Threonine deaminase (TD) catalyzes the conversion of Thr to a-keto butyrate in Ile biosynthesis; however, its dramatic upregulation in leaves after herbivore attack suggests a role in defense. In Nicotiana attenuata, strongly silenced TD transgenic plants were stunted, whereas mildly silenced TD transgenic plants had normal growth but were highly susceptible to Manduca sexta attack. The herbivore susceptibility was associated with the reduced levels of jasmonic acidisoleucine (JA-Ile), trypsin proteinase inhibitors, and nicotine. Adding [ 13 C 4 ]Thr to wounds treated with oral secretions revealed that TD supplies Ile for JA-Ile synthesis. Applying Ile or JA-Ile to the wounds of TD-silenced plants restored herbivore resistance. Silencing JASMONATE-RESISTANT4 (JAR4), the N. attenuata homolog of the JA-Ile-conjugating enzyme JAR1, by virus-induced gene silencing confirmed that JA-Ile plays important roles in activating plant defenses. TD may also function in the insect gut as an antinutritive defense protein, decreasing the availability of Thr, because continuous supplementation of TD-silenced plants with large amounts (2 mmol) of Thr, but not Ile, increased M. sexta growth. However, the fact that the herbivore resistance of both TD-and JAR-silenced plants was completely restored by signal quantities (0.6 mmol) of JA-Ile treatment suggests that TD's defensive role can be attributed more to signaling than to antinutritive defense.

Jasmonic acid (JA)-amino acid conjugates are important JA metabolites that activate JA responses. However, our understanding of their involvement in herbivore defenses is limited. We identified a new Arabidopsis jasmonate resistant 1 (JAR1) homologue in Nicotiana attenuata (N. attenuata) and named it jasmonate resistant 6 (JAR6). JAR6 clustered closely with Arabidopsis JAR1 and the recently reported jasmonate resistant 4 (JAR4), another JAR1 homologue in N. attenuata, in a phylogenic analysis. The strong elicitation of JAR6 transcripts by wounding and treatment with Manduca sexta (M. sexta) oral secretions (OS), which mimics herbivore attack, suggests it plays a role in herbivore defense. Independently silencing JAR4 or JAR6 by transforming N. attenuata with inverted repeat JAR4 or JAR6 constructs significantly reduced levels of not only JA-Ile plus JA-Leu but also JA-Val in OS-elicited leaves, suggesting JAR4 and JAR6 are functionally redundant and their amino acid substrates are not highly specific to individual amino acids. A new JA conjugate, JA-Gln, whose levels are much higher than those of the other JA conjugates in WT plants, was not affected in JAR4- or JAR6-silenced lines, implying that another JA-conjugating enzyme exists in N. attenuata. Neither JA-ACC, the second most abundant JA conjugate in Arabidopsis seedlings, nor JA-Met or JA-Trp, was detectable in N. attenuata. Levels of trypsin proteinase inhibitors (TPIs) in JAR4- and JAR6-silenced plants were significantly reduced, but nicotine levels were normal. We conclude that both JAR4 and JAR6 conjugate JA to Ile, Val, and Leu, and that both positively regulate TPI activity.

It now appears that photosynthetic prokaryotes and lower eukaryotes possess higher plant phytochrome-like proteins. In this work, a second phytochrome-like gene was isolated, in addition to the recently identified Cph1 phytochrome, from the Synechocystis sp. PCC 6803, and its gene product was characterized photochemically. The open reading frame sll0821 (designated cph2 in this work) has structural characteristics similar to those of the plant phytochromes and the Synechocystis Cph1 with high amino acid sequence homology in the N-terminal chromophore binding domain. The predicted Cph2 protein consists of 1276 amino acids with a calculated molecular mass of 145 kDa. Interestingly, the Cph2 protein has two putative chromophore binding domains, one around Cys-129 and the other around Cys-1022. The Cph2 was overexpressed in E. coli as an Intein/CBD (chitin binding domain) fusion and in vitro reconstituted with phycocyanobilin (PCB) or phytochromobilin (PPhiB). Both the Cph2-PCB and Cph2-PPhiB adducts showed the typical photochromic reversibility with the difference spectral maxima at 643/690 and 655/701 nm, respectively. The Cys-129 was confirmed to be the chromophore binding residue by in vitro mutagenesis and Zn(2+) fluorescence. The microenvironment of the chromophore in Cph2 seems to be similar to that in plant phytochromes. The cph2 gene expression was dark-induced and down-regulated to a basal level by light, like the cph1 gene. These observations suggest that Synechocystis species have multiple photosensory proteins, probably with distinct roles, as in higher plants.

SummaryIn eukaryotes, genes carrying premature termination codons (PTCs) are often associated with decreased mRNA levels compared with their counterparts without PTCs. PTC-harboring mRNA is rapidly degraded through the nonsense-mediated mRNA decay (NMD) pathway to prevent the accumulation of potentially detrimental truncated proteins. In a native ecotype of Nicotiana attenuata collected from Arizona (AZ), the mRNA levels of a trypsin proteinase inhibitor (TPI) gene are substantially lower than in plants collected from Utah (UT). Cloning the AZ TPI gene revealed a 6 bp deletion mutation in exon 2 resulting in a PTC and decreased mRNA levels through NMD. Silencing UPF1, 2 and 3 in N. attenuata AZ plants by virus-induced gene silencing (VIGS) enhanced the levels of PTC-harboring TPI mRNA, demonstrating a conserved role for UPF genes in plants. Furthermore, using cell suspension cultures that express variants of the TPI construct, we demonstrate that both intron-containing and intronless genes are subject to NMD in plants; unlike PTCs in mammals, PTCs downstream of introns activate NMD in plants. However, when a PTC is only 4 bp upstream of an intron, the NMD surveillance mechanism is abrogated. We also demonstrate that, in an intronless TPI gene, a PTC located at the beginning or the end of the coding sequence triggers NMD less efficiently than do PTCs located at the middle of the coding sequence. Taken together, these results highlight the complexity of the NMD activation mechanisms in plants.

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