Abiotic stress and phytohormones affect enzymic activity of 1-O-(indole-3-acetyl)–d-glucose: myo-inositol indoleacetyl transferase from rice (Oryza sativa)
Anna Ciarkowska, Maciej Ostrowski∗, Anna Jakubowska
Abstract
Indole-3-acetic acid (IAA) conjugation is a part of mechanism regulating free auxin concentration. 1O-(indole-3-acetyl)–d-glucose: myo-inositol indoleacetyl transferase (IAInos synthase) is an enzyme involved in IAA-ester conjugates biosynthesis. Biotic and abiotic stress conditions can modulate auxin conjugates formation in plants. In this study, we investigated effect of plant hormones (IAA, ABA, SA and 2,4-D) and abiotic stress (drought and salt stress: 150 mM NaCl and 300 mM NaCl) on expression level and catalytic activity of rice IAInos synthase. Enzymic activity assay indicated that all tested phytohormones affected activity of IAInos synthase, but only ABA had inhibiting effect, while IAA, SA and 2,4-D activated the enzyme. Drought and salt stress induced with lower NaCl concentration resulted in decreased activity of IAInos synthase, but 300 mM NaCl had no effect on the enzyme. Despite observed differences in enzymic activities, no changes of expression level, tested by semiquantitative RT-PCR and Western blot, were detected. Based on our results it has been supposed that plant hormones and stress conditions affect IAInos synthase activity on posttranslational level.
Keywords:
IAA
IAA ester conjugates IAInos synthase
SCPL acyltransferase
Oryza sativa s u m m a r y
1. Introduction
Free indole-3-acetic acid (IAA) concentration in plants is tightly regulated by several processes occurring in response to some developmental and environmental signals. One of the mechanisms responsible for auxin homeostasis is formation of IAA conjugates (Bajguz and Piotrowska, 2009). The conjugated IAA is thought to function as auxin storage and also to be involved in auxin transport, protection against peroxidative degradation and auxin excess detoxification (Woodward and Bartel, 2005). Depending on the character of the molecule and bond via which it is conjugated to auxin, IAA conjugates are divided in two groups: ester and amide conjugates (Korasick et al., 2013). Predominant form of IAA conjugates in monocots are its ester conjugates with sugars or myo-inositol (Ludwig-Müller, 2011). Endosperm of maize (Zea mays) kernels contains 97–99% of IAA in the form of ester enzymic activity has only been detected in maize and rice so far (Kesy˛ and Bandurski 1990; Ciarkowska et al., 2013). Based on amino acid sequence analysis this enzyme has been classified as a member of serine carboxypeptidase-like (SCPL) acyltransferases family, a group of glycosylated enzymes which show homology to serine carboxypeptidases (Kowalczyk et al., 2003). Enzymes belonging to this family catalyze transfer of an acyl moiety from energy-rich 1-O-glucose esters to nucleophilic group of acceptor molecule (Mugford and Milkowski, 2012). SCPL acyltransferases play important role in plant secondary metabolism pathways, such as biosynthesis of sinapate esters (Lehfeldt et al., 2000; Shirley et al., 2001; Stehle et al., 2009) and formation of auxin conjugates (Kowalczyk et al., 2003; Starzynska´ and Kowalczyk 2012). They are also involved in regulation of defense responses against biotic and abiotic stress (Liu et al., 2008; Mugford et al., 2009).
In our previous studies we have identified IAInos synthase activity in rice seedlings (Ciarkowska et al., 2013). The activity of this enzyme was easily detectable in 6-days old seedlings, but extremely low in younger plants. We have also found cDNA sequence corresponding to IAInos synthase from rice in UniProt database. Using heterologous expression system we have confirmed that this sequence encodes catalytically active IAInos synthase (unpublished results). In this study we describe how subjecting to abiotic stress (salt stress, drought) and phytohormones: IAA, abscisic acid (ABA), salicylic acid (SA) and 2,4-dichlorophenoxyacetic acid (2,4-D) affects expression and activity of IAInos synthase in 6-days old rice (Oryza sativa) seedlings.
2. Material and methods
2.1. Plant material
Black rice wholemeal (Bio Planet, Poland) was used as plant material. Rice (Oryza sativa) seeds were soaked in distilled water at 37 ◦C for 24 h. Plants were grown in darkness at 27 ◦C on Petri dishes. For the abiotic stress effects, 5-d-old seedlings were transferred to 150 mM or 300 mM NaCl solutions or exposed to drought conditions by transferring them to Petri dishes without water for additional 24 h. For the phytohormone effects, 5-d-old seedlings were incubated for 24 h in 10 M IAA, 10 M ABA, 10 M SA or 0.05 M 2,4-D solutions. Control seedlings were grown in distilled water.
2.2. Tissue homogenization
Rice seedlings were homogenized (1 g tissue: 1 mL buffer) with 50 mM HEPES buffer, pH 7.4, using mortar and pestle. The homogenates were centrifuged at 10,000 × g for 10 min at 4◦ C (Sigma Sartorius 3 K 30 Centrifuge, 12154 rotor, Germany). The supernatant fluid was used for analysis.
2.3. Enzyme activity assay
Enzymic activity of IAInos synthase was determined in a total volume of 8 L containing 27.4 mM HEPES buffer, pH 7.4, 13.2 mM UDPG, 3.5mM IAA, 0.016CiC]IAA (55mCimmol−1), 13.2 mM myo-inositol, 19.4 mM d-gluconic acid lactone, 2.2 mM MgCl2 and 5.5 U of recombinant IAGlc synthase, with 3 L of the supernatant fluid from tissue homogenates. The reaction was stopped after 2 h incubation in 30 ◦C by drying 4 L of aliquots on Silica Gel F260 TLC plate (Merck). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5: 3: 1: 1) as a solvent. For indole compounds visualization, the plate was stained with Van Urk-Salkowski reagent (Ehmann, 1977). Bands corresponding to IAInos were excised and placed in a vial with 2 mL EcoLite (+) scintillation fluid (ICN). Radioactivity level was measured in Wallac 1409 liquid scintillation counter (Turku, Finland). For each experiment we used 3 biological repetitions. Data are presented as mean ± standard deviation (SD).
2.4. RNA isolation and reverse transcription-PCR (RT-PCR) analysis
Total RNA was extracted from the rice seedlings using Gene MATRIX Universal RNA/miRNA Purification Kit (EURx). RNA samples were pretreated with RNA-se free DNase I (Thermo Scientific) to remove any contaminating genomic DNA. Firststrand cDNA synthesis was performed using 1 g of RNA with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. PCRs of 20 L with 1.2 L of template cDNA were performed with 1.2 U of Maxima Hot Start Taq DNA Polymerase (Thermo Scientific). IAInos synthase gene (UniProt EEC73124.1) expression was analyzed using gene-specific primers: F: 5-CGTGCAATGGGAAGTACTGG3; R: 5-AGCAAGGCATGATCTCCACT-3 . Actin gene expression 35 cycles of 94 ◦C for 45 s, 50.7 ◦C (IAInos synthase) or 53 ◦C (actin) for 45 s; 72 ◦C for 90 s, followed by final extension of 72 ◦C for 10 min. Samples obtained with RT-PCR were separated by electrophoresis in 1% agarose gel in TAE buffer using DELFIN electrophoresis apparatus (DNA Gdansk).´
2.5. SDS-PAGE and western blot analysis
A. Ciarkowska et al. / Journal of Plant Physiology 205 (2016) 93–96 95 SDS-PAGE was performed according to the method of Ogita and Markert (1979) in a Mini Protean II electrophoresis apparatus (Bio-Rad) using 12% (w/v) running gel and 6% (w/v) stacking gel. A supernatant fluid of rice seedling extract containing 20 g of protein was subjected to the SDS-PAGE. The protein mixture used as a molecular mass standards was a SpectraTM multicolor broad range protein ladder (10–260 kDa) (Thermo Scientific). For Western blot analysis, separated proteins were transferred onto a Protran BA 83 nitrocellulose membrane (Whatmann, GmBH) by the wet system (Bio-Rad) in the transfer buffer containing 10 mM CAPS/NaOH, pH 11.0 and 10% (v/v) ethanol. After blotting, membrane was stained with Ponceau S. After blocking in TBS buffer containing 5% (w/v) nonfat dry milk, the membrane was incubated with rabbit polyclonal anti-maize IAInos synthase antibodies. The proteins were detected with goat anti-rabbit IgG antibodies conjugated to alkaline phosphatase (1: 20 000) (Sigma-Aldrich) and the blot was visualized using NBT/BCIP Tablets (Roche).
2.6. Protein concentration
Protein concentration was determined by Bradford method (Bradford, 1976) using albumin as a standard.
3. Results and discussion
A strict regulation of free IAA concentration is necessary for proper plant growth and development but also its adaptation to stress conditions, especially since auxin is involved in many phytohormonal interactions, e.g. with hormones linked to biotic and abiotic stress responses. There are many studies indicating that expression of some GH3 (Gretchen Hagen3) encoding enzymes involved in conjugation of IAA to amino acids is affected by stress conditions and plant hormones (Zhang et al., 2009; Du et al., 2012; Ostrowski and Jakubowska, 2013). Activity of indole3-acetylglucose synthase (IAGlc synthase), an enzyme catalyzing first step of IAA-ester conjugate synthesis in maize, has also been reported to be regulated by plant hormones (Kowalczyk et al., 2002; Leznicki and Bandurski 1988). We wanted to know if such factors affect IAInos synthase, so we examined how some phytohormones and abiotic stress (salt stress and drought) affect expression and activity of this enzyme.
For determination of plant hormone effects on catalytic activity of IAInos synthase we used 6-d old rice seedlings which have been incubated in phytohormone solution (IAA, ABA, SA or auxinic herbicide 2,4-D) for 24 h. Some of the tested factors visibly affected rice seedlings growth, resulting in changes in stem length compared to the control (Fig. 1). Drought caused the strongest inhibitory effect on stem growth. Exposition to both concentrations of NaCl, 2,4-D and IAA also resulted in reduced stem length. Only SA increased stem length of rice seedlings.
Incubation with exogenous IAA caused 2.1-fold increase of rice IAInos synthase activity (Fig. 2.). Considering how IAA affects plant growth, higher activity of IAA conjugating enzyme results in deacreased free auxin concentration. It can explain why we observed inhibition of stem growth after IAA treatment (Fig. 1.). Increase of rice IAInos synthase activity is consistent with study by Kowalczyk et al. (2002), which shows that synthetic auxin 1naphtalene acid (NAA) causes increase in maize IAGlc synthase activity and its expression level. Expression of auxin-responsive GH3 genes is also induced by exogenous IAA. Expression of PsGH3.5 from pea increased 60 times and activity of amide synthetase encoded by this gene increased 12,5 times (Ostrowski and Jakubowska, 2013). Moreover, IAA upregulated expression of some GH3 genes in sorghum (Wang et al., 2010) and maize (Feng et al., 2015), but downregulated expression of other genes. It indicates that plants synthesize more IAA conjugates in response to increased auxin concentration.
Other tested phytohormones also affected rice IAInos synthase activity. ABA caused 2.2-fold decrease of the enzyme activity. Maize IAGlc synthase activity is also inhibited by ABA and some other phytohormones, such as zeatin, gibberellic acid (GA3) and kinetin (Leznicki´ and Bandurski, 1988). SA and 2,4-D potentiated rice IAInos synthase activity 2.1-fold and 3.4-fold respectively (Fig. 2.). The effect of SA on maize IAGlc synthase has not been tested but 2,4-D acts as its inhibitor (Leznicki´ and Bandurski, 1988).
Enzymes involved in IAA-amide conjugates synthesis are also affected by plant hormones (Ostrowski and Jakubowska, 2013). Pea amide synthetase activity increased 10 times upon incubation with SA, 3 times with ABA and 20 times with 2,4-D. GA3, zeatin, jasmonic acid (JA) and methyl jasmonate (MeJA) also had activating effect on this enzyme. Beside higher enzymic activity, induction of expression of PsGH3.5 by tested phytohormones has also been observed. There is some evidence for effect of phytohormones on expression of GH3 genes from other plants as well. Brassinosteroids upregulted some of GH3 genes in sorghum (Wang et al., 2010), JA and ABA caused induction of expression of some GH3 genes from maize (Feng et al., 2015). Also expression of GH3.13 from rice was induced by ABA (Zhang et al., 2009; Du et al., 2012). Thus, plant hormones have mostly activating effect on expression of GH3 genes with the exception of some maize genes which were slightly downregulated by SA (Feng et al., 2015). These results show beside auxin, other phytohormones have regulatory effect on synthesis of IAA-ester and amide conjugates.
We have also analyzed effect of phytohormones on the expression of IAInos synthase gene on the transcript and protein level using semiquantitative RT-PCR and Western blot analysis. No changes compared to control have been detected on mRNA level (Fig. 3a.). We obtained similar result by Western blot analysis, with the exception of 300 mM NaCl which caused slight decrease in IAInos synthase expression (Fig. 3c.). It suggests that plant hormones have regulatory effect on IAInos synthase but only on the posttranslational level. Activity of IAInos synthase can possibly be modulated by changes in glycosylation pattern of this protein, which in turn could be triggered by other plant hormones.
For investigation of the abiotic stress effect on IAInos synthase activity rice seedlings were subjected to drought or salt stress (150 mM NaCl or 300 mM NaCl). Higher NaCl concentration had no effect on enzymic activity of rice IAInos synthase but 150 mM NaCl caused strong inhibition of IAInos synthase (Fig. 2.). It has been reported that salt stress modulates expression of GH3 genes in maize (Feng et al., 2015) and sorghum (Wang et al., 2010). Drought caused 2.1-fold decrease in IAInos synthase activity. Dehydration differently affects genes involved in IAA-amide conjugate synthesis and IAInos synthase. Expression of rice GH3.2 and GH3.13 is strongly induced by drought (Du et al., 2012). During dehydration, most of maize GH3 genes was upregulated in roots but downregulated in shoots (Feng et al., 2015). Since we determined IAInos synthase activity only in seedlings, it is possible that activity of this enzyme increases in roots of rice subjected to drought as it was with expression of GH3 genes in maize roots. Similarly to phytohormones, drought and 150 mM NaCl had no effect on expression of rice IAInos synthase on mRNA level (Fig. 3a.). However, higher concentration of 300 mM NaCl caused slight decrease in IAInos synthase expression on protein level (Fig. 3c.).
Almost all tested treatments affected growth of rice seedlings (Fig. 1.). However, it is difficult to compare changes in stem length with IAInos synthase activity because observed phenotype is an overall effect of phytohormone or abiotic stress on the plant.
Western blot analysis of rice seedlings homogenates revealed three protein bands of molecular mass of approximately 52 kDa, 55 kDa and 62 kDa (Fig. 3c.). IAInos synthase from maize was previously isolated and purified to homogeneity by Kowalczyk et al. (2003). SDS-PAGE analysis of purified preparation indicated that maize Sodium 2-(1H-indol-3-yl)acetate IAInos synthase constitutes of two polypeptides of molecular weight of 56,4 kDa and 53,5 kDa. It was hypothesized that molecular mass difference of 3 kDa is probably due to differences in glycosylation level. It could suggest that rice IAInos synthase is very similar to maize enzyme. However, rice enzyme, in contrary to maize IAInos synthase, probably exists in three differently glycosylated forms.
In conclusion, the presented results show that synthesis of IAA ester conjugates in monocots is regulated by some abiotic factors and possibly is involved in plant adaptation to stress conditions.
References
Bajguz, A., Piotrowska, A., 2009. Phytochemistry 70, 957–969.
Bradford, M.M., 1976. Anal. Biochem. 72, 248–254.
Ciarkowska, A., Ostrowski, M., Jakubowska, A., 2013. Acta Biochim. Pol. 60 (Supp. 1), 123.
Du, H., Wu, N., Fu, J., Wang, S., Li, X., Xiao, J., Xiong, L., 2012. J. Exp. Bot. 63,6467–6480.
Ehmann, A., 1977. J. Chromatogr. 132, 267–276.
Feng, S., Yue, R., Tao, S., Yang, Y., Zhang, L., Xu, M., Wang, H., Shen, C., 2015. J. Integr. Plant Biol. 57, 783–795.
Hall, P.J., 1980. Phytochemistry 19, 2121–2123.
Jensen, P.J., Bandurski, R.S., 1994. Plant Physiol. 106, 343–351.
Kesy,˛ J.M., Bandurski, R.S., 1990. Plant Physiol. 94, 1598–1604.
Korasick, D.A., Enders, T.A., Strader, L.C., 2013. J. Exp. Bot. 64, 2541–2555.
Kowalczyk, S., Jakubowska, A., Bandurski, R.S., 2002. Plant Growth Regul. 38, 127–134.
Kowalczyk, S., Jakubowska, A., Zielinska,´ E., Bandurski, R.S., 2003. Physiol. Plant. 119, 165–174.
Leznicki,´ A.J., Bandurski, R.S., 1988. Plant Physiol. 88, 1481–1485.
Lehfeldt, C., Shirley, A.M., Meyer, K., Ruegger, M.O., Cusumano, J.C., Viitanen, P.V., Strack, D., Chapple, C., 2000. Plant Cell 12, 1295–1306.
Liu, H., Wang, X., Zhang, H., Yang, Y., Ge, X., Song, F., 2008. Gene 420, 57–65.
Ludwig-Müller, J., 2011. J. Exp. Bot. 62, 1757–1773.
Michalczuk, L., Bandurski, R.S., 1980. Biochem. Biophys. Res. Commun. 93, 588–592.
Mugford, S.T., Milkowski, C., 2012. Method Enzymol. 516, 279–297.
Mugford, S.T., Qi, X., Bakht, S., Hill, L., Wegel, E., Hughes, R.K., Papadopoulou, K., Melton, R., Philo, M., Sainsbury, F., Lomonossoff, G.P., Roy, A.D., Goss, R.J.M., Osbourn, A., 2009. Plant Cell 21, 2473–2484.
Ogita, Z.I., Markert, C.L., 1979. Anal. Biochem. 99, 233–241.
Ostrowski, M., Jakubowska, A., 2013. J. Plant. Physiol. 170, 361–368.
Shirley, A.M., McMichael, C.M., Chapple, C., 2001. Plant J. 28, 83–94.
Starzynska,´ E., Kowalczyk, S., 2012. Acta Physiol. Plant. 34, 53–63.
Stehle, F., Brandt, W., Stubbs, M.T., Milkowski, C., Strack, D., 2009. Phytochemistery 70, 1652–1662.
Ueda, M., Bandurski, R.S., 1974. Phytochemistry 13, 243–253.
Wang, S., Bai, Y., Shen, C., Wu, Y., Zhang, S., Jiang, D., Guilfoyle, T.J., Chen, M., Qi, Y., 2010. Funct. Integr. Genomics 10, 533–546.
Woodward, A.W., Bartel, B., 2005. Ann. Bot. 95, 707–735.
Zhang, S.W., Li, C.H., Cao, J., Zhang, Y.C., Zhang, S.Q., Xia, Y.F., Sun, D.Y., Sun, Y., 2009. Plant Physiol. 151, 1889–1901.