Growth arrest by trehalose-6-phosphate: an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway

Abstract

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.

Figure 1.

Carbon allocation, growth, and trehalase activity of seedlings on 100 mm trehalose. Seedlings were grown under long-day conditions for 14 d on medium with 100 mm sorbitol osmoticum control (sorb) or trehalose (tre). A, Starch staining. Seedlings were harvested at midday, stained with Lugol, and mounted in chloral hydrate. WT, Wild-type Col-0 seedlings; pgm1, seedlings lacking Plastidic Phosphoglucomutase1 (Caspar et al., 1985); 93-1, seedlings from FOX line 93-1. Bars = 3 mm. B, Root lengths. Average root lengths from more than 20 seedlings of the different genotypes with sd are shown. treF, Seedlings overexpressing E. coli trehalase treF; 128, 89-1, 89-3, 33-1G, 33-1, 93-1, and 93-32, lines from the FOX collection of FOX pools 128, 89, 33, and 93. C, Trehalase activity in extracts of seedlings grown on trehalose for 14 d. The data are averages with sd of three independent extracts. * P < 0.050 by ANOVA.

Figure 2.

Characterization of the independent FOX lines expressing bZIP11 cDNA from pools 33, 93, and 70. A, Trehalose resistance is a dominant trait, as shown for lines 33-1 and 93-1. P, Seedlings from the parental lines: WT, the wild type; 33-1, FOX line 33-1; 93-1, FOX line 93-1. F1 represents the first generation from FOX line crosses with the wild type. B, PCR amplification of the FOX cDNA using DNA template from plants of lines 70, 128, 89, 93-1, and 33-1. MW, Molecular weight marker λ Pst1. C, Expression of bZIP11 in 14-d-old seedlings from FOX lines 70 and 93, the line expressing the E. coli trehalase treF (treF), and wild-type Col-0. D, Expression of the TRE1 trehalase in the genotypes from C. E, Seedlings with altered expression of bZIP1 do not grow on medium with 100 m trehalose (tre) compared with sorbitol (sorb). bZIP1oe and bZIP1as, bZIP1-overexpressing and antisense lines, as described by Kang et al. (2010). F, Expression of bZIP1 in seedlings of FOX lines 70 and 93, the line expressing treF, and the wild type grown on medium with sorbitol or trehalose. Expression was determined by Q-PCR and is given relative to PP2A (At1g13320). Error bars represent sd of three replicates.

Figure 3.

The roles of trehalase (TRE1) and of uORF2 in the FOX lines growing on trehalose. Error bars represent sd of three replicates. A, Expression of TRE1 in wild-type Col-0 (WT) and seedlings from two different plants of the tre1-1 line (Salk 147073c). B, Trehalase activity in flowers from the wild type and several plants from the tre1-1 line (P1–P4). C, tre1-1 and tre1-2 seedling growth compared with their respective wild types. Growth was on MS medium without (MS) or with 25 and 50 mm trehalose (25 mm T and 50 mm T). D, Genotype analysis of long root seedlings in the F2 generation of the cross 93-1 with the wild type. DNA from the wild type and 11 different seedlings (1–10) was used as the template. PCR was carried out to amplify the wild-type sequence of TRE1 (WT TRE1) or the T-DNA insertion at the TRE1 locus (KO TRE1) on the top agarose gel (tre1). PCR was also carried out to amplify the FOX cDNA on the bottom gel (Fox cDNA). E, Unlike on Suc, translational repression of bZIP11 does not occur on trehalose. Seedlings expressing the 5′ mRNA uORFs of the bZIP11 mRNA fused to the GUS gene were grown for 7 d on MS medium, transferred for 48 h to medium with Suc (SUC) or trehalose (TRE) at 0, 20, or 100 mm (0, 20, and 100), and then stained for GUS activity.

Figure 4.

T6P accumulation and in vitro T6P inhibition of SnRK1 in seedlings grown on trehalose. WT, Wild-type Col-0; TreF, seedlings expressing E. coli trehalase treF; 93, 70, and 33, seedlings from FOX lines 93, 70, and 33, respectively; FW, fresh weight. A, Seedlings grown on osmoticum control for 14 d (100 mm sorbitol [sorb]). B, Seedlings grown on 100 mm trehalose for 14 d. C, SnRK1 activity assayed using the AMARA peptide in 14-d-old seedlings grown on 100 mm trehalose from wild-type accession Col-0, FOX lines 93 and 33, and wild-type accession Ler. Error bars represent se of three biological replicates.

Figure 5.

KIN10-overexpressing seedlings grow on trehalose without increased trehalase or bZIP11 expression. Seedlings were grown on trehalose (tre) compared with osmoticum control (sorbitol [sorb]) for 14 d. A, Phenotype of the Ler wild type (WT) and KIN10 overexpression line O1. B, Trehalase activity in extracts from the Ler wild type and the lines overexpressing KIN10 (O1 and O2). C, TRE1 expression determined relative to PP2A by Q-PCR in the genotypes from B. D, bZIP11 expression. The levels are averages of three biological replicates, and error bars represent sd.

Figure 6.

Do KIN10 and bZIP11 act in the same pathway? A, Expression of targets common to KIN10 and bZIP11. Seedlings of the wild type were grown for 14 d on 100 mm either sorbitol (sorb) or trehalose (tre) and collected at midday. Expression was determined by Q-PCR relative to PP2A, then normalized to the level of expression on sorbitol. B, Soluble sugars Suc, Glc, and Fru in the seedlings with the genotype wild type (Col-0), treF expressors (treF), bZIP11 expressors from line 93 (93) and 33 (33), wild-type Ler (Ler), and KIN10-overexpressing lines O1 and O2. In A and B, levels are averages of three biological replicates, and error bars represent sd. FW, Fresh weight. C, Phenotypes of bZIP11-expressing seedlings on trehalose in continuous darkness. After 78 h at 4°C, seed were exposed to light and 22°C for 6 h, then grown for 14 d in continuous darkness on medium with 100 mm either sorbitol or trehalose. WT, Wild type Col-0; 33-1-1 and 33-1-2, seeds from two plants of FOX line 33-1; 93-3-2, seeds from FOX line 93. D, Phenotype of KIN10-expressing seedlings on trehalose in continuous darkness. Seeds were treated as in C. WT, Wild type Ler; KIN10oe, seeds from the O2 line (Baena-González et al., 2007).

Figure 7.

A, Model of interactions affecting growth and starch accumulation on trehalose when T6P accumulates. Glc and Suc feeding cause AGPase redox activation and thus starch synthesis by different pathways that are likely also relevant for the growth responses to these sugars (Tiessen et al., 2003; Michalska et al., 2009; Geigenberger, 2011). When feeding Glc, T6P does not accumulate (T.L. Delatte, P. Sedijani, and H. Schluepmann, unpublished data), and Glc-6-P (G6P) in plastids is shunted through the oxidative part of the pentose phosphate pathway (OPP), generating NADPH for NADPH-thioredoxin reductase C (NTRC)-dependent reduction of AGPase and thus activation. In contrast, feeding Suc or trehalose leads to T6P increase, which acts upon AGPase redox by an unknown mechanism (Schluepmann et al., 2004; Kolbe et al., 2005; Lunn et al., 2006; Michalska et al., 2009). Suc inhibits the translation of bZIP11 by way of uORF2 (Wiese et al., 2004), but trehalose does not. When feeding trehalose, T6P accumulates. T6P accumulation inhibits SnRK1; this inhibition of SnRK1 depends on an intermediary factor I, present in young tissues (Zhang et al. 2009). Possibly, SnRK1 phosphorylation activates bZIP11 transfer to the nucleus or complexing of the transcription factor in such a way that bZIP11 controls part of the SnRK1 output that is required for growth. Thus, when T6P accumulates and inhibits SnRK1 in young tissues, overexpression of bZIP11 may act as a surrogate for SnRK1. B, Antisense SnRK1 restricted to individual pollen of barley in particular (Zhang et al., 2001) but also work in developing potato tubers (Purcell et al., 1998) show that SnRK1 is required in growing heterotrophic cells for growth and starch accumulation. Therefore, it is possible that SnRK1 is needed to respond to nutrient stress so as to make carbon available in growing sinks. SnRK1 inhibition (by artificially increasing T6P when feeding trehalose or by antisense SnRK1) would thus uncouple growth from carbon starvation responses, leading to the swollen cells observed in the growing zones of roots of Arabidopsis seedlings on trehalose (tre) compared with sorbitol (sorb). Root tips were stained with propidium iodide in water immediately prior to visualization with the confocal microscope. [See online article for color version of this figure.]