Low-temperature conditioning attenuates chilling-induced lignification in loquat by modulating the miR164-EjNAC3L-EjLAC7 cascade.

Low-temperature storage induces lignification in red-fleshed loquat fruit, adversely affecting quality, while modified low-temperature conditioning (mLTC, 5°C for 2 days before 0°C storage) alleviates this effect. Here, integrated sRNAome and transcriptome analysis of 'Dahongpao' (DHP) loquat under 0°C and mLTC treatments identified miR164 as a key regulator whose expression is suppressed at 0°C but partially restored under mLTC. As a result, one of its target genes EjNAC3-like (EjNAC3L)'s expression level was upregulated during chilling-induced lignification but was suppressed by mLTC. Molecular evidence has proved that EjNAC3L could activate several lignin biosynthetic genes, especially for EjLAC7 (a homolog of AtLAC7), which was also strongly induced by 0°C but not mLTC. Mechanistically, EjNAC3L indirectly regulated EjLAC7 by forming heterodimers with EjNAC5/6, which directly bound the EjLAC7 promoter. Functional assays and transient overexpression in tobacco leaves and loquat fruits confirmed the function of MIR164, EjNAC3L, and EjLAC7 in lignin biosynthesis. Furthermore, stable overexpression of EjNAC3L in Arabidopsis and EjLAC7 in liverwort both led to an increase in lignin content and may have also altered the cold resistance in the transgenic plants. Meanwhile, we obtained stable transgenic loquat roots expressing STTM164 by using Agrobacterium rhizogenes, confirming the function of the miR164-EjNAC3L-EjLAC7 regulatory module. Collectively, our findings demonstrate that mLTC treatment mitigates chilling-induced lignification partly through restoring miR164 expression, like the effect of a vaccine, thus providing a novel insight and potential strategy for improving post-harvest cold tolerance in horticultural crops.