научная статья по теме THE LEVEL OF MRNA NAD-SDH IS REGULATED THROUGH RNA SPLICING BY SUGARS AND PHYTOHORMONES Биология

Текст научной статьи на тему «THE LEVEL OF MRNA NAD-SDH IS REGULATED THROUGH RNA SPLICING BY SUGARS AND PHYTOHORMONES»

ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 2, с. 301-304

КРАТКИЕ СООБЩЕНИЯ

УДК 581.1

THE LEVEL OF mRNA NAD-SDH IS REGULATED THROUGH RNA SPLICING

BY SUGARS AND PHYTOHORMONES1

© 2015 N. Wongkantrakorn, S. Duangsrisai

Department of Botany, Faculty of Science, Kasetsart University, Jatuchak, Ladyoa, Bangkok, Thailand

Received January 13, 2014

Unspliced (L-SDH) and spliced (S-SDH) transcripts of NAD+-dependent sorbitol dehydrogenase (NAD-SDH) of strawberry (Fragaria ananassa Duch., cv. Nyoho) were investigated. Fructose, mannitol, and IAA increased S-SDH transcript level. Sorbitol decreased the level of L-SDH transcript 2.5-fold. ABA and benzyladenine (BA) decreased S-SDH transcript level and increased L-SDH transcripts. Ratio of S-SDH to L-SDH was increased by sorbitol (2.7-fold), fructose (2.2-fold), and IAA (1.5-fold), and decreased by BA (4-fold) and ABA (1.7-fold) as compared to control. These results suggest that sorbitol, fructose, and IAA stimulated splicing of pre-mRNA of NAD-SDH, but BA and ABA repressed this process.

Keywords: Fragaria ananassa — NAD-SDH — splicing — sugar and hormonal treatments

DOI: 10.7868/S0015330315010169

INTRODUCTION

In many Rosaceae fruit trees, sorbitol is a major translocated sugar [1], which is important for fruit growth and development. In these plants, sorbitol is synthesized by sorbitol-6-phosphate dehydrogenase (S6PDH; EC 1.1.1.200) in leaves, translocated into fruits, and then catabolized to fructose by NAD+-de-pendent sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14). NAD-SDH catalyzes the oxidation of sorbitol and the reduction of fructose [2]. The enzyme has been characterized [3], and its gene has been cloned [4, 5] for many Rosaceae fruit trees. It has been reported that NAD-SDH plays an important role in sorbitol breakdown mechanism in many Rosaceae fruit trees [5—7]. In general, NAD-SDH activity would favor the conversion of sorbitol to fructose.

Strawberry, which belongs to Rosaceae family and does not have S6PDH activity in its leaves and fruits, contains only small amounts of sorbitol in fruits [8]. Probably, NAD-SDH in strawberry fruits catalyzes the reduction of fructose and is responsible for sorbitol accumulation in fruits. The same was suggested for ger-

1 This text was submitted by the authors in English.

Abbreviations'. BA — benzyladenine; GA3 — gibberellin 3; NAD-SDH - NAD+-dependent sorbitol dehydrogenase; RT-PCR -reverse-transcribed polymerase chain reaction; UTR — untranslated region.

Corresponding author. Duangsrisai Sutsawat. Department of Botany, Faculty of Science, Kasetsart University, Jatuchak, Ladyoa, Bangkok, 10900 Thailand; fax. +66-2-562-5555 ext 1322/+66-94-495-3781; e-mail. sutsawatying@gmail.com

minating soybean seeds [9] and for developing maize endosperm [10].

In apple and Japanese pear, sorbitol and some other sugars increased NAD-SDH mRNA level and enzyme activity, but fructose did not [11, 12]. In strawberry NAD-SDH enzyme activity was enhanced about 2.5-fold by both sorbitol and fructose; the activity was also enhanced by IAA [13]. All treatments by sugars and hormones did not change the transcript level. However, primers used in these works could not differentiate between unspliced and mature transcripts. In this study, we used primers that let us to trace levels of intron-con-taining and spliced transcripts independently. We studied the effects ofvarious sugars and phytohormones and discussed the significance of our findings.

MATERIALS AND METHODS

Strawberry (Fragaria ananassa Duch., cv. Nyoho) plants were grown under natural light in a greenhouse of Nagoya University. Strawberries at 15—20 days after pollination were harvested. Fruit discs were prepared by removing the head and the end of the fruit and cutting into 5-mm slides. To lower sugar concentration in tissues before treatments, fruit discs were pre-incubat-ed for 16 h at 25°C in five volumes of buffer A (10 mM Mes—KOH, pH 6.5, 2 mM DTT, and 2 mM CaCl2).

For treatments, fruit discs were incubated in buffer A with addition of 100 mM sugars (sorbitol, fructose, glucose, sucrose, or mannitol) or 100 ^M phytohormones (IAA, ABA, GA3, or BA) at 25°C for 24 h. After incubation, the discs were washed twice, frozen

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RNA cDNA

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Fig. 1. RT-PCR using RNA from control and sorbitol treatments and cDNA from sorbitol treatment as a template.

in liquid nitrogen, and stored at —80°C until used for RNA extraction. Total RNA was isolated from the discs by using the modified phenol-SDS method [14] combined with the cetyltrimethylammonium bromide method [15].

Total RNA was reverse-transcribed using an oligo (dT)20 primer, as recommended by the TaKaRa RNA PCR Kit (AMV) v. 2.1 ("TaKaRa", Japan). Primer for NAD-SDH was designed from the sequence of FaSDH (GenBank accession no. AB268587). Forward primer was nested to exon 1 (5'-ACATGGAGATGACCAA-CAAGAGAACATGGC-3') and reverse primer was nested to exon 3 (5'-GGCATAGATTGTATCGACCT-TCTTTGCAAG-3'). The housekeeping gene of glycer-aldehyde-3-phosphate dehydrogenase (FaGAPDH, GenBank accession no. AB363963) was used as a control. Primers: GAPDHsense (5'-CTGCCACCCA-GAAGACTGTTG-3') and GAPDHantisense (5'-CTCCAGTGCTGCTAGGAATG-3'). The program for semi-quantitative RT-PCR was: 94°C for 5 min, then by 24 (GAPDH) or 26 (NAD-SDH) cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. PCR products were separated on 1% (w/v) agarose gels in 0.5x Tris—borate buffer (pH 8.0) containing 0.5 mg/mL ethidium bromide. The amount of each transcript (upper or lower band) was quantified by densitometry, then normalized to GAPDH.

PCR products were purified by phenol-chloroform extraction from a low-melting-temperature agarose gel. The purified products were cloned into a vector and sequenced by the dideoxy chain-termination method using an ABI BigDye terminator cycle sequencing Kit ("Applied Biosystems", United States) and automatic 3130 ABI Genetic Analyzer ("Applied Biosystems").

RESULTS

Presence of different transcripts for NAD-SDH in strawberry

Using primers designed from 5'-region of NAD-SDH, two PCR products of 600 and 350 bp were obtained (fig. 1). Sequencing analysis revealed that 350 bp fragment was a part of NAD-SDH. The nucle-otide sequence of 600 bp fragment was practically identical to that of 350 bp, but contained still two insertions, which as judged from their characteristics are introns. To exclude the possibility of genomic DNA contamination, RT-PCR was conducted using RNA extracted from the control and sorbitol-treated strawberry fruit discs as a template. 35 cycles of PCR were performed. No band appeared in any lane when RNA was used as a template, but two bands appeared in the lane when cDNA was used as a template (fig. 1).

Full-length cDNAs of 350 and 600 bp fragments were cloned by RACE method. The longer transcript (L-SDH) had 1843 nucleotides, whereas a shorter one (S-SDH) had 1273 nucleotides. The S-SDHwas identical to strawberry NAD-SDH cDNA (FaSDH; AB268587). The identical region covered 1083 nucleotides of coding region as well as 29 nucleotides at 5'-untranslated sequence and 159 nucleotides at 3'-untranslated sequence. Compared with S-SDH, L-SDH contained an insertion of 115 nucleotides at position 110, 175 nucleotides at position 178, 152 nucleotides at position 415, and 127 nucleotides at position 894 relative to the ATG. These insertions contained stop codons, which blocked translation process for L-SDH.

Changes in transcript levels of S-SDH and L-SDH

The amounts of two NAD-SDH transcripts were analyzed by semi-quantitative RT-PCR. The S-SDH was present in relatively higher levels at all sugar treatments compared with the control, whereas L-SDH was proportionally less abundant in sorbitol treatment (fig. 2a). Fructose and mannitol increased S-SDH transcripts to the highest level among sugar treatments. Sorbitol and glucose increased transcript levels to a lesser extent (fig. 2b). The level of L-SDH transcript decreased 2.5-fold by sorbitol treatment only, and it was not changed significantly by other sugars (fig. 2c). The ratio of S-SDH to L-SDH was increased by sorbitol treatment 2.7-fold and by fructose treatment 2.2-fold as compared to control.

Among phytohormonal treatments (fig. 3), IAA and to a lesser extent GA3 stimulated the accumulation of mature transcript (S-SDH) and decreased the level on unsliced transcript (L-SDH), leading to the increase in the ratio of S-SDH to L-SDH (insignificant for GA3). In contrast, ABA and BA treatments decreased S-SDH level and increased L-SDH level, lead-

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THE LEVEL OF mRNA NAD-SDH IS REGULATED THROUGH RNA SPLICING

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Fig. 2. The effect of sugars on the ratio of S-SDH to L-SDH.

a — the semiquantitive RT-PCR; b—d — quantification of bands normalized to GAPDH mRNA level; b - S-SDH; c - L-SDH; d - the ratio of S-SDH to L-SDH.

Fig. 3. The effects of phytohormones on the ratio of S-SDH to L-SDH.

a — the semiquantitive RT-PCR; b-d — quantification of bands normalized to GAPDH mRNA level; b - S-SDH; c - L-SDH; d - the ratio of S-SDH to L-SDH.

ing to the great reduction in the ratio of S-SDH to L-SDH (fig. 3d).

DISCUSSION

In this study, two types of NA

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