Expression of sucrose synthase genes involved in enhanced elongation of pondweed (Potamogeton distinctus) turions under anoxia

Abstract

Background and aims: Overwintering buds (turions) of the monocot aquatic pondweed species (Potamogeton distinctus) are highly tolerant to anoxic stress. Sucrose metabolism accompanied by enhanced activity of sucrose synthase (SuSy) operates actively during anaerobic elongation of pondweed turions. The aim of this study is to isolate SuSy genes from the turions and to investigate their transcriptional changes in response to anoxia and other stimuli.

Methods: SuSy genes were isolated from pondweed turions by PCR methods and transcript levels of SuSy genes were examined in response to anoxia, sugars and plant hormones. In addition, the effects of anoxia on SuSy activity were examined both in the soluble fraction and in the microsomal fraction.

Key results: cDNAs of two SuSy genes (PdSUS1 and PdSUS2) were cloned from pondweed turions. The levels of PdSUS1 transcripts increased under anoxia but did not with sugar treatments. Anoxia-stimulated elongation of turions was further enhanced by 2,4-dichlorophenoxyacetic acid (2,4-D) and suppressed by treatments with sorbitol, 2-deoxyglucose (2-dGlc) and abscisic acid (ABA). The levels of PdSUS1 transcripts were increased by 2,4-D and decreased by sorbitol under anoxia. The levels of PdSUS2 transcripts were not significantly affected by anoxia and any other treatments. SuSy activity of turions under anoxia was enhanced in the soluble fraction, but not in the microsomal fraction.

Conclusions: Up-regulation of PdSUS1 transcription under anoxia may not be attributed to sugar starvation under anoxia. A positive correlation between stem elongation and the level of PdSUS1 transcripts was observed in turions treated with anoxic conditions, 2,4-D and sorbitol. The increase in SuSy activity in the cytosol may contribute to sugar metabolism and sustain stem elongation under anoxia.

Fig. 1.

Predicted amino acid sequences of PdSUS1 and PdSUS2. The alignment was made using GENETIX-MAC Ver. 8.0. Identical amino acids are shaded and gaps are indicated by dashes. An arrowhead shows a conserved serine residue (see text). The characteristic N-terminal extension of sucrose synthase (thin underline) and a glycosyl transferases group 1 domain (broken underline) were identified by NCBI Conserved Domain Search. PdSUS2 has an alanine-rich region at the C-terminal region (bold underline). A primer for 3′ RACE of SuSy genes was designed from nucleotides encoding amino acid residues, indicated by an arrow.

Fig. 2.

Phylogenetic tree of plant SuSy proteins. Predicted amino acid sequences were aligned using Clustal W. SuSys from Arabidopsis were named according to Baud et al. (2004). EMBL accession numbers (AGI ID for SuSys from Arabidopsis thaliana and gene number assigned by RiceGAAS for OsSUS4, OsSUS5 and OsSUS6) are given in parentheses. Al (X92378) from Alnus glutinosa; AtSUS1 (At5g20830), AtSUS2 (At5g49190), AtSUS3 (At4g02280), AtSUS4 (At3g43190), AtSUS5 (At5g37180) and AtSUS6 (At1g73370) from Arabidopsis thaliana; BoSUS1 (AF412039) and BoSUS2 (AF412037) from Bambusa oldhamii; BvSUS1 (X81974) and BvSUS2 (AY457173) from Beta vulgaris; Cl (AB018561) from Citrullus lanatus; CpSUS1 (AJ131999) and CpSUS2 (AJ132000) from Craterostigma plantagineum; Cr (X82504) from Chenopodium rubrum; CuSUS1 (AB022092) and CuSUSA (AB022091) from Citrus unshiu; DcSUS1 (X75332) and DcSUS2 (Y16091) from Daucus carota; Gh (U73588) from Gossypium hirsutum; Gm (AF030231) from Glycine max; HvSUS1 (X65871) and HvSUS2 (X69931) from Hordeum vulgare; LeSUS1 (L19762) and LeSUS2 (AJ011319) from Lycopersicon esculentum; Ms (AF049487) from Medicago sativa; Mt (AJ131943) from Medicago truncatula; My (AF530568) from Mokara ‘Yellow’; Og (AF530567) from Oncidium ‘goldiana’; OsSUS1 (X59046), OsSUS2 (X64770), OsSUS3 (L03366), OsSUS4 (OSJNBb0050D18_1·01), OsSUS5 (OSJNBb0026I12·04) and OsSUS6 (OJ1149_C12·08) from Oryza sativa; PdSUS1 (AB193515) and PdSUS2 (AB193516) from Potamogeton distinctus (this study); Pt (AY341026) from Populus tremuloides; Pv (AF315375) from Phaseolus vulgaris; PsSUS1 (AJ012080), PsSUS2 (AJ001071) and PsSUS3 (AJ311496) from Pisum sativum; So (AY118266) from Saccharum officinarum; StSUS3 (U24088) and StSUS4 (U24087) from Solanum tuberosum; TaSUS1 (AJ001117) and TaSUS2 (AJ000153) from Triticum aestivum; TgSUS1 (X96938) and TgSUS2 (X96939) from Tulipa gesneriana; Vf (X69773) from Vicia faba; Vr (D10266) from Vigna radiata; ZmSH1 (X02400), ZmSUS1 (L22296) and ZmSUS3 (AY124703) from Zea mays. SuSys that are reported to be enhanced in gene or protein expression at low oxygen concentrations are indicated by asterisks according to Baud et al. (2004), except for OsSUS2 and OsSUS3 in Huang et al. (1996) and StSUS3 and StSUS4 in Bologa et al. (2003).

Fig. 3.

Effects of anoxia on the levels of PdSUS1 and PdSUS2 transcripts. RNA was extracted from turion segments incubated in air or under anoxia at 25 °C in the dark for 0 d, 1 d and 3 d. The amount of transcript was determined by RT–PCR. The level of 26S rRNA transcript was used as an internal control. A typical example in three independent experiments is shown.

Fig. 4.

Effects of exogenous sugars on stem elongation and on the levels of PdSUS1 and PdSUS2 transcripts in turion segments. (A) Stem elongation was determined after 3 d of incubation in air (open bars) or under anoxia (shaded bars). Data for elongation are means ± s.e. of more than six turion segments. (B) The level of transcripts was determined at 3 d of incubation by RT–PCR and an example in three independent experiments is shown. (C) Northern blot analysis of PdSUS1 transcripts. Total RNA was extracted from turion segments incubated under each condition for 3 d. Ten µg of total RNA were applied to each lane and hybridized with RNA probes for PdSUS1. rRNA stained with ethidium bromide is also shown as a loading control. Significantly different from water control in air at: ^*P < 0·0001, ^**P < 0·01. Significantly different from water control in anoxia at: ^***P < 0·05 (Mann–Whitney test).

Fig. 5.

Effects of auxin and abscisic acid on stem elongation and on the levels of PdSUS1 and PdSUS2 transcripts in turion segments. (A) Stem elongation was determined after 3 d of incubation in air (open bars) or under anoxia (shaded bars). Data for elongation are means ± s.e. of more than 18 turion segments. (B) The level of transcripts was determined at 3 d of incubation by RT–PCR and an example in three independent experiments is shown. (C) Northern blot analysis of PdSUS1 transcripts. Total RNA was extracted from turion segments incubated under each condition for 3 d under anoxia. Ten µg of total RNA were applied to each lane and hybridized with RNA probes for PdSUS1. rRNA stained with ethidium bromide is also shown as a loading control. Significantly different from water control in air at: ^*P < 0·05. Significantly different from water control in anoxia at: ^**P < 0·05 (Mann–Whitney test).

Fig. 6.

Effects of anoxic conditions on SuSy activity in soluble and membrane fractions extracted from pondweed turions. SuSy was extracted from pondweed turions incubated at 25 °C in the dark in air (open bars) and under anoxia (shaded bars) for 3 d. Total activity of SuSy was determined in the supernatant after centrifugation of the extract at 7000 g for 20 min. The activity of soluble SuSy and membrane-bound SuSy was determined in the soluble fraction and in the microsomal fraction separated by ultracentrifugation at 95 000 g for 1 h. SuSy activity was measured by the method described in Materials and Methods. Data are means ± s.e. of three separate replicates. Significantly different from the activity in air at: ^*P < 0·1, ^**P < 0·05 (t-test).