Powdery mildew, caused by the fungus Erysiphe pulchra, is one of the most serious diseases affecting the popular ornamental tree flowering dogwood (Cornus florida). Employing gene editing techniques such as CRISPR to introduce powdery mildew resistance by inactivating the Mildew Locus O (MLO) gene requires an efficient genetic transformation system. This novel research will fill a critical gap in our knowledge of flowering dogwood biotechnology. Previous research efforts have genetically transformed embryogenic dogwood cultures, now we are aiming to produce transgenic plantlets. Recent research focused on using the RITA® temporary immersion bioreactor system for testing germination of somatic embryos and conversion to plants. The primary test was the impact of the plant growth regulator (PGR) gibberellic acid (GA3). For this experiment, we analyzed the impact of different environmental light exposures on dogwood embryo stress levels by observing anthocyanin production. The starting material was unwashed callus transformed with the GUS reporter gene and subjected to one of three RITA® treatments to examine the influence of varying light levels. Treatments included full darkness, continuous low light, and a combination of both light conditions for different durations of time. Few somatic embryos germinated from the treatment with 60 days of full darkness, but the anthocyanin stress was absent. We observed the same result for constant low light exposure except for exponential growth of the transgenic callus. The treatment in which the somatic embryos were in full darkness for 30 days and switched to low light for another 30 days showed a higher germination rate, but there were increased signs of anthocyanin stress. Implementation of this research will assist in the optimization of the production of plantlets from dogwood cultures transformed with a CRISPR-Cas9 construct that can inactivate the MLO gene to obtain powdery mildew resistance.
Fresh fruits and vegetables are invaluable for human health, but their quality deteriorates before reaching consumers during distribution due to ongoing biochemical processes and compositional changes. The current lack of any objective indices for defining “freshness” of fruits or vegetables limits our capacity to control product quality and leads to food loss and waste. In this work, we undertook interdisciplinary research to address plant science challenges related to food security and human health. This work has leveraged machine learning technologies and multi-omics tools to understand post-harvest senescence and microbial spoilage of fresh produce for the purpose of developing a simple imaging “FreshID” device to evaluate fruit and vegetable quality. In essence, we are proposing a comprehensive research program to identify proteins and compounds as “freshness-indicators” and to aid development of an innovative and easy-to-use accessibility tool to accurately estimate the freshness and/or contamination of produce. The goal of the proposed research will be advances in both basic research and applied science. Such a tool would allow a new level of post-harvest logistics, supporting availability of high-quality, nutritious, fresh produce.
Plant natural promoters are always very long and contain many different promoter motifs, providing complex expression patterns, while synthetic promoters can be constructed to be very short in sequence and very strong in promoter strength. Bioinformatics-assisted de novo promoter motif discovery searches for statistically overrepresented motifs without the inclusion of biological information, leading to limited prediction efficiency. To overcome this limitation, we have developed a novel ensemble approach by mapping the motifs detected by a set of selected bioinformatics tools back to the promoter sequences and looking for overlapping motif regions among the detected motifs. Using this approach, we searched and identified novel constitutive promoter motifs from the soybean genome. Seven user-friendly bioinformatics tools, including BioProspector, CisGenome, HOMER, MEME, MotifSuite, RSAT Plants, and Weeder were employed for the de novo discovery of constitutive motifs among 11 published soybean constitutive promoters. A total of 62 promoter motifs were detected among the 11 soybean constitutive promoters by at least four of the seven bioinformatics tools. A tetramer (4×) of each promoter motif was cloned in front of the minimal 35S promoter driving GUS reporter gene expression, and used for tobacco leaf agroinfiltration and stable Arabidopsis transformation. Quantitative GUS activity assays following transient tobacco leaf agroinfiltration identified 26 of the 62 promoter motifs that drove GUS expression significantly higher than the basal level conferred by the minimal 35S promoter. Histochemical GUS analysis of stable transgenic Arabidopsis seedlings found that 16 of the 26 promoter motifs were 19 ~ 60 bp in length and exhibited constitutive expression with variable promoter strength, and 7 of the 26 promoter motifs showed strong constitutive expression which was comparable to (slightly weaker than) the 35S promoter. Thus, these novel constitutive motifs can be used to drive constitutive gene expression in dicot species.
In sweetpotato (Ipomoea batatas L.), the sink strength of developing adventitious roots limits storage root formation. Sucrose synthase (SuSy) has been identified as a marker for sink strength in developing storage roots. In model systems, declining nitrogen (N) availability has been associated with increased carbohydrate allocation to root systems. To test the hypothesis that N limitation triggers increased SuSy activity that leads to storage root formation, we subjected sweetpotato cv. ‘Beauregard’ to progressively declining N treatments in a split-root system. SuSy expression and root system architecture were evaluated over 15 days, and storage root formation was assessed at 50 days. Declining N availability enhanced SuSy activity in the root base tissue across all time points and was associated with increased lateral root count at 15 days. Previous work has shown that the anatomical cue of the onset of storage root formation, the appearance of anomalous cambia, is initially limited to the root base tissue. The omission of N was associated with decreased root base SuSy activity and an overall reduction in root architectural attributes. These data support the hypothesis that declining N could be a critical switch for storage root formation in sweetpotato. Our findings have profound implications for increasing N fertilizer efficiency and enhancing our understanding of the intrinsic and environmental variables that mediate storage root formation and productivity in this globally important crop.
