Thierry L. Delatte

Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate

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 μm). 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.

Wheat Grain Development Is Characterized by Remarkable Trehalose 6-Phosphate Accumulation Pregrain Filling: Tissue Distribution and Relationship to SNF1-Related Protein Kinase1 Activity

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.

Growth Arrest by Trehalose-6-Phosphate: An Astonishing Case of Primary Metabolite Control over Growth by Way of the SnRK1 Signaling Pathway

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.

Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability

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.

The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation

The T6P/SnRK1 mechanism of growth regulation responds to sink growth restriction and recovery following low-temperature limitation. 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.

The sucrose-regulated Arabidopsis transcription factor bZIP11 reprograms metabolism and regulates trehalose metabolism

• The Arabidopsis basic region-leucine zipper transcription factor 11 (bZIP11) is known to be repressed by sucrose through a translational inhibition mechanism that requires the conserved sucrose control peptide encoded by the mRNA leader. The function of bZIP11 has been investigated in over-expression studies, and bZIP11 has been found to inhibit plant growth. The addition of sugar does not rescue the growth inhibition phenotype. Here, the function of the bZIP11 transcription factor was investigated. • The mechanism by which bZIP11 regulates growth was studied using large-scale and dedicated metabolic analysis, biochemical assays and molecular studies. • bZIP11 induction results in a reprogramming of metabolism and activation of genes involved in the metabolism of trehalose and other minor carbohydrates such as myo-inositol and raffinose. bZIP11 induction leads to reduced contents of the prominent growth regulatory molecule trehalose 6-phosphate (T6P). • The metabolic changes detected mimic in part those observed in carbon-starved plants. It is proposed that bZIP11 is a powerful regulator of carbohydrate metabolism that functions in a growth regulatory network that includes T6P and the sucrose non-fermenting-1 related protein kinase 1 (SnRK1).

Up-regulation of biosynthetic processes associated with growth by trehalose 6-phosphate

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 kinases1. 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 vivo1. 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.

Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 microM (r(2)>0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100mM trehalose by different mechanisms.

Regulation of growth by the trehalose pathway: relationship to temperature and sucrose.

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.