First, in either the aerial or root tissues, GMPase activity levels in the 3 genotypes all were significantly lower when grown on NO3+ NH4+medium compared with the corresponding values when NO3medium used for the culture (Fig. in the growth inhibition ofArabidopsisby NH4+. Keywords:glycosylation, NH4+toxicity, unfolded protein response, L-ascorbic acid Ammonium (NH4+) is an essential ion in living cells. NH4+and nitrate (NO3) form the major sources of nitrogen nutrition for plants and microorganisms. Furthermore, NH4+is also an indispensable intermediate in the biosynthesis of essential cellular components. However, NH4+is toxic to cellular organisms when present in excess amounts (1). In fact, NH4+sensitivity is a widespread phenomenon in animals, plants, and fungi, although the levels of sensitivity differ considerably among different species (1). Plants, being unable to escape from harmful environments, are especially prone to NH4+-induced growth inhibition. Owing to the application of large quantities of nitrogen fertilizers in intensive agriculture, high levels of NH4+accumulation Aloe-emodin are becoming more common in many natural and agricultural soils (2). Consequently, NH4+toxicity has been linked to plant species extinction and decline of forest under certain ecological conditions in recent years (3). The molecular mechanisms underlying plant sensitivity to this ion are still unclear. Past studies have revealed important physiological changes (for example, acidification of external growth environment, disturbance in the acid/base balance, or excessive energy consumption in pumping the toxic level of NH4+out of cells) accompanying NH4+uptake and toxicity symptoms (1,2). However, the genetic determinant of NH4+sensitivity in plants remains unknown. Interestingly, recent biochemical investigations in animal cells have shown that NH4+sensitivity is linked with reduced efficiencies in protein glycosylation and the correct Aloe-emodin processing and secretion of glycoproteins (46). But, again, the genetic trigger for these changes has not been determined. From the fact that different plant families, or different species in the same plant family, can differ considerably in their growth response to NH4+(7), we deduce that NH4+sensitivity is genetically controlled. Identification of a Aloe-emodin genetic determinant should provide valuable clues for understanding the molecular mechanisms of NH4+sensitivity. To this end, we have taken a forward genetics approach by identifying anArabidopsis thalianamutant showing enhanced NH4+sensitivity than WT control and have characterized the hypersensitive response ofhsn1and its allelic mutantvtc1to NH4+. We found thathsn1was the result of a point mutation in the gene encoding GDP-mannose pyrophosphorylase (GMPase; EC 2.7.7.22), which has been established to be essential for synthesizing the vital cellular metabolite GDP-mannose inArabidopsis(8). We provide molecular genetic and biochemical evidence on the inhibition of GMPase by NH4+and describe the consequential downstream molecular events likely to be important for the inhibition ofArabidopsisgrowth by NH4+. The relevance of our finding on further studies of the mechanisms underlying NH4+sensitivity in higher plants is discussed. == Results == == Morphological and Physiological Characterization ofhsn1Mutant. == Previous studies have shown that Brassicaceae is one of the NH4+-sensitive plant families (1) and that the growth of the Col-0 ecotype ofArabidopsis, which is a member of Brassicaceae, is inhibited by physiological concentrations of NH4+(9). When cultured on defined media with both NO3and NH4+as nitrogen sources, the growth ofArabidopsis, especially the elongation of its roots, is increasingly inhibited by rising concentrations of NH4+(9). Based on these findings, we screened 20,000 ethylmethanesulfonate-mutagenized M2seedlings on a half-strength MurashigeSkoog (MS) medium (with 20 mM NO3and 10 mM NH4+as FASLG nitrogen sources, hereafter referred as NO3+ NH4+medium), and isolated a mutant exhibiting hypersensitivity to NH4+(named ashsn1, hypersensitive to NH4+1). Thehsn1mutant showed dramatically reduced growth of both aerial and root organs in the media containing NH4+irrespective of the presence or absence of NO3as an additional nitrogen source (Fig. 1). Moreover, the level of growth retardation ofhsn1, especially that of its root, increased substantially with rising NH4+concentrations (Fig. 1AandB), demonstrating that the growth ofhsn1(particularly its root) is more sensitive to NH4+than that of WTArabidopsis. A typical comparison of WT andhsn1seedlings germinated on NO3+ NH4+medium or a modified 1/2 MS medium with NO3as the sole nitrogen source (henceforth named as NO3medium) is shown inFig. 1C. Quantitative analysis confirmed that the primary root ofhsn1was markedly shorter than that of WT control on both NO3+ NH4+and NO3medium, with the difference in primary root length between the 2 genotypes being much larger on NO3+ NH4+medium (Fig. 1D). Consistent with Aloe-emodin previous findings (9), the primary root length of WTArabidopsisseedlings was also significantly reduced on the medium.