Disproportion of reticulon-like 16 (RTNLB16) splice variants expression disrupts growth and decreases sensitivity to ABA and senescence in Arabidopsis

The Reticulon family proteins (RTNs) are membrane-spanning proteins found in the endoplasmic reticulum (ER) with diverse functions, such as ER membrane morphogenesis, vesicle formation, and trafficking. The plant-specific reticulon-like protein family (RTNLBs) comprises multiple members, yet their functions remain poorly understood. The Arabidopsis RTNLB16 gene has seven splice variants, each encoding seven distinct protein isoforms. We identified an Arabidopsis mutant (Salk_122275/rtnlb16-1) as a knockout for the upper coding frame, isoform 7, of RTNLB16 while overexpressing the other six isoforms through the CaMV 35S promoter at the left border of the T-DNA insertion. rtnlb16-1 exhibits distinctive growth retardation and reduced chlorophyll levels. Under photoperiodic long day (16:8 h) conditions, activation of the 35S promoter intensifies RTNLB16 expression in the mutant, resulting in profound growth inhibition. Conversely, growth under continuous low-light (CLL) conditions restrains the overexpression and significantly mitigates rtnlb16-1 phenotype. Confocal microscopy experiments revealed the localization of RTNLB16:GFP in the tubular ER network, plasmodesmata, and potentially in Golgi bodies. Peculiarly, RTLB16/rtnlb16 heterozygote plants exhibit non-Mendelian reduced fertility, suggesting potential involvement of RTNLB16 in reproductive development. Transcriptomics comparisons between rtnlb16-1 and the wild type under CLL and 16:8h conditions revealed differential gene expression involved in salicylic acid, jasmonic acid, and abscisic acid responses, indicating activation of defense and osmotic stress responses contributing to the growth inhibition in the mutant. We further demonstrate that rtnlb16 has decreased sensitivity to abscisic acid and enhanced tolerance to darkness-induced senescence. Our findings highlight the importance of balanced expression among RTNLB16 isoforms for normal cellular and physiological activities in Arabidopsis. Additionally, our study underscores the significance of employing T-DNA mutants to investigate genes with multiple splice variants.