Identification and Functional Characterization of Arabidopsis icl Mutant Under Trehalose Feeding in Light and Dark Conditions

Document Type: Research Article

Authors

Department of Biology, Faculty of Sciences, Golestan University, Gorgan, Iran

Abstract

Trehalose is a non-reducing sugar that plays an important role in plant growth and development. To study the role of trehalose on lipid metabolism and gluconeogenesis, Arabidopsis thaliana wild type (WT) and TreF (a line expressing trehalase) were grown on ½ MS medium with or without 100 mM sucrose and or trehalose in light or continuous darkness. In dark, trehalose leads skotomorphogenesis in WT seedlings and inhibits hypocotyl elongation without altering root growth. Then, a knock out mutant of icl (defective in isocitrate lyase/ICL) was identified in the SALK T-DNA insertion line, yet plants of this line were not altered with regard to growth on MS medium supplemented with or without trehalose in light condition, compared to WT. But the hypocotyl length of icl seedling was shorter than WT when grown on trehalose in darkness. The current data revealed that trehalose feeding altered seedling establishment in both WT and icl mutant. ICL enzyme activity measurement showed that the patterns of changes were similar in all treatments. Meanwhile, trehalose feeding reduced icl gene expression and enzyme activity. Trehalose fed seedlings demonstrated a high accumulation of total lipids in darkness. Also fatty acids level was higher in seedlings grown in darkness, compared with the light condition. Therefore, trehalose may inhibit lipid utilization by suppressing icl gene expression and enzyme activity and thus restrict the supply of carbon sources to the growing seedling. These observations confirm that trehalose regulates plant metabolism in both light and dark.

Keywords


Aeschbacher RA, Muller J, Boller T, Wiemken A. 1999. Purification of the trehalse GMTRE1 from soybean nodules and clioning of its cDAN. GMTRE1 is expressed at a low level in multiple tissues. Plant physiol 119:489-496.

Aghdasi M. 2007. Analysis of trehalose-6-phosphate control over carbon allocation and growth in plants. Unpublished Ph.D. Thesis, Utrecht University, Utrecht, Netherlands.

Aghdasi M, Smeekens S, Schluepmann H. 2010. Characterization of Arabidopsis seedlings growth and development under trehalose feeding. J Cell Mol Res 1:1-9

Blazquez M, Santos E, Flores CI, Martinez-Zapater, Salinas J, and Gancedo C. 1998. Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. Plant J 5: 685-689.

Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J. 2001. Growth stage–based phenotypic analysis of Arabidopsis a model for high throughput functional genomics in plants. Plant Cell 13:1499-1510.

Chakrabarty J, Banerjee D, Pal D, De J, Ghosh A, Majumder GC. 2007. Shedding off specific lipid constituents from sperm cell membrane during cryopreservation. Cryobiology 54: 27-35.

Comai L, Dietrich RA, Maslyar DJ, Baden CS, Harada JJ. 1989. Co-ordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L. Plant Cell 1: 293-300.

Cornah JE, Germain V, Ward JL, Beale MH, Smith SM. 2004. Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase. J Biol Chem 279:42916-42923

Cooper TG, Beevers H. 1969. β-Oxidation in glyoxysomes from castor bean endosperm. J Biol Chem 244:3514-3520

Dellata TL, Sedijani P, Kondou Y, Matsui M, de jong GJ, Sosmen GW, Wiese-Klinkenberg A, Primavesi LF, Paul MJ, Schluepmann H (2011) Growth arrest by Trehalose-6-phosphate: an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway. J Plant Physiol 157: 160-174.

Elbein AD. 1974. The metabolism of alpha,alpha-trehalose. Adv Carbohydr Chem Biochem 30:227-256.

Elbein AD, Pan YT, Pastuszak I, Carroll D. 2003. New insights on trehalose: a multifunctional molecule. Glycobiology 13:17-27. 

Eastmond PJ, Germain V, Lange PR, Bryce JH, Smith SM, Graham IA. 2000. Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle. Proc Natl Acad Sci 97: 5669-5674.

Figueroa CM, Feil R, Ishihara H, Watanabe M, Kolling K, Krause U, HohneM, Encke B, Plaxton WC, Zeeman SC, Li Z, Schulze WX, Hoefgen R, Stitt M, Lunn J. 2016. Trehalose-6-phosphate coordinates organic and amino acid metabolism with carbon availability. Plant J 85:410-423.

Finkelstein RR, Lynch TJ. 2000. Abscisic acid inhibition of radicle emergence but not seedling growth is suppressed by sugars. Plant Physiol 122:1179-1186.

Gómez LD, Baud S, Gilday A, Li Y, Graham IA. 2006. Delayed embryo development in the Arabidopsis trehalose-6 phosphate synthase 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation. Plant J 46: 69-84

Graham IA, Smith LM, Leaver, CJ, Smith SM. 1990. Developmental regulation of expression of the malate synthase genes in transgenic plants. Plant Mol Biol 15:539-549.

Gut H, Matile P. 1988. Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley. Planta 176: 548-550.

Handel EV. 1968. Direct micro determination of sucrose. Anal Biochem 22:280-283.

Kolbe A, Tiessen A, Schluepmann H, Paul M, UlrichM, Geigenberger P. 2005. Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase. Proc Natl Acad Sci 102:11118-11123.

Lancashire P, Bleiholder H, Van Den Boom T, Langeluddke P, Stauss R, Weber E, Witezenberger A, 1991. A uniform decimal code for growth stages of crops and weeds. Ann Appl Biol 119: 561-601.

Lunn JE, Feil R, Hendriks JHM, Gibon Y, Morcuende R, Osunda D, Scheible W, Petronia Carillo P, Hajirezaei MR, Stitt M. 2006. Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADP-glucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana. Biochem J 397:139-148.

McKinney JD, Höner zu Bentrup K, Muñoz-Elías EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR JR, Russell DG. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735-738.

Muller J, Aeschbacher RA, Wingler A, Boller T, Wiemken A 2001. Trehalose and Trehalase in Arabidopsis. Plant Physiol 125:1086-1093.

Nounjan N, Nghia PT, Theerakulpisut P. 2012. Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 6: 596-604.

Oszvald M, Primavesi L, Griffiths CA, Cohn J, Basu SS, Nuccio ML, Paul M. 2018. Trehalose 6-phosphate regulates photosynthesis and assimilate partitioning in reproductive tissue. Plant Physiol 176: 2623-2630.

Paul MJ, Jhurreea D, Zhang Y, Primavesi LF, Delatte T, Schluepmann H, Wingler A. 2010. Upregulation of biosynthetic processes associated with growth by trehalose 6-phosphate. Plant Signal Behavior 5: 386-392.

Pellny Tk, Ghannoum O, Conroy JP, Schlupmann H, Smeekens S, Andralojc J, Krause KP, Goddijn O, Paul MJ. 2004. Genetic modification of photosynthesis with E. coli genes for trehalose synthesis. Plant Biotech J 2:71-82.

Penfield S, Li Y, Gilday AD, Graham S, Graham IA. 2006. Arabidopsis ABA INSENSITIVE 4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm. Plant Cell 18:1887-1899.

Pellicer MT, Fernandez C, Badia J, Aguilar J, Lin ECC, Baldoma L. 1999. Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection: characterization of the glc promoter. J Biol Chem 274:1745-1752.

Pracharoenwattana I, Cornah JE, Smith SM. 2005. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell 17:2037-2048.

Prado DE, Gonzales JA, Boero C, Sampietro AR. 1998. A simple method for reducing sugars in plant tissues. Application to quantify the sugar content in Quinoa (Chenopodium quinoa wild) seedlings. Phytochem Ann 9:58-63.

Pritchard SL, Charlton WL, Baker A, Graham IA. 2002. Germination and storage reserve mobilization are regulated independently in Arabidopsis. Plant J 31:639-647.

Ramon M, Rolland F, Johan M, Thevelein JM, Van Dijck P, Leyman B. 2007. ABI4 mediates the effects of exogenous trehalose on Arabidopsis growth and starch breakdown. Plant Mol Biol 63:195-206.

Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B. 2003. An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverses genetics. Plant Mol Biol 53:247-259.

Rylott EL, Hooks MA, Graham IA. 2001. Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana. Biochem Soc Trans 29:283-287.

Sadeghipour HR, Bhatla SC. 2002. Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings. Plant Cell Physiol 43:1117-1126.

Sarah CJ, Graham IA, Reynolds SJ, Leaver CJ, Smith SM. 1996. Distinct cis-acting elements direct the germination and sugar responses of the cucumber malate synthase gene. Mol Gen Genet 250:153-161.

Sambrook J, Russell DW. 2001. Purification of RNA from cells and tissues by acid phenol-guanidinium thiocyanate–chloroform extraction Molecular cloning: a laboratory manual cold spring Harbor laboratory Press, NewYork.

Schluepmann H, Pellny T, Van Dijken A, Smeekens S, Paul M. 2003. Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc Natl Acad Sci 11:6849-6854.

Schluepmann H, Van Dijken A, Aghdasi M, Wobbes B, Paul M, Smeekens S. 2004. Trehalose mediated growth inhibition of Arabidopsis seedlings is due to Trehalose-6-phosphate accumulation. Plant Physiol 135:879-890.

Siloto RM, Findlay K, Lopez-Villalobos A, Yeung EC, Nykiforuk CL, Moloney MM. 2006. The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis. Plant Cell 18:1961-1974.

Van Dijken AJ, Schuepmann H, Smeekens S. 2004. Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering. Plant Physiol 135:969-977.

Wahl V, Ponnu J, Schlereth A, Arrivault S, Langenecker T, Franke A, Feil R, Lunn JE, Stitt M, Schmid M. 2013. Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana. Science 339: 704-707.

Wingler A, Fritzius T, Wiemken A, Boller T, Aeschbacher RA. 2000. Trehalose induces the ADP-Glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis. Plant Physiol 124:105-114.

Yavada UP, Ivakov A, Feil R, Duan GY, Dirk Walther D, Giavalisco P, Piques M, Carillo P, Hubberten HM, Stitt M, Lunn JE. 2014. The sucrose–trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P. J Exp Bot 65:1051-1068.

Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RA, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ. 2009. Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol 4:1860-1871.

Zhan Li, Yue G, Yuchan Z, Cheng L, Dongting G, Yajing G, Jin H. 2018. Reactive oxygen species and gibberellin acid mutual induction to regulate tobacco seed germination. Fron Plant Sci 9:1-14.