No abstract

The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants.

Winter rye (Secale cerealeL.) is used as a cover crop because of the weed suppression potential of its mulch. To gain insight into the more effective use of rye as a cover crop we assessed changes in benzoxazinone (BX) levels in rye shoot tissue over the growing season. Four rye varieties were planted in the fall and samples harvested at intervals the following spring. Two different measures of phytotoxic compound content were taken. Seed germination bioassays were used as an estimate of total phytotoxic potential. Dilutions of shoot extracts were tested using two indicator species to compare the relative toxicity of tissue. In addition, BX (DIBOA, DIBOA-glycoside, and BOA) levels were directly determined using gas chromatography. Results showed that rye tissue harvested in March was the most toxic to indicator species, with toxicity decreasing thereafter. Likewise the BX concentration in rye shoot tissue increased early in the season and then decreased over time. Thus, phytotoxicity measured by bioassay and BX levels measured by GC have a similar but not identical temporal profile. The observed decrease in phytotoxic potential and plant BX levels in rye later in the season appears to correlate with the transition from vegetative to reproductive growth.

Climate change and geography affect all the living organisms. To date, the effects of climate and geographical factors on plant metabolome largely remain open for worldwide and local investigations. In this study, we designed field experiments with tobacco (Nicotiana tabacum) in India versus USA and used untargeted metabolomics to understand the association of two weather factors and two different continental locations with respect to tobacco metabolism. Field research stations in Oxford, North Carolina, USA, and Rajahmundry, Andhra Pradesh India were selected to grow a commercial tobacco genotype (K326) for 2 years. Plant growth, field management, and leaf curing followed protocols standardized for tobacco cultivation. Gas chromatography–mass spectrometry based unbiased profiling annotated 171 non-polar and 225 polar metabolites from cured tobacco leaves. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that two growing years and two field locations played primary and secondary roles affecting metabolite profiles, respectively. PCA and Pearson analysis, which used nicotine, 11 other groups of metabolites, two locations, temperatures, and precipitation, revealed that in North Carolina, temperature changes were positively associated with the profiles of sesquiterpenes, diterpenes, and triterpenes, but negatively associated with the profiles of nicotine, organic acids of tricarboxylic acid, and sugars; in addition, precipitation was positively associated with the profiles of triterpenes. In India, temperature was positively associated with the profiles of benzenes and polycyclic aromatic hydrocarbons, but negatively associated with the profiles of amino acids and sugar. Further comparative analysis revealed that nicotine levels were affected by weather conditions, nevertheless, its trend in leaves was independent of two geographical locations and weather changes. All these findings suggested that climate and geographical variation significantly differentiated the tobacco metabolism.

Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacteriummediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/ or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.Stevia rebaudiana (Bertoni) is a self-incompatible plant species, and one of a few species in the Stevia genus whose leaves produce and accumulate high quantities of sweet steviol glycoside (SG) compounds 1 . SGs exist as a complex mixture of related compounds, with certain SG species conferring favorable sweetness characteristics while others are associated with a bitter or metallic taste 2 . As a natural sweetener that cannot be further metabolized in the human digestive tract, Stevia and the SGs have been rapidly gaining popularity in the food and beverage industries. The abundance and ratios of the SGs vary greatly among Stevia cultigens. Even within the same Stevia cultigen, the concentration and diversity of the SGs can vary in response to environmental factors such as light, water and nutrient availability 1,3-5 .The biosynthesis of SGs in Stevia has been extensively investigated and reviewed 2,6 . Figure 1 shows a simplified version of the pathway based on our current understanding. Most of the key reactions of SG biosynthesis are catalyzed by cytosolic UDP-dependent glycosyltransferases (UGTs). In the first characterization of the genes encoding UGTs of the SG biosynthetic pathway, UGT85C2, UGT74G1 and UGT76G1 were identified as being central to the production of the SGs 7 . Using E. coli extracts containing recombinant UGT proteins, it was shown that UGT85C2 functioned at the beginning of the pathway to catalyze the synthesis of steviol-13-O-glucoside from steviol. UGT74G1 displayed a broader substrate specificity, catalyzing the formation of steviol-monoside (presumably 19-O-glucoside) from steviol, a steviolbioside (presumably rubusoside) from steviol-13-O-glucoside, and st...