Article Figures & Data
Figures
- 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.]
Tables
- Table I. Genes jointly controlled by SnRK1 and bZIP11
Two hundred sixty-one differentially expressed genes at 2 h after nuclear transfer of bZIP11 (Hanson et al., 2008) were compared with the 1,021 genes altered by 6 h of transient KIN10 expression in protoplasts (Baena-González et al., 2007). The commonly regulated genes are listed with The Arabidopsis Information Resource annotations (May 2011).
Identifier Annotation Induced At1g02660 α/β-Hydrolase superfamily protein; putative triglyceride lipase activity At1g10070 BCAT-2 chloroplast branched-chain amino acid aminotransferase At1g18460 α/β-Hydrolase superfamily putative lipase family At1g32170 XTR4 xyloglucan endotransglycosylase-related protein At1g62510 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein At1g64660 MGL cytosolic Met γ-lyase At2g25200 Plant protein of unknown function (DUF868) At2g30600 BTB/POZ domain-containing protein; involved in cell adhesion At2g32150 Haloacid dehalogenase-like hydrolase (HAD) superfamily protein; nitrate responsive At2g33380 CALEOSIN3 calcium binding, induced by NaCl, abscisic acid, and desiccation At2g36220 Expressed protein At2g38400 AGT3 Ala:glyoxylate aminotransferase 2 homolog At2g39570 ACT domain-containing protein; functions in amino acid binding At2g47770 TSPO (outer membrane Trp-rich sensory protein)-related At3g13450 DIN4 branched-chain α-keto acid dehydrogenase E1 β At3g26510 Octicosapeptide/Phox/Bem1p family protein At3g30775a ERD5, PRO1, PRODH, Pro dehydrogenase At3g47340a ASN1 Gln-dependent Asn synthetase At3g48360a BT2 (AtBT-2) component of the TAC1-mediated telomerase activation pathway At3g57520 RS2, SIP2 raffinose-specific α-galactosidase At3g61060 PP2-A13, phloem protein 2-A13 At3g61890 HB-12, homeobox-Leu zipper protein HB-12 At4g15530 PPDK pyruvate,orthophosphate dikinase At4g28040 Nodulin MtN21/EamA-like transporter family protein (drug/metabolite transporter) At4g35770 SEN1 senescence-associated protein At5g04040 SDP1, triacylglycerol lipase that is involved in storage lipid breakdown At5g18670 BMY3, BAM9 glycosyl hydrolase family 14 (β-amylase) At5g22920a CHY-type/CTCHY-type/RING-type zinc finger protein At5g49360 BXL1, bifunctional β-d-xylosidase/α-l-arabinofuranosidase At5g53590 SAUR-like auxin-responsive protein family At5g66170 STR18, encodes a thiosulfate sulfurtransferase/rhodanase At5g66650 Protein of unknown function DUF607 Repressed At1g26770 EXP10 (expansins[α-expansin gene family]): expansin 10 At1g64060 RbohF(respiratory burst oxidase family, cytochrome b558-H+channel) At1g69530 EXP1 (α-expansin gene family) At1g70230 TBL27, trichome birefringence-like, plant-specific DUF231 At1g76790 IGMT5, indole glucosinolate O-methyl transferase 5 At2g16660 Major facilitator superfamily protein, endomembrane system At2g38170 CAX1, high-affinity vacuolar calcium antiporter At2g38940 PHT1;4, member of the Pht1 family of phosphate transporters At3g09270 GSTU8 glutathione transferase belonging to the τ-class of GSTs Oppositely regulated At2g14170 ALDH6B2, methylmalonate-semialdehyde dehydrogenase At3g57040 ARR9 response regulator A type; cytokinin signaling At5g17760 P-loop-containing nucleoside triphosphate hydrolases superfamily protein a Also in the list of seven genes that are induced by bZIP11 under the control of its own promoter.
Supplemental Data
Supplemental Tables and Figures
Files in this Data Supplement:
- Adobe PDF - 180442Supplemental_material_Metabolite_control_over_growth.pdf - Supplemental Tables I and II and Supplemntal Figures 1-3
