Susan D. Lawrence

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants

SummaryA method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage (≤ 5%) of the shoots regenerated in the presence of 100 μg/ml kanamycin were GUS+. Polymerase chain reaction (PCR) analysis confirmed that GUS+ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.

The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes.

Abstract Unscheduled DNA synthesis of cultured hepatocytes in response to the direct acting carcinogen, methyl methanesulfonate, and to the procarcinogen, 2-acetylaminofluorene, was markedly increased when these cells were cultured in medium containing 25 mM nicotinamide. This effect of nicotinamide was apparently unrelated to the maintenance of intracellular nicotinamide coenzyme levels. The increase in unscheduled DNA synthesis mediated by 2-acetylaminofluorene in nicotinamide-treated hepatocytes could be partially accounted for by the maintenance of higher microsomal capacity for the metabolism of this procarcinogen. The ability of adult rat hepatocytes to respond to nicotinamide by increased unscheduled DNA synthesis was lost within a few hours after plating or upon short withdrawal of this vitamin from the culture medium.

Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus.

Citrus tristeza virus (CTV) contains approximately 20,000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25,000 Mr that has previously been reported to consist of at least two size variants, cp1 and cp2. In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. Five immunopositive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein. Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons. The encoded protein has a predicted Mr of 24,909 and an amino acid composition consistent with that previously reported for the CTV coat protein. Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively. These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA.

Potato, Solanum Tuberosum, Defense Against Colorado Potato Beetle, Leptinotarsa Decemlineata (Say): Microarray Gene Expression Profiling of Potato by Colorado Potato Beetle Regurgitant Treatment of Wounded Leaves

Colorado potato beetle (CPB) is a leading pest of solanaceous plants. Despite the economic importance of this pest, surprisingly few studies have been carried out to characterize its molecular interaction with the potato plant. In particular, little is known about the effect of CPB elicitors on gene expression associated with the plant’s defense response. In order to discover putative CPB elicitor-responsive genes, the TIGR 11,421 EST Solanaceae microarray was used to identify genes that are differentially expressed in response to the addition of CPB regurgitant to wounded potato leaves. By applying a cutoff corresponding to an adjusted P-value of <0.01 and a fold change of >1.5 or <0.67, we found that 73 of these genes are induced by regurgitant treatment of wounded leaves when compared to wounding alone, whereas 54 genes are repressed by this treatment. This gene set likely includes regurgitant-responsive genes as well as wounding-responsive genes whose expression patterns are further enhanced by the presence of regurgitant. Real-time polymerase chain reaction was used to validate differential expression by regurgitant treatment for five of these genes. In general, genes that encoded proteins involved in secondary metabolism and stress were induced by regurgitant; genes associated with photosynthesis were repressed. One induced gene that encodes aromatic amino acid decarboxylase is responsible for synthesis of the precursor of 2-phenylethanol. This is significant because 2-phenylethanol is recognized by the CPB predator Perillus bioculatis. In addition, three of the 16 type 1 and type 2 proteinase inhibitor clones present on the potato microarray were repressed by application of CPB regurgitant to wounded leaves. Given that proteinase inhibitors are known to interfere with digestion of proteins in the insect midgut, repression of these proteinase inhibitors by CPB may inhibit this component of the plant’s defense arsenal. These data suggest that beyond the wound response, CPB elicitors play a role in mediating the plant/insect interaction.

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.

A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.

Cytokinin Primes Plant Responses to Wounding and Reduces Insect Performance

We report a potential role of endogenous cytokinin supply in priming plant defense against herbivory. Cytokinin priming significantly reduced weight gain by insect larvae. Unlike previously described priming by volatile compounds, priming by cytokinin did not overcome vascular restrictions on systemic wound signaling. However, similar to priming by volatile compounds, cytokinin priming occurred upstream of accumulation of jasmonic acid and its precursor linolenic acid in mechanically wounded source leaves. Cytokinin priming significantly altered wound-induced accumulation of transcripts encoding homologs of allene oxide synthase, trypsin inhibitor, and chitinase. Cytokinin priming may reflect coordination between aboveground inducible defense against herbivory and belowground processes such as nutrient availability. These findings should encourage investigations of how genetic alterations in cytokinin signaling and response pathways may affect plant–herbivore interactions.

A vegetative storage protein homolog is expressed in the growing shoot apex of hybrid poplar

Abstract. The ability of poplars (Populus deltoides Bartr. ex Marsh., and Populus trichocarpa Torr. and Gray) to sequester nitrogen in stems in preparation for winter has been associated with the massive accumulation of protein bodies in the bark and xylem ray parenchyma. These protein bodies contain a bark storage protein (BSP) that can account for up to 30% of the total soluble bark protein during the winter months. Perhaps the plants ability to efficiently cycle nitrogen through BSP is an important aspect of its growth potential. Sequence analysis of BSP led to the identification of a leaf-associated homolog, win4, which was initially isolated because its transcript increased in abundance upon mechanical wounding. The goal of this work was to characterize this putative leaf-associated vegetative storage protein, and determine whether it might perform a storage role in vivo. Antibodies, produced against protein synthesized upon over-expression of the win4 coding region in Escherichia coli, were used to examine the relative abundance of WIN4 protein in response to supplemental nitrogen, and during development. The transcript and protein were most abundant in the youngest leaves and also increased with nitrogen fertilization. Immunolocalization of the protein was performed and showed that WIN4 was associated with cells surrounding the vasculature, and cells of the lower epidermis and stipules of immature leaves. Under moderate nitrogen fertilization regimes, WIN4 accounted for only about 2% of total soluble leaf protein; however, given the cellular specificity and enhancement with nitrogen, the protein is regulated in a manner similar to other vegetative storage proteins. Since poplar is amenable to DNA transformation and regeneration, it is now possible to ask direct questions about the role these proteins play in nitrogen storage in rapidly expanding or in dormant tissue. This type of analysis could determine whether these proteins mainly ameliorate the toxic effects of excess nitrogen, if they are instrumental in controlling nitrogen allocation or if they simply represent an efficient method for sequestering this valuable nutrient.

DNA damage induced by the antihistaminic drug methapyrilene hydrochloride

Treatment of primary cultures of rat hepatocytes with the antihistaminic drug, methapyrilene hydrochloride, stimulated DNA-repair synthesis up to 7-fold and caused the formation of alkaline-labile lesions in hepatocellular DNA. These data clearly demonstrate that methapyrilene hydrochloride is a DNA damaging agent. In view of a recent report and our own findings we suggest that this antihistamine has the properties of a complete carcinogen.