SUMMARYThe Persian walnut (Juglans regia L.), a diploid species native to the mountainous regions of Central Asia, is the major walnut species cultivated for nut production and is one of the most widespread tree nut species in the world. The high nutritional value of J. regia nuts is associated with a rich array of polyphenolic compounds, whose complete biosynthetic pathways are still unknown. A J. regia genome sequence was obtained from the cultivar 'Chandler' to discover target genes and additional unknown genes. The 667-Mbp genome was assembled using two different methods (SOAPdenovo2 and MaSuRCA), with an N50 scaffold size of 464 955 bp (based on a genome size of 606 Mbp), 221 640 contigs and a GC content of 37%. Annotation with MAKER-P and other genomic resources yielded 32 498 gene models. Previous studies in walnut relying on tissue-specific methods have only identified a single polyphenol oxidase (PPO) gene (JrPPO1). Enabled by the J. regia genome sequence, a second homolog of PPO (JrPPO2) was discovered. In addition, about 130 genes in the large gallate 1-b-glucosyltransferase (GGT) superfamily were detected. Specifically, two genes, JrGGT1 and JrGGT2, were significantly homologous to the GGT from Quercus robur (QrGGT), which is involved in the synthesis of 1-O-galloyl-b-D-glucose, a precursor for the synthesis of hydrolysable tannins. The reference genome for J. regia provides meaningful insight into the complex pathways required for the synthesis of polyphenols. The walnut genome sequence provides important tools and methods to accelerate breeding and to facilitate the genetic dissection of complex traits.

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, PPOsilenced 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.

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ABSTRACISalicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K1'PP of about 10 micromolar after 1 to 3 hours incubation. Inhibition appeared noncompetitive. Among 22 related phenolic compounds tested, only acetylsalicylic acid showed similar levels of inhibition. Inhibition by salicylic acid was inversely dependent on the pH of the culture medium and did not require a continuous external supply of salicylate. When compared to known inhibitors of the ethylene forming enzyme, cobalt, n-propyl gallate, and dinitrophenol, inhibition by salicylic acid most closely resembled that by dinitrophenol but salicylic acid did not produce the same degree of respiratory stimulation. Results are discussed in terms of other known effects of salicylic acid on plants, pH-dependency, and the possible influence of salicylic acid on electron transport.Applications of SA2 and ASA to plants have been shown to influence a wide variety of biological processes including flower stimulation (12), vegetative bud formation (4), adventitious root initiation (13), disease resistance (25), stomate function (15), and heat production (24). Recently Leslie and Romani (16) further demonstrated that these compounds strongly reduce the conversion of ACC to ethylene in pear cell suspension cultures, suggesting they inhibit EFE, the putative terminal enzyme in ethylene biosynthesis. Rapid inhibition, proportional to the concentration of SA or ASA in the medium, maximized within 2 h and was followed by a slower reversal requiring a period of hours to days. This paper further characterizes this inhibition and compares SA activity to that of several previously demonstrated EFE inhibitors. under continuous light at room temperature. Experimental additions or manipulations were made following a minimum 1-h equilibration period. ACC was routinely added to increase ethylene production and amplify the effects of SA or other inhibitors. It has been shown, however, that SA inhibits both endogenous and ACC-stimulated ethylene production (16). MATERIALS AND METHODSIn experiments calling for pH adjustment all culture aliquots were supplemented with 40 mm phosphate buffer and pH was adjusted with HCI or KOH as needed. At this level the P04 did not itselfaffect ethylene production. For SA removal, the cultures were centrifuged (International model HN, swinging bucket rotor) at 1000g for 5 min, the supernatant discarded, and the cells gently resuspended in medium of appropriate pH without SA.Ethylene measurements were a modification of the procedure used by Puschman and Romani (21). The 125 mL culture flasks were flushed with a vigorous air flow and capped for 30 min with rubber septa. Six mL head space samples were collected by syringe and ethylene concentrations measured by flame ionization gas chromatography using a Carle model 211 analytical gas chromatograph fitted with an alumina column held at 80°C and employing N2 as a carrier gas.Respiration readings employed an IR CO2 analyzer (H...

Background The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Findings Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Conclusion Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.

Summary Over the last 20 years, global production of Persian walnut ( Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome‐wide association ( GWA ) studies and genomic selection ( GS ) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high‐density 700K single nucleotide polymorphism ( SNP ) array in Persian walnut. Over 609K high‐quality SNP s have been thoroughly selected from a set of 9.6 m genome‐wide variants, previously identified from the high‐depth re‐sequencing of 27 founders of the Walnut Improvement Program ( WIP ) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNP s (55.7%) fell in the highest quality class of ‘ Poly High Resolution ’ ( PHR ) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent‐offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems™ Axiom™ J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.

One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm × J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profiles; the accessions with identical profiles appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identification, verification of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections.

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotianatabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP+ to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI–MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP+ as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-011-9739-3) contains supplementary material, which is available to authorized users.