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GDP-Mannose Pyrophosphorylase Is a Genetic Determinant of Ammonium Sensitivity in Arabidopsis Thaliana

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GDP-Mannose Pyrophosphorylase Is a Genetic Determinant of Ammonium Sensitivity in Arabidopsis Thaliana GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana Cheng Qina, Weiqiang Qianb, Wenfeng Wanga, Yue Wua, Chunmei Yub, Xinhang Jianga, Daowen Wangb,1, and Ping Wua,1 aThe State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China; and bThe State Key Laboratory of Plant Cell and Chromosomal Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved September 30, 2008 (received for review June 28, 2008) Higher plant species differ widely in their growth responses to genetically controlled. Identification of a genetic determinant ؉ ammonium (NH4 ). However, the molecular genetic mechanisms should provide valuable clues for understanding the molecular ؉ ϩ underlying NH4 sensitivity in plants remain unknown. Here, we mechanisms of NH4 sensitivity. To this end, we have taken a report that mutations in the Arabidopsis gene encoding GDP- forward genetics approach by identifying an Arabidopsis thaliana ϩ mannose pyrophosphorylase (GMPase) essential for synthesizing mutant showing enhanced NH4 sensitivity than WT control and ؉ GDP-mannose confer hypersensitivity to NH4 . The in planta activ- have characterized the hypersensitive response of hsn1 and its ؉ ϩ ities of WT and mutant GMPases all were inhibited by NH4 , but the allelic mutant vtc1 to NH4 . magnitude of the inhibition was significantly larger in the mutant. We found that hsn1 was the result of a point mutation in the Despite the involvement of GDP-mannose in both L-ascorbic acid gene encoding GDP-mannose pyrophosphorylase (GMPase; EC (AsA) and N-glycoprotein biosynthesis, defective protein glycosyl- 2.7.7.22), which has been established to be essential for synthe- ation in the roots, rather than decreased AsA content, was linked sizing the vital cellular metabolite GDP-mannose in Arabidopsis ؉ to the hypersensitivity of GMPase mutants to NH4 . We conclude (8). We provide molecular genetic and biochemical evidence on ؉ ϩ that NH4 inhibits GMPase activity and that the level of GMPase the inhibition of GMPase by NH4 and describe the consequen- activity regulates Arabidopsis sensitivity to NH؉. Further analysis tial downstream molecular events likely to be important for the 4 ϩ showed that defective N-glycosylation of proteins, unfolded pro- inhibition of Arabidopsis growth by NH . The relevance of our 4 ϩ tein response, and cell death in the roots are likely important finding on further studies of the mechanisms underlying NH4 downstream molecular events involved in the growth inhibition of sensitivity in higher plants is discussed. ؉ Arabidopsis by NH4 . Results ͉ ϩ ͉ ͉ glycosylation NH4 toxicity unfolded protein response L-ascorbic acid Morphological and Physiological Characterization of hsn1 Mutant. Previous studies have shown that Brassicaceae is one of the ϩ ϩ ϩ mmonium (NH ) is an essential ion in living cells. NH and NH4 -sensitive plant families (1) and that the growth of the Col-0 Ϫ 4 4 ecotype of Arabidopsis, which is a member of Brassicaceae, is Anitrate (NO3 ) form the major sources of nitrogen nutrition ϩ ϩ inhibited by physiological concentrations of NH4 (9). When for plants and microorganisms. Furthermore, NH4 is also an Ϫ ϩ indispensable intermediate in the biosynthesis of essential cel- cultured on defined media with both NO3 and NH4 as nitrogen ϩ lular components. However, NH is toxic to cellular organisms sources, the growth of Arabidopsis, especially the elongation of 4 ϩ its roots, is increasingly inhibited by rising concentrations of when present in excess amounts (1). In fact, NH4 sensitivity is ϩ a widespread phenomenon in animals, plants, and fungi, al- NH4 (9). Based on these findings, we screened 20,000 ethyl- methanesulfonate-mutagenized M2 seedlings on a half-strength though the levels of sensitivity differ considerably among dif- Ϫ Murashige–Skoog (MS) medium (with 20 mM NO and 10 mM ferent species (1). Plants, being unable to escape from harmful ϩ 3 Ϫ ϩ ϩ ϩ environments, are especially prone to NH -induced growth NH4 as nitrogen sources, hereafter referred as NO3 NH4 4 medium), and isolated a mutant exhibiting hypersensitivity to inhibition. Owing to the application of large quantities of ϩ ϩ ϩ nitrogen fertilizers in intensive agriculture, high levels of NH NH4 (named as hsn1, hypersensitive to NH4 1). The hsn1 4 mutant showed dramatically reduced growth of both aerial and accumulation are becoming more common in many natural and ϩ ϩ root organs in the media containing NH4 irrespective of the agricultural soils (2). Consequently, NH4 toxicity has been Ϫ linked to plant species extinction and decline of forest under presence or absence of NO3 as an additional nitrogen source (Fig. 1). Moreover, the level of growth retardation of hsn1, certain ecological conditions in recent years (3). ϩ The molecular mechanisms underlying plant sensitivity to this especially that of its root, increased substantially with rising NH4 concentrations (Fig. 1 A and B), demonstrating that the growth ion are still unclear. Past studies have revealed important ϩ physiological changes (for example, acidification of external of hsn1 (particularly its root) is more sensitive to NH4 than that growth environment, disturbance in the acid/base balance, or of WT Arabidopsis. A typical comparison of WT and hsn1 ϩ excessive energy consumption in pumping the toxic level of NH ϩ 4 out of cells) accompanying NH uptake and toxicity symptoms 4 ϩ Author contributions: C.Q., D.W., and P.W. designed research; C.Q., W.Q., W.W., Y.W., X.J., (1, 2). However, the genetic determinant of NH4 sensitivity in and P.W. performed research; P.W. contributed new reagents/analytic tools; C.Q., C.Y., and plants remains unknown. Interestingly, recent biochemical in- P.W. analyzed data; and C.Q., D.W., and P.W. wrote the paper. ϩ vestigations in animal cells have shown that NH4 sensitivity is The authors declare no conflict of interest. linked with reduced efficiencies in protein glycosylation and the This article is a PNAS Direct Submission. correct processing and secretion of glycoproteins (4–6). But, 1To whom correspondence may be addressed. E-mail: [email protected] or again, the genetic trigger for these changes has not been deter- [email protected]. mined. From the fact that different plant families, or different This article contains supporting information online at www.pnas.org/cgi/content/full/ species in the same plant family, can differ considerably in their 0806168105/DCSupplemental. ϩ ϩ growth response to NH4 (7), we deduce that NH4 sensitivity is © 2008 by The National Academy of Sciences of the USA 18308–18313 ͉ PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806168105 20 mM NO - No NO - A 3 B 3 + + + + + + + 0 mM NH4 0.1 mM NH4 1 mM NH4 10 mM NH4 0.1 mM NH4 1 mM NH4 10 mM NH4 WT hsn1 WT hsn1 WT hsn1 WT hsn1 WT hsn1 WT hsn1 WT hsn1 C NO - + NH + NO - D E 100 3 4 3 6 WT, aerial WT, NO - + NH + hsn1 3 4 hsn1, aerial WT WT hsn1 5 hsn1 - + 80 , NO3 + NH4 WT, root WT, NO - hsn1, root 4 3 hsn1, NO - 60 3 3 40 2 20 1 FW) (µmol/g Content + 0 4 0 NH - + - Primary root length (cm) length root Primary 246810 NO3 + NH4 NO3 DAG Fig. 1. Phenotypes of hsn1mutant. (A and B) Comparisons of seedling growth of WT Arabidopsis and hsn1 on modified MS media at 10 days after germination Ϫ ϩ (DAG). In A, the nitrogen source included 20 mM NO3 with increasing concentrations of NH4 (from 0 to 10 mM). In B, the nitrogen source was provided solely ϩ Ϫ ϩ ϩ Ϫ by ascending concentrations of NH4 (from 0.1 to 10 mM). (Bars: 1 cm.) (C) A typical example of WT and hsn1 seedlings cultured on NO3 NH4 or NO3 medium. Ϫ ϩ ϩ Ϫ (Bars: 1 cm.) (D) Comparisons of postgerminative root growth between WT Arabidopsis and hsn1 on NO3 NH4 and NO3 medium. Statistically significant Ͻ ϩ ␮ differences (P 0.05) were found between the 4 data points at 8 or 10 DAG. (E)NH4 contents (expressed as mol/g of fresh weight) in the aerial and root tissues Ϫ ϩ ϩ Ϫ Ͻ ϩ of WT Arabidopsis and hsn1 seedlings (10 DAG) grown on NO3 NH4 or NO3 medium. Significant differences (P 0.01) were found between the NH4 contents Ϫ ϩ ϩ Ϫ of aerial and root tissues for both WT Arabidopsis and hsn1 grown on either NO3 NH4 or NO3 medium. Ϫ ϩ seedlings germinated on NO ϩ NH medium or a modified 1/2 GMPase is a highly conserved protein in eukaryotic organ- Ϫ 3 4 MS medium with NO3 as the sole nitrogen source (henceforth isms, with nearly 60% identity among Arabidopsis, human, and Ϫ Ͼ named as NO3 medium) is shown in Fig. 1C. Quantitative yeast orthologs and 85% identity between the orthologs from analysis confirmed that the primary root of hsn1 was markedly dicot and monocot plant species (data not shown). GMPase is Ϫ ϩ ϩ Ϫ shorter than that of WT control on both NO3 NH4 and NO3 necessary for synthesizing GDP-mannose, which is indispensable medium, with the difference in primary root length between the for proper N-glycosylation of proteins in eukaryotic cells (11). In Ϫ ϩ ϩ 2 genotypes being much larger on NO3 NH4 medium (Fig. higher plants, GDP-mannose is also an important intermediate 1D). Consistent with previous findings (9), the primary root for the biosynthesis of ascorbic acid (AsA) through the length of WT Arabidopsis seedlings was also significantly reduced Smirnoff–Wheeler pathway (12).
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