RNAi Suppression of LEAFY Gives Stable Floral Sterility, and Reduced Growth Rate and Leaf Size, in Field-Grown Poplars

Abstract

The central floral development gene LEAFY (LFY), whose mutation leads to striking changes in flowering and often sterility, is commonly expressed in non-floral structures; however, its role in vegetative development is poorly understood. Sterility associated with suppression of LFY expression is an attractive means for mitigating gene flow by both seeds and pollen in vegetatively propagated forest trees, but the consequences of its suppression for tree form and wood production are unclear. To study the vegetative effects of RNAi suppression of LFY, we created a randomized, multiple-year field study with 30–40 trees (ramets) in each of two sterile gene insertion events, three transgenic control events, and a wild-type control population. We found that floral knock-down phenotypes were stable across years and propagation cycles, but that several leaf morphology and productivity traits were statistically and often substantially different in sterile vs. normal flowering RNAi-LFY trees. Though trees with suppressed LEAFY expression looked visibly normal, they appear to have reduced growth and altered leaf traits. LFY appears to have a significant role in vegetative meristem development, and evaluation of vegetative impacts from LFY suppression would be prudent prior to large-scale use for genetic containment.

Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering

“…These LFY targeted trees showed an absence of well-formed anthers or carpels, no evidence of male or female gamete production, and the presence of flat leaf-like structures. In our male clone, the LFY- mutated trees often formed replicated floral structures, which was not a predicted outcome given that our field trial of natural flowering with RNAi targeting of LFY in poplar gave reduced catkin size and very underdeveloped flowers [ 5 , 6 ]. However, we observed a similar re-iterative floral form in eucalypts with CRISPR targeting of the LFY homolog under floral acceleration with a constitutive promoter [ 10 ].…”

Mobility of FLOWERING LOCUS T protein as a systemic signal in trifoliate orange and its low accumulation in grafted juvenile scions

“…The lack of early flowering in trifoliate orange grafts may be caused by dilution or attenuation of FT products during long-distance transport to the non-transgenic recipient scion through the graft junction. Second, the flowering of woody plants is an extraordinarily complicated process that requires the regulation of multiple genes [ 6 , 35 , 61 – 64 ]. Interlocking feedback loops may regulate the dynamic behavior of the floral transition in trifoliate orange [ 65 , 66 ].…”

Advances and Perspectives of Transgenic Technology and Biotechnological Application in Forest Trees

“…In sweetgum, RNAi was used to inhibit LEAFY gene expression, generating sterile transgenic plants (Qiao et al, 2007). A vector with the RNAi-LFY cassette was transferred into P. alba, which markedly altered flower morphology and led to female flower sterility (Klocko et al, 2021). However, in asexual forest trees, sterility associated with LFY expression inhibition can alleviate the gene flow of seeds and pollen, although the effects on tree shape and wood production are unclear (Klocko et al, 2021).…”

“…A vector with the RNAi-LFY cassette was transferred into P. alba, which markedly altered flower morphology and led to female flower sterility (Klocko et al, 2021). However, in asexual forest trees, sterility associated with LFY expression inhibition can alleviate the gene flow of seeds and pollen, although the effects on tree shape and wood production are unclear (Klocko et al, 2021). However, the use of LFY suppressor genes could be costly, and further research is needed.…”