A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening

Abstract

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES (LOB) TF, SlLOB1, up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 (EXP1) are strongly suppressed in SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.

Keywords: SlLOB1; cell wall; ripening; softening; transcription factor.

Fig. 1.

Fruit gene expression and phenotypes following SlLOB1 repression. (A) Expression of SlLOB1 in WT tissues leaves (Le), root (R), and stem (St) of 2-wk-old seedlings, anthers (An), floral buds (B), prepollination carpel (Ca), sepals (Se), petals (Pe), seed (S), breaker stage (BR), immature green (IMG), mature green (MG), breaker + 7 d (B7), and breaker + 15 d (B15). (B) Relative transcript abundance of SlLOB1 in WT and two RNAi lines (LOB #3 and #6) at stages MG, BR, B7, and B15 of pericarp and locule (gel). (C) Fruit firmness measured by fruit compression and pericarp penetration at the indicated developmental stages. *P < 0.05, **P < 0.01. (D) WT and SlLOB1 RNAi (LOB #3) fruit at indicated developmental stages (see A) with pericarp partially removed to see locule development. Error bars indicate SE.

Fig. 2.

Postharvest shelf life and water loss of T2 SlLOB1 RNAi fruit. (A) WT and LOB #3 and #6 fruit were harvested at B15 and stored at room temperature (25 °C) and photographed at the indicated days postharvest. (B) The same fruit shown in A were weighed at the indicated days postharvest to measure water loss (**P < 0.01). Error bars indicate SE.

Fig. 3.

Transcriptome analysis, cell wall gene expression, and EXP1 protein of SlLOB1 RNAi lines. (A) Overview of DEGs in SlLOB1 repression lines. (B) Venn diagram of overlapping down-regulated genes between SlLOB1 repression lines LOB #3 and LOB #6 pericarp and locule (gel) compared to WT. (C) Venn diagram of overlapping up-regulated genes of tissues as in B. (D) Subset of the 10 cell wall–associated DEGs in B, annotation, functional categories, and expression (log 2 of reads per kilobase million). (E) Time course qRT-PCR of EXP1 in pericarp and locule (gel) of WT and SlLOB1 RNAi fruit. MG, BR, B7, and B15 tissues were compared to MG WT as reference (defined as 1). (F) Detection of EXP1 protein in SlLOB1 RNAi and control Breaker fruit. (Upper) Total protein ponceau staining. (Bottom) Immunoblotting. 1, WT MG pericarp (peri); 2, WT BR peri; 3, WT BR locule (gel); 4, LOB #3 BR peri; 5, LOB #3 BR gel; 6, LOB #6 BR peri; 7, LOB #6 BR gel. (G) qRT-PCR validation of selected cell wall genes at MG, BR, B7, and B15 with WT MG used as reference (defined as 1). Error bars indicate SE.

Fig. 4.

Effects of SlLOB1 overexpression fruit. (A) Premature locule liquefaction occurs in locule of SlLOB1 overexpression fruit. WT (Left) and SlLOB1 OE2 (Right). (B) Softening as defined by pericarp penetration. (C) Softening as defined by whole fruit compression (*P < 0.05, **P < 0.01). (D) SlLOB1 expression in 35S: LOB1 OE2 and WT fruit tissues. (E) Heat map of cell wall gene expression in 35S: LOB1 OE2 leaves and fruit (log10 of reads per kilobase million [RPKM]). Error bars indicate SE. (F) Dual luciferase assay. EV indicates empty vector with output defined as 1. The dotted line indicates LUC/REN = 3. Error bars indicate SE.