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Abstract7-Azaindole is the chromophoric moiety of 7-azatryptophan, which is an alternative to tryptophan as an optical probe of protein structure and dynamics. The great power of the 7-azaindole chromophore is that it is red shifted both in absorption and emission from tryptophan, that its fluorescence decay is single exponential in water under appropriate conditions, and that its emission is sensitive to solvent. In addition, 7-azatryptophan can be incorporated into synthetic peptides and bacterial protein. In this article, the interactions of 7-azaindole with its environment are discussed. Special attention is directed to the difference in its fluorescence properties in water as opposed to nonaqueous solvents. The sensitivity of 7-azaindole to its environment is demonstrated and then exploited by studying it and its analogs in peptides and in complexes with larger proteins containing many tryptophan residues. Departments of Chemistry and Biochemistry and Biophysics Iowa State UniVersity, Ames, Iowa 50011ReceiVed: October 1, 1996; In Final Form: January 7, 1997 X 7-Azaindole is the chromophoric moiety of 7-azatryptophan, which is an alternative to tryptophan as an optical probe of protein structure and dynamics. The great power of the 7-azaindole chromophore is that it is red shifted both in absorption and emission from tryptophan, that its fluorescence decay is single exponential in water under appropriate conditions, and that its emission is sensitive to solvent. In addition, 7-azatryptophan can be incorporated into synthetic peptides and bacterial protein. In this article, the interactions of 7-azaindole with its environment are discussed. Special attention is directed to the difference in its fluorescence properties in water as opposed to nonaqueous solvents. The sensitivity of 7-azaindole to its environment is demonstrated and then exploited by studying it and its analogs in peptides and in complexes with larger proteins containing many tryptophan residues.

Plants offer metabolically rich floral nectar to attract visiting pollinators. The composition of nectar includes not only sugars, but also amino acids. We have examined the amino acid content of the nectar of ornamental tobacco and found that it is extremely rich (2 mM) in proline. Because insect pollinators preferentially utilize proline during the initial phases of insect flight and can reportedly taste proline, we determined whether honeybees showed a preference for synthetic nectars rich in proline. We therefore established an insect preference test and found that honeybees indeed prefer nectars rich in the amino acid proline. To determine whether this was a general phenomenon, we also examined the nectars of two insect-pollinated wild perennial species of soybean. These species also showed high levels of proline in their nectars demonstrating that plants often produce proline-rich floral nectar. Because insects such as honeybees prefer proline-rich nectars, we hypothesize that some plants offer proline-rich nectars as a mechanism to attract visiting pollinators.

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A potato inhibitor II gene (UK) was isolated from a library of potato genes in X bacteriophage. An 8-kilobase-pair (kbp) insert was identified using a tomato inhibitor II cDNA as a hybridization probe, and a 2.6-kbp fragment containing the gene was subcloned into the plasmid pUC13 and characterized. The nucleotide sequence of the isolated gene exhibited 87% identity with the wound-inducible tomato inhibitor II cDNA sequence. The amino acid sequence of inhibitor IUK, deduced from the potato gene, exhibited 84% identity with the tomato inhibitor II protein. A 1000-bp restriction fragment from the 5' flanking region of the gene was fused to the open reading frame of the chloramphenicol acetyltransferase (CAT) gene. This fusion was terminated in two ways: (i) with a terminator sequence from the potato inhibitor II gene and (ii) with a terminator from the 6b gene of Ti plasmid pTiA6. These chimeric genes were transferred into tobacco cells via a binary Ti vector system, and transgenic plants were regenerated. The CAT gene was expressed in leaves of transformed plants in response to wounding when fused with the inhibitor IHK promoter and terminator regions. The chimeric gene containing the 6b terminator did not express CAT in response to wounding. The wound-inducible expression of CAT activity was systemic and was induced in tissues distal to the wounded tissues. The time course of wound induction of CAT activity in transgenic tobacco leaves is similar to that found for woundinducible inhibitor I and II mRNAs in tomato leaves. These results demonstrate that sequences necessary and sufficient for wound inducibility are present within 1000 bp of the control regions of the inhibitor IIK genes and that wound-inducible components of tobacco leaf cells can regulate these sequences.Leaves of plants from the Solanaceae and Leguminosae families accumulate serine proteinase inhibitor proteins when severely damaged by attacking insects or other mechanical agents (1-3). The response is systemic and is triggered by a putative signal or "wound hormone" that is released from the sites of damage (4). Fragments of the plant's cell wall, released by endogenous polygalacturonase activity, have been shown to activate the response in detached leaves and may be part of the signaling process of the plant that induces the expression of the proteinase inhibitor genes (5, 6). The accumulation of proteinase inhibitors is thought to be a defensive response that interferes with the digestive processes of attacking pests (7,8).In potato and tomato leaves, two small gene families of serine proteinase inhibitors are wound inducible (9, 10). From wound-inducible mRNAs coding for the two families of inhibitors, cDNAs were isolated (11, 12) and utilized as probes to identify genes from potato and tomato gene libraries (9, 10). In this report, we describe the isolation of the potato inhibitor IIK gene that has been identified as a wound-inducible gene based on its similarity to a woundinducible cDNA from tomato leaves and the strong hybridizatio...

Tobacco floral nectaries undergo changes in form and function. As nectaries change from green to orange, a new pigment is expressed. Analysis demonstrated that it is β-carotene. Plastids undergo dramatic changes. Early in nectary development, they divide and by stage 9 (S9) they are engorged with starch. About S9, nectaries shift from quiescent anabolism to active catabolism resulting in starch breakdown and production of nectar sugars. Starch is replaced by osmiophilic bodies, which contain needle-like carotenoid crystals. Between S9 and S12, amyloplasts are converted to chromoplasts. Changes in carotenoids and ascorbate were assayed and are expressed at low levels early in development; however, following S9 metabolic shift, syntheses of β-carotene and ascorbate greatly increase in advance of expression of nectar redox cycle. Transcript analysis for carotenoid and ascorbate biosynthetic pathways showed that these genes are significantly expressed at S6, prior to the S9 metabolic shift. Thus, formation of antioxidants β-carotene and ascorbate after the metabolic shift is independent of transcriptional regulation. We propose that biosynthesis of these antioxidants is governed by availability of substrate molecules that arise from starch breakdown. These processes and events may be amenable to molecular manipulation to provide a better system for insect attraction, cross pollination, and hybridization.

We have isolated and characterized the cDNA encoding the ornamental tobacco (Nicotiana langsdorffii X N. sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This transcription factor was robustly expressed at Stage 12 of nectary development but was only weakly expressed in the earlier Stage 6 nectaries. The ornamental tobacco MYB305 contains a conserved R2R3 MYB DNA binding domain with 76 amino acids in the activation domain. A green fluorescent protein-MYB305 fusion localized to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrated that it functions as a transcription activator. A conserved 23-amino acid C-terminal domain is required to activate gene expression. The coding region of the myb305 cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and was purified to homogeneity. This protein shows binding to two consensus MYB binding sites on the ornamental tobacco Nectarin I (nec1) promoter as well as to the single site located on the Nectarin V (nec5) promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduced expression in nectary tissues. Temporally, MYB305 expression precedes that of nec1 and nec5, as would be expected if the MYB305 factor regulates expression of the nec1 and nec5 genes. Ectopic expression of MYB305 in foliage was able to induce expression of both nec1 and nec5, as well as two flavonoid biosynthetic genes in the foliage. Finally, RNA interference knockdown of MYB305 resulted in reduced expression of both nectarins and flavonoid biosynthetic genes. We conclude that expression of MYB305 regulates expression of the major nectarin genes in the floral nectary.