Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum

Abstract

Thioredoxins (TRXs) f and m are key components in the light regulation of photosynthetic metabolism via thiol-dithiol modulation in chloroplasts of leaves; however, little is known about the factors modulating the expression of these proteins. To investigate the effect of sugars as photosynthetic products on the expression of PsTRX f and m1 genes, sucrose and glucose were externally supplied to pea plants during the day. There was an increase in the mRNA levels of PsTRX f and m1 genes in response mainly to glucose. When leaf discs were incubated for up to 4h in the dark, glucose also led to an increase in both mRNA and protein levels of TRXs f and m, while sucrose had no substantial effect. Expression of PsDOF7, a carbon metabolism-related transcription factor gene, was also induced by glucose. Protein-DNA interaction showed that PsDOF7 binds specifically to the DOF core located in PsTRX f and m1 gene promoters. Transient expression in agroinfiltrated pea leaves demonstrated that PsDOF7 activated transcription of both promoters. The incubation of leaf discs in dithiotreitol (DTT) to increase the redox status led to a marked increase in the mRNA and protein levels of both TRXs within 4h. The increase in TRX protein levels occurred after 1h DTT feeding, implying a rapid effect of the thiol status on TRX f and m1 protein turnover rates, while transcriptional regulation took 3h to proceed. These results show that the protein levels of both TRXs are under short-term control of the sugar and thiol status in plants.

Fig. 8.

(a) PsDOF7 protein sequence. The underlined sequence corresponds to the DOFmotif and Cs in bold show the cysteine residues involved in the zinc-finger domain binding to DNA. (b) Phylogenetic tree of plant DOF proteins. PsDOF7 is clustered with carbon-regulated metabolism transcription factors DAG1 and AtDOF4.1.

Fig. 1.

Changes in the intracellular content of glucose, fructose, and sucrose after supplying buffer (2mM MES) (a), glucose (b), fructose (c), or sucrose (d) via the roots of pea plants. The leaves were collected at 2, 4, 8, and 14h (2h dark) of the photoperiod. The results are the mean ±SE from three individual plant leaves of three different experiments.

Fig. 2.

Relative expression of (a) PsTRX f, (b) PsTRX m1, and(c) PsDOF7 of pea plants supplied with 100mM sorbitol, 100mM glucose, 100mM fructose, or 100mM sucrose for 2, 4, 8, and14h (2h dark). Levels of mRNA were analysed by RT-PCR with specific primers. The values were normalized to the sorbitolcontrol. Each value is the mean ±SE from three determinationsof three cDNA preparations. Asterisks mark significantdifferences (P ≤ 0.05) as determined by Student’st-test comparing the treatment inrelation to control.

Fig. 3.

Short-term feeding of sugars to leaf discs of pea plants at the end of the night leads to increased mRNA levels of TRX f and m. Leaf discs were incubated for 1, 2, 3, and 4h in 100mM sorbitol (control), 100mM glucose or 100mM sucrose, before samples were frozen to analyse TRX f (a) and TRXm (b) mRNA levels by real-time PCR using specific oligonucleotides. The values were normalized to the sorbitol control. Each value is the mean ±SE from three determinations of three cDNA preparations. The transcript amounts are represented as arbitrary units in relation to the control level, time 0h set to 1.0.

Fig. 4.

Short-term feeding of sugars to leaf discs of pea plants at the end of the night leads to an increased TRX f protein level. Leaves of pea plants were harvested at the end of the night to prepare discs that were immediately incubated in the dark for 1, 2, 3, and 4h in buffer (control), 100mM sorbitol (control), 100mM glucose, or 100mM sucrose, before samples were frozen to analyse the Trx f protein level using western blots. t₀, leaf tissue before incubation. Western blot images are representativeof three independent experiments.

Fig. 5.

Short-term feeding of sugars to leaf discs of pea plants at the end of the night leads to an increased TRX m protein level. Leaves of pea plants were harvested at the end of the night to prepare discs that were immediately incubated in the dark for 1, 2, 3, and 4h in buffer, 100mM sorbitol (control), 100mM glucose, or 100mM sucrose, before samples were frozen to analyse the Trx m protein level using western blots. t₀, leaf tissue before incubation. Western blot images are representative of three independent experiments.

Fig. 6.

Short-term feeding of sugars to leaf discs of pea plants at the end of the night leads to increased PsDOF7 gene expression. Leaves of pea plants were harvested at the end of the night to prepare discs that were immediately incubated in the dark for 1, 2, 3, and 4h in buffer, 100mM sorbitol (control), 100mM glucose, or 100mM sucrose. mRNA levels were analysed by real-time PCR using specific oligonucleotides. The values were normalized to the sorbitol control. Each value is the mean ±SE three determinations of three cDNA preparations. The transcript amounts are represented as arbitrary units in relation to the control level, time 0h set to 1.0.

Fig. 7.

Dithiotreitol (DTT) feeding leads to increased TRX f and m1 expression in leaves. Leaves of pea plants were harvested at the end of the night to prepare discs that were immediately incubated on zero and 5mM DTT for 1–4h in the dark. Discs were then quickly rinsed in buffer to remove external DTT before freezing in liquid nitrogen to analyse (a) TRX mRNA levels using real-time PCR or (b and c) TRX protein levels using western blots. The expression values were normalized to the buffer control. Each value of transcript is the mean ±SE from three determinations of three cDNA preparations. The transcript amounts are represented as arbitrary units in relation to the control level, time 0h set to 1.0.

Fig. 9.

Functional properties of the pea DOF motif present in PsTRX f and PsTRX m1 promoters. EMSA of the recombinant PsDOF7 with oligonucleotide probes derived from (a) PsTRX f and (b) PsTRX m promoters. The probe without recombinant PsDOF7 is designated Free Probe (lane 1). Competition experiments were performed using increasing amounts of either the non-specific competitor [poly(dI–dC)] (lanes 2–4) or the unlabelled probe (lanes 5–7). Increasing molar amounts 100, 300, and 500ng are indicated by triangles.(a) A probe of 29bp (LumF) derived from the PsTRX f promoters was ³²P labelled 5'-AGTGAAAAAAAAAAGAGATATTCGAAGGG-3'. (b) A probe of 41bp (LumM) derived from the PsTRX m1 promoters was ³²P labelled 5'-TTGACATAAGTAGATATTGAAAGCA-
AGATTGAAAAAAATGT-3'.

Fig. 10.

Transient GUS expression in pea leaves driven by PsTRX f and PsTRX m1 promoters. Agrobacterium tumefaciens-mediated transient transformation was conducted on near fully expanded leaves still attached to 2-week-old intact pea plants. Bacterial suspensions were infiltrated into mesophyll (Yang et al., 2000). PsDOF was used as the control (A). TRX f-GUS (B) and TRX m-GUS (C) were infiltrated into the right-hand side of the leaf, while the co-infiltration of TRX f-GUS (B) and TRX m-GUS (C) with DOF7 was into the left-hand side of the leaf. Histochemical assays were performed 3 d after infiltration. After GUS staining, plant tissues were incubated in 70% (v/v) ethanol at 37 ºC. The leaves in the figure are representative images of three different leaves of three independent experiments.