Nitrate-Regulated Glutaredoxins Control Arabidopsis Primary Root Growth.

Nitrogen is an essential soil nutrient for plants, and lack of nitrogen commonly limits plant growth. Soil nitrogen is typically available to plants in two inorganic forms: nitrate and ammonium. To better understand how nitrate and ammonium differentially affect plant metabolism and development, we performed transcriptional profiling of the shoots of ammonium-supplied and nitrate-supplied Arabidopsis (Arabidopsis thaliana) plants. Seven genes encoding class III glutaredoxins were found to be strongly and specifically induced by nitrate. RNA silencing of four of these glutaredoxin genes (AtGRXS3/4/5/8) resulted in plants with increased primary root length (approximately 25% longer than the wild type) and decreased sensitivity to nitrate-mediated inhibition of primary root growth. Increased primary root growth is also a well-characterized phenotype of many cytokinin-deficient plant lines. We determined that nitrate induction of glutaredoxin gene expression was dependent upon cytokinin signaling and that cytokinins could activate glutaredoxin gene expression independent of plant nitrate status. In addition, crosses between "long-root" cytokinin-deficient plants and "long-root" glutaredoxin-silenced plants generated hybrids that displayed no further increase in primary root length (i.e. epistasis). Collectively, these findings suggest that AtGRXS3/4/5/8 operate downstream of cytokinins in a signal transduction pathway that negatively regulates plant primary root growth in response to nitrate. This pathway could allow Arabidopsis to actively discriminate between different nitrogen sources in the soil, with the preferred nitrogen source, nitrate, acting to suppress primary root growth (vertical dimension) in concert with its well-characterized stimulatory effect on lateral root growth (horizontal dimension).

Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut.

The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants.

Nitrogen is the only macronutrient that is commonly available to plants in both oxidized and reduced forms, mainly nitrate and ammonium. The physiological and molecular effects of nitrate supply have been well studied, but comparatively little is known about ammonium nutrition and its differential effects on cell function and gene expression. We have used a physiologically realistic hydroponic growth system to compare the transcriptomes and redox status of the roots of ammonium- and nitrate-supplied Arabidopsis thaliana plants. While approximately 60% of nitrogen-regulated genes displayed common responses to both ammonium and nitrate, significant 'nitrate-specific' and 'ammonium-specific' gene sets were identified. Pathways involved in cytokinin response and reductant generation/distribution were specifically altered by nitrate, while a complex biotic stress response and changes in nodulin gene expression were characteristic of ammonium-supplied plants. Nitrate supply was associated with a rapid decrease in H(2)O(2) production, potentially because of an increased export of reductant from the mitochondrial matrix. The underlying basis of the nitrate- and ammonium-specific patterns of gene expression appears to be different signals elaborated from each nitrogen source, including alterations in extracellular pH that are associated with ammonium uptake, downstream metabolites in the ammonium assimilation pathway, and the presence or absence of the nitrate ion.

Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses.

pH is a highly variable environmental factor for the root, and plant cells can modify apoplastic pH for nutrient acquisition and in response to extracellular signals. Nevertheless, surprisingly few effects of external pH on plant gene expression have been reported. We have used microarrays to investigate whether external pH affects global gene expression. In Arabidopsis thaliana roots, 881 genes displayed at least twofold changes in transcript abundance 8 h after shifting medium pH from 6.0 to 4.5, identifying pH as a major affector of global gene expression. Several genes responded within 20 min, and gene responses were also observed in leaves of seedling cultures. The pH 4.5 treatment was not associated with abiotic stress, as evaluated from growth and transcriptional response. However, the observed patterns of global gene expression indicated redundancies and interactions between the responses to pH, auxin and pathogen elicitors. In addition, major shifts in gene expression were associated with cell wall modifications and Ca(2+) signalling. Correspondingly, a marked overrepresentation of Ca(2+)/calmodulin-associated motifs was observed in the promoters of pH-responsive genes. This strongly suggests that plant pH recognition involves intracellular Ca(2+). Overall, the results emphasize the previously underappreciated role of pH in plant responses to the environment.

Mitochondrial NAD(P)H oxidation pathways and nitrate/ammonium redox balancing in plants.

Plant mitochondrial oxidative phosphorylation is characterised by alternative electron transport pathways with different energetic efficiencies, allowing turnover of cellular redox compounds like NAD(P)H. These electron transport chain pathways are profoundly affected by soil nitrogen availability, most commonly as oxidized nitrate (NO3-) and/or reduced ammonium (NH4+). The bioenergetic strategies involved in assimilating different N sources can alter redox homeostasis and antioxidant systems in different cellular compartments, including the mitochondria and the cell wall. Conversely, changes in mitochondrial redox systems can affect plant responses to N. This review explores the integration between N assimilation, mitochondrial redox metabolism, and apoplast metabolism.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

Agrobacterium tumefaciens as an agent of disease.

Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer. Since this discovery, A. tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange. It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology. These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research. Now, the diverse areas of A. tumefaciens research are again converging because new discoveries in transformation biology and the use of A. tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.

The AtGRXS3/4/5/7/8 glutaredoxin gene cluster on Arabidopsis thaliana chromosome 4 is coordinately regulated by nitrate and appears to control primary root growth.

Nitrate and ammonium are the 2 most common forms of inorganic nitrogen available to plants in the soil. We previously identified a group of class III glutaredoxin genes whose expression is strongly upregulated by nitrate, but not ammonium, in Arabidopsis thaliana shoots and roots. A reverse genetics approach was used to functionally characterize a subset of these nitrate-regulated glutaredoxins, and we found that the AtGRXS3,4,5, and 8 genes function as negative regulators of primary root growth. AtGRXS3/4/5/8 are arranged in a tandem array on Arabidopsis chromosome 4, and these genes show very high levels of sequence similarity. Interestingly, there is one additional glutaredoxin, AtGRXS7, in this same gene cluster, but this gene was not identified as nitrate-responsive in our previous studies. We show here that AtGRXS7 is upregulated by nitrate and shows strong co-expression with the other glutaredoxins in this gene cluster. Further, AtGRXS7 was effectively silenced by the RNAi construct used to target AtGRXS3/4/5/8 for previous functional analyses. Overall, it appears that the 5 genes in the AtGRX3/4/5/7/8 cluster share virtually identical sequences, regulatory patterns, and functions, collectively acting to regulate primary root growth in response to soil nitrate.

An alternatively spliced domain of the NDC1 NAD(P)H dehydrogenase gene strongly influences the expression of the ACTIN2 reference gene in Arabidopsis thaliana.

In plant respiratory chains, alternative pathways for NAD(P)H oxidation are mediated by type II NAD(P)H dehydrogenases belonging to the NDA, NDB, and NDC families. For the latter type, Arabidopsis thaliana contains a single gene, NDC1, whose functional role has not previously been analyzed in the plant. We found that A. thaliana NDC1 is alternatively spliced. Four base pairs at the 3' end of intron 5 are spliced out in NDC1-1, but retained in the NDC1-2 mRNA, which therefore contains a truncated reading frame. Both variants are conserved in dicotyledonous and monocotyledonous plants and their relative abundance varies between organs and in response to light. Three analyzed NDC1 T-DNA insertion lines all displayed an early bolting phenotype. A dramatic upregulation of ACTIN2 was characteristic of two lines containing T-DNA inserts upstream of intron 5, whereas a line with an insertion downstream of the NDC1-2 reading frame had an ACTIN2 expression level identical to the wildtype. Thus, the alternatively spliced 5' domain of NDC1 strongly influences the expression of the functionally unrelated ACTIN2, which is a common reference gene for quantitative RT-PCR. Also for other reference genes, strong expressional effects were observed when comparing various mutants and wildtypes in microarray databases.

Reorganization of the alternative pathways of the Arabidopsis respiratory chain by nitrogen supply: opposing effects of ammonium and nitrate.

The mitochondrial oxidative phosphorylation system in plants possesses a variety of alternative pathways that decrease respiratory ATP production. These alternative pathways are mediated by three classes of bypass proteins: the type II NAD(P)H dehydrogenases (which circumvent complex I of the electron transport chain), the alternative oxidases (AOXs; which circumvent complexes III and IV) and the uncoupling proteins (which circumvent ATP synthase). We have monitored the expression of all genes encoding respiratory bypass proteins in Arabidopsis thaliana growing with different sources of inorganic nitrogen (N). Resupply of nitrate (NO) to N-limited seedling cultures caused a decrease in the transcript abundance of several type II NAD(P)H dehydrogenase and AOX genes, while resupply of ammonium (NH) led to broad increases in expression in the same gene families. Similar results were observed upon switching between nitrate and ammonium in the absence of N stress. Nitrate signalling was found to be mediated primarily by the nitrate ion itself, whereas ammonium regulation was dependent upon assimilation and affected by changes in apoplastic pH. Corresponding alterations in alternative respiratory pathway capacities were apparent in seedlings supplied with either nitrate or ammonium as an N source and in mitochondria purified from the seedlings. Specifically, AOX capacity and protein abundance, as well as calcium-dependent external NADH oxidation, were substantially elevated after growth on ammonium. The increased capacity of respiratory bypass pathways after switching from nitrate to ammonium was correlated to an overall respiratory increase.

Light regulation of the Arabidopsis respiratory chain. Multiple discrete photoreceptor responses contribute to induction of type II NAD(P)H dehydrogenase genes.

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism.