A Splice-Variant Imbalance of Reticulon-like Protein 16 (RTNLB16) Disrupts Growth and Decreases Sensitivity to ABA and Dark-Induced Senescence in Arabidopsis

Reticulon-like proteins shape the endoplasmic reticulum (ER) membrane network, yet the developmental and physiological roles of individual plant reticulon isoforms remain poorly understood. Here, we characterize an Arabidopsis RTNLB16 T-DNA allele, rtnlb16-1, that exhibits severe photoperiod-dependent growth retardation and chlorosis. Molecular analysis revealed that rtnlb16-1 is not a simple loss-of-function mutant: the T-DNA insertion deletes the 5′ region required for RTNLB16 splice variant 7, while a CaMV35S enhancer associated with the insertion drives overexpression of the remaining splice variants. This misexpression is enhanced under long-day photoperiods and reduced under continuous low light, paralleling the severity of the mutant phenotype and its partial rescue. RTNLB16.5-GFP localized mainly to the tubular ER network and punctate cell-boundary structures consistent with plasmodesmata-associated ER. Neither overexpression of RTNLB16 isoforms 1–6 nor CRISPR-Cas9 disruption of major RTNLB16 isoforms reproduced the rtnlb16-1 phenotype, supporting a model in which altered splice-variant stoichiometry, rather than simple loss or gain of function, underlies the developmental defects. Transcriptome profiling showed that rtnlb16-1 undergoes extensive photoperiod-dependent transcriptional reprogramming, including changes in defense, hormone-response, senescence, photosynthesis, and iron/redox-associated gene networks. Physiologically, rtnlb16-1 displayed enhanced recovery from dark-induced senescence, while both rtnlb16-1 and rtnlb16-2 showed reduced sensitivity to exogenous abscisic acid during germination. Together, these findings suggest that balanced expression of RTNLB16 splice variants is important for normal growth and for coordinating ER-associated stress, hormone, and senescence responses in Arabidopsis.