Nigel M. Crawford
Professor
Section of Cell and Developmental Biology, UCSD

e-mail: ncrawford@ucsd.edu
Lab Homepage: Crawford Lab

    Plants have evolved elaborate regulatory networks that allow rapid responses 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 nitric oxide and are determining how these two molecules regulate plant metabolism and growth using microarray and mutant analyses in Arabidopsis. We have identified genes involved in nitrate uptake and reduction and have generated comprehensive datasets of nitrate-responsive genes.  We have also identified a gene that regulates nitric oxide synthesis or accumulation and affects flower timing.

    Our work on nitrate uptake has focused on the role 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. The lab of Yi-Fang Tsay has shown that AtNRT1.1 is a dual-affinity nitrate transporter that is regulated by phosphorylation. Second, AtNRT1.1 is preferentially expressed in actively growing regions of the plant (root tips, young leaves and flowers) and contributes to their growth. Lastly and most remarkable, AtNRT1.1 is 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 genomic 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, some of the observed responses could be due to downstream metabolites. To identify genes that respond specifically to nitrate, we constructed a nitrate reductase-null mutant 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. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there is substantial organ-specificity to the nitrate response. Lastly, we have identified many potential regulatory genes that respond to nitrate. We are studying this suite of genes in the hope of uncovering components in the nitrate signaling system.

    Besides nitrate, there is another inorganic form of nitrogen that plays a very important role in signaling. This molecule is nitric oxide (NO). NO is a reactive, free radical that functions in plant development, metabolism and disease responses by acting as an intermediate in signal transduction pathways that mediate hormone responses, programmed cell death, and defense gene induction. The source of NO for these signaling events has been quite a mystery. There has been accumulating evidence that NO can be made in plants by a nitric oxide synthase activity (i.e. from arginine) similar to what has been found in animal systems; however, no gene or protein that could account for this activity had been uncovered.  We have discovered a gene AtNOA1 (originally named AtNOS1) that controls NO synthesis or accumulation. Mutations in this gene reduce NO accumulation in shoots and roots in response to ABA and pathogen infection, and many NO-regulated processes (seed germination, shoot and root growth, fertility and flower timing) are impaired in the mutant.  Leaves from AtNOA1 mutants senesce earlier than wildtype plants in the dark, indicating that AtNOA1 protects plants from senescence.  The protein is targeted to mitochondria and affects cellular oxidation processes.  We are continuing our studies of NO synthesis and signaling in Arabidopsis with special focus on elucidating the mechanism(s) of NO synthesis under aerobic conditions.

A complete list of our publications is available on our lab web page: http://www.biology.ucsd.edu/labs/crawford/

Information on our Division's Plant Systems Biology Training Grant for graduate students can be found at: http://www-biology.ucsd.edu/psbigert/

Wang, R., X. Xing and N. Crawford (2007) Nitrite acts as a transcriptome signal at micromolar concentrations in Arabidopsis roots. Plant Physiol., 145: 1735-1745.

Tischner, R., M. Galli, Y.M. Heimer, S. Bielefeld, M. Okamoto, A. Mack and N.M. Crawford (2007) Interference of the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts. FEBS Journal, 274: 4238-4245.

Guo, F.-Q. and N.M. Crawford (2005) Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence. Plant Cell, 17: 3436-3450.

Wang, R., R. Tischner, R.A. Gutierrez, M. Hoffman, X. Xing, M. Chen, G. Coruzzi and N.M. Crawford (2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiol. 136: 2512-2522.

Guo, F.-Q., J. Young and N.M. Crawford (2003) The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell, 15:107-117.

    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.