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.

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 ∼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 H₂O₂ 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.

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).

The enzyme polyphenol oxidase (PPO) is nearly ubiquitous in Kingdom Plantae and catalyzes the oxidation of phenolic compounds into highly reactive quinones. Although the functional importance of PPO in plants remains uncertain, a putative antipathogen role for walnut ( Juglans regia ) PPO was posited as early as 1911. However, despite the rich diversity of phenolics present in walnut leaves and hulls, walnut PPO has been little studied since the early 1900s. We cloned a PPO-encoding gene from a walnut pistillate flower cDNA library and designated the gene jrPPO1 . Genomic Southern analysis demonstrated that jrPPO1 is the sole PPO gene in walnut. Transgenic tobacco ( Nicotiana tabacum ) plants expressing jrPPO1 display greater than 10-fold increases in leaf PPO activity compared with wild-type tobacco, demonstrating that jrPPO1 encodes a functional enzyme. The jrPPO1 protein is expressed primarily in the leaves, hulls, and flowers of walnut trees and is not regulated by wounding or methyl jasmonate. To examine whether walnut PPO could affect pathogen resistance, tobacco plants expressing jrPPO1 were challenged with Pseudomonas syringae pv. tabaci . Based on both symptom development and quantitative analyses of bacterial growth in planta, the PPO-expressing plants did not display increased resistance to this pathogen. Leaf extract browning assays indicated that tobacco leaves lack the endogenous phenolic substrates required for significant jrPPO1 activity and quinone production in planta.

Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, and Arabidopsis thaliana has 21 class III GRXs, which remain largely uncharacterized. About 80% of A. thaliana class III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate‐induced GRX genes, AtGRXS5 and AtGRXS8, defining their roles in signaling and development in the A. thaliana root. We demonstrated that AtGRXS5 and AtGRXS8 are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo‐cytosolic subcellular localization. Ectopic expression of AtGRXS8 in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots of AtGRXS8‐overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high‐affinity nitrate transporters. Correspondingly, high‐affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised in AtGRXS8‐overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate in A. thaliana roots. Overall, these results suggest that AtGRXS8 acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors.

SUMMARY: Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate fundamental questions related to Agrobacterium biology and post‐transcriptional gene silencing (PTGS). The oncogene‐silenced transgenic tomato line 01/6 was used to characterize A. tumefaciens growth in planta and to screen for iaaM and ipt sequence variants. Even in the absence of macroscopic and microscopic indications of tumorigenesis, A. tumefaciens is capable of long‐term survival in the hypocotyl tissues of the 01/6 line. A. tumefaciens growth, however, is significantly reduced in the 01/6 line, with populations decreased by 96% relative to wild‐type at 52 days post‐inoculation. In addition, the 01/6 line displayed suppression of tumorigenesis against all 35 tested strains of A. tumefaciens . High target homology is an absolute requirement of PTGS, therefore this result suggests that regions of the iaaM and ipt oncogenes are very highly conserved across most A. tumefaciens strains. Finally, graft transmissibility of oncogene silencing was assessed by grafting various non‐silenced tomato genotypes on to the 01/6 line. Phenotypic and molecular evidence (tumorigenesis and absence of small interfering RNAs, respectively) suggest that oncogene silencing is not graft‐transmissible, at least to wild‐type and antisense iaaM ‐over‐expressing genotypes.