Susan I. Gibson

ABA and sugar interactions regulating development: cross-talk or voices in a crowd?

Plant growth and development are controlled by the concerted action of many signaling pathways that integrate information from environmental signals with that from developmental and metabolic cues. Physiological studies have demonstrated that abscisic acid and sugars have both similar and antagonistic effects on diverse processes, including seed development, germination, and seedling growth. Recent genetic studies have identified several loci that are involved in both sugar and hormonal responses. It is rarely clear whether these apparent linkages reflect direct or indirect interactions between sugar and hormone signaling pathways, but the identification of gene products that are encoded at these loci is allowing these possibilities to be tested.

Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana.

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated [omega]-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized [omega]-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased [omega]-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.

Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species

Abstract. Photoassimilates are used by plants for production of energy, as carbon skeletons and in transport of fixed carbon between different plant organs. Many studies have been devoted to characterizing the factors that regulate photoassimilate concentrations in different plant species. Most studies examining photoassimilate concentrations in C3 plants have focused on analyzing starch and soluble sugars. However, work presented here demonstrates that a number of C3 plants, including the popular model organism Arabidopsis thaliana (L.) Heynh., and agriculturally important plants, such as soybean, Glycine max (L.) Merr., contain significant quantities of fumaric acid. In fact, fumaric acid can accumulate to levels of several milligrams per gram fresh weight in Arabidopsis leaves, often exceeding those of starch and soluble sugars. Fumaric acid is a component of the tricarboxylic acid cycle and, like starch and soluble sugars, can be metabolized to yield energy and carbon skeletons for production of other compounds. Fumaric acid concentrations increase with plant age and light intensity in Arabidopsis leaves. Moreover, Arabidopsis phloem exudates contain significant quantities of fumaric acid, raising the possibility that fumaric acid may function in carbon transport.

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast [omega]6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known [omega]3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a [delta]12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized [omega]6 desaturase corresponding to the Arabidopsis fad2 gene.

Mobilization of seed storage lipid by Arabidopsis seedlings is retarded in the presence of exogenous sugars

BackgroundSoluble sugar levels must be closely regulated in germinating seeds to ensure an adequate supply of energy and building materials for the developing seedling. Studies on germinating cereal seeds indicate that production of sugars from starch is inhibited by increasing sugar levels. Although numerous studies have focused on the regulation of starch metabolism, very few studies have addressed the control of storage lipid metabolism by germinating oilseeds.ResultsMobilization of storage lipid by germinating seeds of the model oilseed plant Arabidopsis thaliana (L.) Heynh. occurs at a greatly reduced rate in the presence of exogenous glucose or mannose, but not in the presence of equi-molar 3-O-methylglucose or sorbitol. The sugar-insensitive5-1/abscisic acid-insensitive4-101 (sis5-1/abi4-101) mutant is resistant to glucose inhibition of seed storage lipid mobilization. Wild-type seedlings become insensitive to glucose inhibition of storage lipid breakdown within 3 days of the start of imbibition.ConclusionsGrowth in the presence of exogenous glucose significantly retards mobilization of seed storage lipid in germinating seeds from wild-type Arabidopsis. This effect is not solely due to the osmotic potential of the media, as substantially higher concentrations of sorbitol than of glucose are required to exert significant effects on lipid breakdown. The inhibitory effect of glucose on lipid breakdown is limited to a narrow developmental window, suggesting that completion of some critical metabolic transition results in loss of sensitivity to the inhibitory effect of glucose on lipid breakdown.

Isolating plant genes

The genetic transformation of most agriculturally important plant species is now possible. However, the application of this technology to rational plant-improvement is currently limited by a shortage of cloned genes for important traits. Recent technological advances in plant-gene isolation and identification, such as map-based cloning, insertional mutagenesis and large-scale cDNA sequencing, have accelerated the rate of gene isolation and significantly expanded the opportunities for genetic engineering of crop plants.

Characterization of an inducible promoter system in Catharanthus roseus hairy roots.

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid‐inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage‐dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12–18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage‐dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 μM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid‐inducible promoter in transgenic C. roseus hairy roots.

The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway.

BackgroundThe terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized.ResultsTranscriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors.ConclusionsResults presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

Transient effects of overexpressing anthranilate synthase α and β subunits in Catharanthus roseus hairy roots

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback‐insensitive anthranilate synthase α subunit under the control of the glucocorticoid‐inducible promoter system and the anthranilate synthase β subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.