The systemic accumulation of both hydrogen peroxide (H 2 O 2 ) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H 2 O 2 -generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H 2 O 2 was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H 2 O 2 acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H 2 O 2 signals for systemic signaling in tomato plants in response to wounding. INTRODUCTIONReactive oxygen species (ROS) are common components of the defense responses of plants against pathogen and herbivore attacks. Inoculation of plant tissues with pathogens or treatment of cell cultures with microbial elicitors causes an oxidative burst characterized by the rapid generation of hydrogen peroxide (H 2 O 2 ; reviewed by Low and Merida, 1996;Lamb and Dixon, 1997; Bolwell, 1999). Similarly, ROS are generated in plant tissues in response to wounding (Angelini et al., 1990; Bradley et al., 1992;Olson and Varner, 1993; Felton et al., 1994; Bi and Felton, 1995; OrozcoCárdenas and Ryan, 1999). Mechanical stimulation of isolated cells (Yahraus et al., 1995; Gus-Mayer et al., 1998) and the treatment of cell suspension cultures with plant cell wallderived oligogalacturonic acid (OGA; Legendre et al., 1993;Stennis et al., 1998) also generate H 2 O 2 accumulation. Woundinduced H 2 O 2 accumulation is observed both locally and systemically in leaves of several plant species, apparently caused by OGA that was released by a systemically woundinduced polygalacturonase (PG; Bergey et al., 1999; OrozcoCárdenas and Ryan, 1999).H 2 O 2 can act as a local signal for hypersensitive cell death and also as a diffusible signal for the induction of defensive genes in adjacent cells (Alvarez et al., 1998). For example, transgenic potato plants that overexpress a fungal gl...

Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wound- Willmitzer [19931 Planta 191: 123-128), are shown here t o be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 1 3s-hydroperoxy linolenic acid t o 12-0x0-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step i n the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes.

Leucine aminopeptidase A (LapA) is a late wound-response gene of tomato (Solanum lycopersicum). To elucidate the role of LapA, transgenic plants that overexpressed or abolished LapA gene expression were used. The early wound-response gene RNA levels were similar in wild-type and Lap-silenced (LapA-SI), -antisense (LapA-AS), and -overexpressing (LapA-OX) plants. By contrast, late wound-response gene RNA levels and protection against Manduca sexta damage were influenced by LapA RNA and protein levels. While LapA-OX plants had elevated levels of LapA RNAs and protein, ectopic expression of LapA was not sufficient to induce Pin (Ser proteinase inhibitor) or PPO (polyphenol oxidase) transcripts in nonwounded leaves. M. sexta larvae damaged less foliage and displayed delays in growth and development when feeding on LapA-OX plants. By contrast, LapA-SI and LapA-AS lines had lower levels of Pin and PPO RNAs than wild-type controls. Furthermore, larvae consumed more foliage and attained larger masses when feeding on LapA-SI plants. Jasmonic acid (JA) did not complement the wound-signaling phenotype of LapA-SI plants. Based on root elongation in the presence of JA, JA perception appeared to be intact in LapA-SI lines. Collectively, these data suggested that LAP-A has a role in modulating essential defenses against herbivores by promoting late wound responses and acting downstream of JA biosynthesis and perception.

Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 59-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.