6P plays a central role in the coordination of metabolism with development. The discovery of trehalose metabolism has been one of the most exciting developments in plant metabolism and plant science in recent years. The field is fast moving and this review highlights the most recent insights.
Assuntos
Plantas/genética , Plantas/metabolismo , Trealose/fisiologia , Dissacarídeos/química , Dissacarídeos/metabolismo , Modelos Moleculares , Mutação , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Transdução de Sinais , Sacarose/metabolismo , Trealose/metabolismo , Leveduras/metabolismoRESUMO
The strong regulation of plant carbon allocation and growth by trehalose metabolism is important for our understanding of the mechanisms that determine growth and yield, with obvious applications in crop improvement. To gain further insight on the growth arrest by trehalose feeding, we first established that starch-deficient seedlings of the plastidic phosphoglucomutase1 mutant were similarly affected as the wild type on trehalose. Starch accumulation in the source cotyledons, therefore, did not cause starvation and consequent growth arrest in the growing zones. We then screened the FOX collection of Arabidopsis (Arabidopsis thaliana) expressing full-length cDNAs for seedling resistance to 100 mm trehalose. Three independent transgenic lines were identified with dominant segregation of the trehalose resistance trait that overexpress the bZIP11 (for basic region/leucine zipper motif) transcription factor. The resistance of these lines to trehalose could not be explained simply through enhanced trehalase activity or through inhibition of bZIP11 translation. Instead, trehalose-6-phosphate (T6P) accumulation was much increased in bZIP11-overexpressing lines, suggesting that these lines may be insensitive to the effects of T6P. T6P is known to inhibit the central stress-integrating kinase SnRK1 (KIN10) activity. We confirmed that this holds true in extracts from seedlings grown on trehalose, then showed that two independent transgenic lines overexpressing KIN10 were insensitive to trehalose. Moreover, the expression of marker genes known to be jointly controlled by SnRK1 activity and bZIP11 was consistent with low SnRK1 or bZIP11 activity in seedlings on trehalose. These results reveal an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway involving T6P, SnRK1, and bZIP11.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , DNA Complementar , Plantas Geneticamente Modificadas , Biossíntese de Proteínas , Trealose/metabolismoRESUMO
Trehalose 6-phosphate (T6P) is a sugar signal that regulates metabolism, growth, and development and inhibits the central regulatory SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11). To better understand the mechanism in wheat (Triticum aestivum) grain, we analyze T6P content and SnRK1 activities. T6P levels changed 178-fold 1 to 45 d after anthesis (DAA), correlating with sucrose content. T6P ranged from 78 nmol g(-1) fresh weight (FW) pregrain filling, around 100-fold higher than previously reported in plants, to 0.4 nmol g(-1) FW during the desiccation stage. In contrast, maximum SnRK1 activity changed only 3-fold but was inhibited strongly by T6P in vitro. To assess SnRK1 activity in vivo, homologs of SnRK1 marker genes in the wheat transcriptome were identified using Wheat Estimated Transcript Server. SnRK1-induced and -repressed marker genes were expressed differently pregrain filling compared to grain filling consistent with changes in T6P. To investigate this further maternal and filial tissues were compared pre- (7 DAA) and during grain filling (17 DAA). Strikingly, in vitro SnRK1 activity was similar in all tissues in contrast to large changes in tissue distribution of T6P. At 7 DAA T6P was 49 to 119 nmol g(-1) FW in filial and maternal tissues sufficient to inhibit SnRK1; at 17 DAA T6P accumulation was almost exclusively endospermal (43 nmol g(-1) FW) with 0.6 to 0.8 nmol T6P g(-1) FW in embryo and pericarp. The data show a correlation between T6P and sucrose overall that belies a marked effect of tissue type and developmental stage on T6P content, consistent with tissue-specific regulation of SnRK1 by T6P in wheat grain.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Sementes/metabolismo , Sacarose/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Triticum/metabolismo , Sequência de Aminoácidos , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Especificidade de Órgãos , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Sacarose/análise , Fosfatos Açúcares/análise , Trealose/análise , Trealose/metabolismo , Triticum/genética , Triticum/crescimento & desenvolvimentoRESUMO
The composition of proanthocyanidins in the testa (seed coat) of bread wheat was analyzed by thiolysis of PA oligomers from developing grain and found to consist of (+)-catechin monomers, with a small amount of (+)-gallocatechin. The average chain length of soluble PA stayed relatively constant between 10 and 20 days post-anthesis, whereas that of unextractable PA increased over the same period, suggesting that increases in chain length might account for the insolubility of PAs from mature wheat grain. We carried out RNA-Seq followed by differential expression analysis from dissected tissues of developing grain from red- and white-grained near-isogenic lines differing in the presence of an active R gene that encodes a MYB transcription factor involved in control of PA biosynthesis. In addition to genes already identified encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and dihydroxyflavonoid 4-reductase, we showed that wheat genes encoding phenylalanine ammonia lyase, flavonoid 3',5'-hydroxylase, leucoanthocyanidin reductase, and a glutathione S-transferase (the orthologue of maize Bronze-2) were more highly expressed in the red NIL. We also identified candidate orthologues of other catalytic and regulatory components of flavonoid biosynthesis in wheat.
RESUMO
Like most angiosperms, wheat (Triticum aestivum) shows maternal inheritance of plastids. It is thought that this takes place by cytoplasmic stripping at fertilisation rather than the absence of plastids in sperm cells. To determine the fate of plastids during sperm cell development, plastid-targeted green fluorescent protein was used to visualise these organelles in nuclear transgenic wheat lines. Fewer than thirty small 1-2-µm plastids were visible in early uninucleate pollen cells. These dramatically increased to several hundred larger (4 µm) plastids during pollen maturation and went through distinct morphological changes. Only small plastids were visible in generative cells (n = 25) and young sperm cells (n = 9). In mature sperm cells, these green fluorescent protein (GFP)-tagged plastids were absent. This is consistent with maternal inheritance of plastids resulting from their degradation in mature sperm cells in wheat.
Assuntos
Plastídeos/metabolismo , Pólen/citologia , Pólen/metabolismo , Triticum/citologia , Diferenciação Celular , Proteínas de Fluorescência Verde/metabolismo , Pólen/crescimento & desenvolvimentoRESUMO
Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Zea mays/genética , Zea mays/fisiologia , Adaptação Biológica/genética , Biotecnologia , Secas , Monoéster Fosfórico Hidrolases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Zea mays/metabolismoRESUMO
SnRK1 of the SNF1/AMPK group of protein kinases is an important regulatory protein kinase in plants. SnRK1 was recently shown as a target of the sugar signal, trehalose 6-phosphate (T6P). Glucose 6-phosphate (G6P) can also inhibit SnRK1 and given the similarity in structure to T6P, we sought to establish if each could impart distinct inhibition of SnRK1. Other central metabolites, glucose 1-phosphate (G1P), fructose 6-phosphate and UDP-glucose were also tested, and additionally ribose 5-phosphate (R5P), recently reported to inhibit SnRK1 strongly in wheat grain tissue. For the metabolites that inhibited SnRK1, kinetic models show that T6P, G1P and G6P each provide distinct regulation (50% inhibition of SnRK1 at 5.4 µM, 480 µM, >1 mM, respectively). Strikingly, G1P in combination with T6P inhibited SnRK1 synergistically. R5P, in contrast to the other inhibitors, inhibited SnRK1 in green tissues only. We show that this is due to consumption of ATP in the assay mediated by phosphoribulokinase during conversion of R5P to ribulose-1,5-bisphosphate. The accompanying loss of ATP limits the activity of SnRK1 giving rise to an apparent inhibition of SnRK1. Inhibition of SnRK1 by R5P in wheat grain preparations can be explained by the presence of green pericarp tissue; this exposes an important caveat in the assessment of potential protein kinase inhibitors. Data provide further insight into the regulation of SnRK1 by metabolites.
Assuntos
Glucofosfatos/farmacologia , Proteínas de Plantas/metabolismo , Fosfatos Açúcares/farmacologia , Trealose/análogos & derivados , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ribulosefosfatos/metabolismo , Trealose/farmacologia , Triticum/efeitos dos fármacos , Triticum/metabolismoRESUMO
Trehalose 6-phosphate (T6P), the precursor of trehalose, is a signaling molecule in plants with strong effects on metabolism, growth and development. We recently showed that in growing tissues T6P is an inhibitor of SnRK1 of the SNF1-related group of protein kinases. SnRK1 acts as transcriptional integrator in response to carbon and energy supply. In microarray experiments on seedlings of transgenic Arabidopsis with elevated T6P content we found that expression of SnRK1 marker genes was affected in a manner to be predicted by inhibition of SnRK1 by T6P in vivo. A large number of genes involved in reactions that utilize carbon, e.g., UDP-glucose dehydrogenase genes involved in cell wall synthesis, were upregulated. T6P was also found to affect developmental signaling pathways, probably in a SnRK1-independent manner. This includes upregulation of genes encoding UDP-glycosyltransferases that are involved in the glycosylation of hormones. In addition, genes involved in auxin response and light signaling were affected. Many of these genes belong to pathways that link the circadian clock to plant growth and development. The overall pattern of changes in gene expression supports a role for T6P in coordinating carbon supply with biosynthetic process involved in growth and development.
RESUMO
Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1-20 microM). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Redes e Vias Metabólicas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fosfatos Açúcares/farmacologia , Trealose/análogos & derivados , Trifosfato de Adenosina/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosiltransferases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Software , Fatores de Transcrição/metabolismo , Trealose/farmacologiaRESUMO
The binding kinetics of disaccharides trehalose and trehalose-6-phosphate to repressor protein TreR have been determined using STD NMR and shed light on the contrasting biological roles of these two sugars.
Assuntos
Proteínas de Escherichia coli/química , Espectroscopia de Ressonância Magnética , Proteínas Repressoras/química , Fosfatos Açúcares/química , Trealose/análogos & derivados , Trealose/química , Sítios de Ligação , Simulação por Computador , Proteínas de Escherichia coli/metabolismo , Cinética , Ligação Proteica , Proteínas Repressoras/metabolismoRESUMO
The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.