Crawford, Nigel - University of California, San Diego

个人简介

Nigel Crawford received his Ph.D. from the Massachusetts Institute of Technology. He was an NSF Postdoctoral Fellow in the Department of Biochemistry at Stanford University.

研究领域

Plants have evolved elaborate regulatory networks to respond rapidly to changes in the environment. Environmental and internal signals such as nitrogen and carbon metabolites, phytohormones, light, and circadian rhythms dramatically affect metabolic and developmental programs in plants. We are studying the metabolism of nitrate and are determining how it regulates plant metabolism and growth using microarray and mutant analyses in Arabidopsis. We have identified genes involved in nitrate uptake, reduction and regulation and have generated comprehensive datasets of nitrate-responsive genes. We have also studied nitric oxide metabolism and signaling and identified several genes that regulate nitric oxide accumulation and affect plant development and stress responses. Our work on nitrate uptake has focused on the functions of the AtNRT1.1 (CHL1) gene. AtNRT1.1 encodes a nitrate transporter whose expression is induced by nitrate, acid pH and carbon. For over twenty years, AtNRT1.1 was thought to function only in low affinity nitrate uptake in roots. Our work has shown that AtNRT1.1’s function is much broader. First, AtNRT1.1 contributes to high affinity nitrate uptake in young plants and thus spans both high and low affinity systems. AtNRT1.1's contribution to each system depends on the environmental conditions in which the plants are grown and the age of the plants. Second, AtNRT1.1 is induced by auxin and is preferentially expressed in actively growing regions of the plant (root tips, young leaves and flowers) and contributes to their growth. AtNRT1.1 is also expressed and functions in guard cells. Compared to wildtype plants, Atnrt1.1 mutants have reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO2 in the dark. At the cellular level, Atnrt1.1 mutants show reduced nitrate accumulation in guard cells during stomatal opening and fail to show a nitrate-induced depolarization of guard cells. In wildtype guard cells, nitrate induces depolarization, and nitrate concentrations increase three-fold during stomatal opening. These results identify the first anion transporter that functions in stomatal opening and suggest that nitrate plays an important role in stomatal movements and plant transpiration. In addition to serving as a nutrient and as an osmolyte, nitrate also acts as a signal. Nitrate reprograms nitrogen and carbon metabolism and influences root and shoot growth. To understand how plants perceive and respond to nitrate, we performed transcriptome analyses in Arabidopsis. Using ATH1 microarrays containing over 22,000 probe sets, we have uncovered over a thousand genes that are rapidly induced or repressed by nitrate treatment. In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase and enzymes in the pentose phosphate pathway), genes involved in glycolysis, trehalose-6-P biosynthesis, iron transport/metabolism, and sulfate uptake/reduction are also induced by nitrate. Because nitrate can be metabolized to nitrite, ammonium and amino acids, it is possible that downstream metabolites of nitrate could be the signals that induce some of these responses. To identify genes that respond specifically to nitrate, we constructed a nitrate reductase-null mutant in Arabidopsis. Microarray analysis revealed that almost 600 genes respond to nitrate (5 mM nitrate for 2 h) in both WT and mutant plants. This group of genes is over-represented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was nitrate reductase-independent, nitrate and not a downstream metabolite served as the signal for these genes. A different suite of genes was identified that require nitrate reduction for their response, implicating a downstream metabolite as the signal. We have shown that nitrite, the product of nitrate reduction, can act as a signal that is more potent than nitrate itself. To elucidate the genes and mechanisms that mediate these responses, we have developed several tools to facilitate our studies of nitrate signaling. We have developed a transient expression system to assay nitrate induction of gene expression and used it to identify a powerful cis-regulatory module that contains three nitrate enhancer elements (Plant Phys 154: 423-432). We used this regulatory module to drive GFP/YFP and GUS expression in transgenic plants to identify mutants defective in nitrate signaling. The first two genes identified encoded a transcription factor (AtNLP7) and the nitrate transporter AtNRT1.1 (Plant Phys. 151: 472-478). Atnrt1.1 mutants are defective in nitrate signaling under conditions where nitrate uptake is replete; thus, AtNRT1.1 serves as a nitrate regulator as well as transporter. Independently, the lab of Dr. Yi-Fang Tsay has shown that AtNRT1.1 transport function can be separated from its signaling function indicating that AtNRT1.1 acts as a nitrate transceptor (Cell 138: 1184-1194). In collaboration with Dr. Yong Wang’s lab at Shandong Agricultural University, we identified a third gene encoding a novel, nuclear-localized protein (NRG2) that functions upstream of NRT1.1 to regulate nitrate-responsive genes (Plant Cell 28: 485-504). Lastly, we used the nitrate enhancer DNA in a one-hybrid screen of transcription factors. We identified a member of a large, plant-specific gene family (TCP20) that plays a role in nitrate foraging in plant roots, specifically in the systemic response system. These tools are making it possible to identify and study numerous genes that function in nitrogen signaling.

近期论文

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Wang, R., X. Xing, Y. Wang, A. Tran, and N.M. Crawford (2009) A genetic screen for nitrate-regulatory mutants captures the nitrate transporter gene NRT1.1. Plant Physiol., 151: 472-478. PMID: 19633234 Krouk, G., N.M. Crawford, G.M. Coruzzi, Y.-F. Tsay (2010) Nitrate signaling: adaptation to fluctuating environments. Current Opinion Plant Biology, 13: 265-272. PMID: 20093067 Wang, Y., A. Ries, K. Wu, A. Yang, N.M. Crawford (2010) The Arabidopsis prohibitin gene PHB3 functions in nitric oxide-mediated responses and in hydrogen peroxide-induced nitric oxide accumulation. Plant Cell, 22: 249-259. PMID: 20068191 Wang, R., P. Guan, M. Chen, X. Xing, Y. Zhang, N.M. Crawford (2010) Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer. Plant Phys. 154: 423-432. PMID: 20668061 Krouk, G., S. Ruffel, R.A. Gutiérrez, A. Gojon, N.M. Crawford, G.M. Coruzzi, B. Lacombe (2011) A framework integrating plant growth with hormones and nutrients. Trends Plant Sci., 16: 178-182. PMID: 21393048 Guan, P., R. Wang, P. Nacry, G. Breton, S.A. Kay, J.L. Pruneda-Paz, A. Davani, N.M. Crawford (2014) Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway. Proc. Natl. Acad. Sci., 111: 15267- 15272. PMID: 25288754 Medici, A., A. Marshall-Colon, E. Ronzier, W. Szponarski, R. Wang, A. Gojon, N.M. Crawford, S. Ruffel, G.M. Coruzzi, G. Krouk (2015) AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. Nature Communications, 6: 6274, doi:10.1038/ncomms7274. PMID: 25723764 Xu, N., R. Wang, L. Zhao, C. Zhang, Z. Li, Z. Lei, F. Liu, P. Guan, Z. Chu, N.M. Crawford, Y. Wang (2016) The Arabidopsis NRG2 protein mediates nitrate signaling and interacts with and regulates key nitrate regulators. The Plant Cell, 28: 485-504. PMID: 26744214