Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using b-glucuronidase fusion constructs with the promoters of both genes. As evaluated by b-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 doubleknockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.

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.

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The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software.

Plants respond to pathogens with both active and passive defense mechanisms. These defense responses include the induction of defense or defense-related genes such as polyphenol oxidase (PPO) and pathogenesis-related (PR) proteins. The role of PPO in the interaction between bacterial blight [Xanthomonas arboricola pv. juglandis (Xaj)] and walnut (Juglans regia) was studied. JrPPO-1 and P14a genes were identified in two walnut cultivars, Chandler and Serr, using standard polymerase chain reaction (PCR) to understand their inducible ability in response to Xaj. ‘Serr’ and ‘Chandler’ were inoculated with Xaj strain 417. PPO activity in leaves was assayed at 0, 24, 72, 96, 120, and 144 hours after inoculation. Results showed a steady increase in activity commencing within 24 hours of inoculation. Increase in PPO activity was close to 2-fold greater in ‘Chandler’ than in ‘Serr’ at all time points examined. Real-time PCR analysis showed differences between cultivars in PPO gene expression. The JrPPO-1 gene was highly expressed in both cultivars 24 hours after inoculation but expression in ‘Serr’ was much greater than in ‘Chandler’. Significant expression of P14a gene was observed in both cultivars within 24 hours. Expression in ‘Serr’ was strong and maximized with a significant increase at 96 hours. Expression in ‘Chandler’ was far weaker than ‘Serr’ at 24 hours and did not increase further. Our results imply that the walnut–bacterial blight interaction induces the expression of JrPPO-1 and P14a as well as the activity of PPO.

The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software.

Plant structural traits can act as barriers for herbivore attachment, feeding, and oviposition. In particular, epicuticular waxes (EWs) on the aerial surfaces of many land plants offer protection from biotic and abiotic stresses. In rice (Oryza sativa L.), mutations that reduce EWs have been previously reported. However, whether such mutations affect rice water weevil (Lissorhoptrus oryzophilus Kuschel) and fall armyworm (Spodoptera frugiperda Smith) performance has not been investigated yet. These pests cause significant economic problems in important rice-producing areas of the United States. The aim of our study was to characterize the EWs of EW mutants and wild-type rice plants by gas chromatography–mass spectrometry and compare the resistance of mutant and wild-type plants against rice water weevil and fall armyworm. We hypothesized that mutants with reduced EWs would have weaker resistance to pests than wild-type plants. Three mutant lines (6-1A, 7-17A, and 11-39A) and their wild-type parent (cv. ‘Sabine’) were used to test this hypothesis. Levels of EWs were significantly lower in mutant lines than in the wild-type, and qualitative differences in EW composition were also observed. Reduction in EWs significantly affected performance of insects in experiments conducted under greenhouse conditions. Experiments with rice water weevils were conducted in arenas in which females were given a choice of the mutants and the wild-type for oviposition. Number of first instars emerging from the three EW mutants (an indication of oviposition preference) was higher on the three EW mutants than on wild-type plants with normal wax levels. Similarly, in no-choice experiments using whole plants or detached leaves, weight gains of armyworms on leaves were higher on the mutant lines than on the wild-type. These results indicate that EW traits are involved in rice resistance to weevils and armyworms. Understanding the plant traits that contribute to resistance to rice pests will be helpful for the development of resistant varieties for reducing pest insect damage.

We showed previously that gallic acid is produced in walnut from 3-dehydroshikimate by a shikimate dehydrogenase (JrSkDH). This study focuses on the next step in the hydrolysable tannin pathway, the formation of 1-O-galloyl-β-D-glucose from the phenolic gallic acid and UDP glucose by a glycosyltransferase. JrGGT1 (UGT84A73) and JrGGT2 (UGT84A74) are predicted to be two such glycosyltransferases, which we expressed in tobacco plants. GC-MS analysis of the transgenic tobacco revealed moderate, yet significant alterations in plant secondary metabolism, such as depleted phenolic acids, including gallic acid. We postulate that these effects are due to JrGGT1 and JrGGT2 activity, as JrGGT orthologs glycosylate these phenolic compounds in vitro. Moreover, JrGGT expression in tobacco caused upregulation of shikimic acid pathway metabolites and differing responses in phenylpropanoids, such as phenolic acids and flavonoids. In transcriptome analysis of walnut pellicle tissues, both JrGGTs showed substantial and significant expression correlations with the gallic acid-producing JrSkDHs and were highly coexpressed with the genetic circuits constituting the shikimic acid and phenylpropanoid biosynthetic pathways. Verification of JrGGT gene expression by transcriptome analysis of 20 walnut tissues revealed striking similarities with that of the pellicle data, with the greatest expression in roots, wood, buds, and leaves of Juglans regia cv. Chandler: tissues that typically accumulate hydrolysable tannins. Like the transgenic tobacco, pellicle metabolomic analyses revealed that many phenylpropanoids correlated negatively with JrGGT expression, while shikimic acid pathway metabolites correlated positively with JrGGT expression. This research supports the hypothesis that JrGGT1 and JrGGT2 play non-trivial roles in metabolism of phenolic acids, flavonoids, and ostensibly, tannins.