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. We used this regulatory module to drive GFP/YFP and GUS expression in transgenic plants to identify mutants defective in nitrate signaling. Two mutants have been identified so far: a transcription factor (AtNLP7) and the nitrate transporter AtNRT1.1. 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 signaling function indicating that AtNRT1.1 acts as a nitrate transceptor (Cell 138: 1184-1194).
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. Using both forward and reverse genetics approaches, we have identified two genes that impact NO accumulation. AtNOA1 (originally named AtNOS1) 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 second gene we identified is AtPHB3. Atphb3 mutants have reduced NO accumulation after H2O2, ABA, light or salt treatment. Prohibitins are mitochondrial proteins that function in many processes including development, defense, oxidative stress and ethylene signaling. How they function in these processes is not known but our results show that many of these effects are likely to be mediated by NO.
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.