The Simultaneous Repression Of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana

Cinnamoyl CoA reductase （CCR） and cinnamyl alcohol dehydrogenase （CAD） catalyze the last steps of mono- lignol biosynthesis. In Arabidopsis, one CCR gene （CCR1, Atlg15950） and two CAD genes （CAD C At3g19450 and CAD D At4g34230） are involved in this pathway. A triple cad c cad d ccrl mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther de- hiscence and of pollen release. The cc~ hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR （CCR2, Atlg80820） and another CAD （CAD1, At4g39330） does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

Macroscale fluorescence imaging against autofluorescence under ambient light

Macroscale fluorescence imaging is increasingly used to observe biological samples.However,it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support.In this manuscript,we built a simple and inexpensive fluorescence macroscope,which has been used to evaluate the performance of Speed OPIOM(Out of Phase Imaging after Optical Modulation),which is a reference-free dynamic contrast protocol,to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light.By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection,we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10,respectively,over direct fluorescence observation under constant illumination.Then,we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths.Finally,we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves,even under the interference of incident light at intensities that are comparable to sunlight.The proposed approach is expected to find multiple applications,from biological assays to outdoor observations,in fluorescence macroimaging.