Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans.

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult "hidden" in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times.

Field pennycress (Thlaspi arvense L.) is currently being developed as a new cold-tolerant oilseed crop. In natural populations, pennycress, like many Brassicaceae relatives, can exhibit either a winter or spring annual phenotype. Pennycress is a diploid relative of Arabidopsis thaliana, a model species that has been used to study many adaptive phenotypes, including flowering time and developmental timing. In Arabidopsis and other Brassicaceae species, mutations in negative regulators of flowering, including FLOWERING LOCUS C and FRIGIDA can cause the transition to a spring annual habit. The genetics underlying the difference between spring and winter annual pennycress lines are currently unknown. Here, we report the identification of four natural alleles of FLC in pennycress that confer a spring annual growth habit identified through whole genome sequencing, cosegregation analyses, and comparative genomics. The global distribution of these spring annual alleles of FLC suggests that the spring annual growth habit has arisen on several independent occasions. The two spring annual FLC alleles present in European accessions were only identified in North American accessions collected in southern Montana, which indicates accessions harboring these two alleles were introduced to North America, likely after pennycress became a widespread species on the continent. These findings provide new information on the natural history of the introduction and spread of spring annual pennycress accessions from Europe into North America. At the molecular level, these findings are important for the ongoing development of pennycress as a winter annual crop. An enhanced understanding of the regulation of flowering in this species should allow for the fine-tuning of flowering in commercial varieties.