AUREA maintains the balance between chlorophyll synthesis and adventitious root formation in tomato

Abstract

Flooding tolerance is an important trait for tomato breeding. In this study, we obtained a recessive mutant exhibiting highly enhanced submergence resistance. Phenotypical analyses showed that this resistant to flooding (rf) mutant displays slightly chlorotic leaves and spontaneous initiation of adventitious roots (ARs) on stems. The mutation was mapped to the phytochromobilin synthase gene AUREA (AU), in which a single amino acid substitution from asparagine to tyrosine occurred. In addition to the classic function of AU in phytochrome and chlorophyll biogenesis in leaves, we uncovered its novel role in mediating AR formation on stems. We further observed temporal coincidence of the two phenotypes in the rf mutant: chlorosis and spontaneous AR formation and revealed that AU functions by maintaining heme homeostasis. Interestingly, our grafting results suggest that heme might play roles in AR initiation via long-distance transport from leaves to stems. Our results present genetic evidence for the involvement of the AU-heme oxygenase-1-heme pathway in AR initiation in tomato. As fruit production and yield in the rf mutant are minimally impacted, the mutation identified in this study may provide a target for biotechnological renovation of tomato germplasm in future breeding.

Keywords: Flooding; Gene regulation; Plant breeding; Plant molecular biology; Plant signalling.

Fig. 1. rf mutant is flooding resistant.

a, b Flooding resistance of wild-type (WT) and rf. Both WT (a) and rf (b) at the three-leaf stage were treated by flooding for 7 days. Seedlings of WT (a) and rf (b) at the same stage without the flooding treatment were used as the control. Bars = 1 cm. c Quantification of survival and mortality rate at 7 days after flooding treatment. The results are based on two independent treatments (n = 17 and 26, respectively. Mean ± SD). d AR formation in the hypocotyl of WT and rf exposed to 0 or 3 days of flooding treatment. Bar = 1 cm. e Quantification of adventitious roots in WT and rf exposed to 0 or 3 days of flooding treatment. Error bars represent SD (n = 15). AR adventitious root, DPF days post flooding

Fig. 2. Phenotypic analysis and map-based cloning of the candidate gene.

a Flowering plants of WT and rf grown under normal conditions. The yellow dotted boxes indicate the hypocotyl or distal stem internodes that enlarged in the corresponding insets. Bar = 5 cm. b Histological sections of hypocotyl ARs in WT and rf. The yellow arrows point to ARs initiated in the pericycle-like layer. Bar = 1 mm. c SNP-index peak map of the mutation in rf by BSA-seq. The red circle indicates the collection of points with a 100% SNP index. d Location of the single-nucleotide mutation in the third exon of the candidate gene. The dotted box indicates the mutated codon. e Schematic structure of the AU protein and the position of the single amino acid substitution in the Fe-bilin-red domain

Fig. 3. Genetic complementation of rf.

a, b The AR phenotype in WT and rf. Hypocotyl-derived ARs (c) and histological sections of AR primordia in OE-AU/rf (d). T1-47, 56, and 93 represent three T1 lines from different independent T0 plants. e Test of flooding resistance in OE-AU/rf lines at 1, 3, and 5 DPF and 1 and 10 DPR (days post recovery). Seedlings were treated with 5 days of flooding and then transferred to normal conditions for recovery. Bar = 1 cm

Fig. 4. Dual roles of AU in chlorophyll synthesis and AR primordia initiation.

a Schematic diagrams of AU function in the heme pathway in tetrapyrrole metabolism. b Histological analysis of AR primordia in the hypocotyl of rf at the three- to five-leaf stage. Five-leaf-stage WT plants were used as controls. The red circle shows the AR primordium, and the number below each figure represents the corresponding leaf stage. Bar = 0.2 mm. c The temporal expression pattern of SlHEMA1 in WT and rf. The number below each column represents the corresponding leaf stage. Error bars represent SE (n > 3). d Comparison of HO-1 activity between WT and rf at the two- to four-leaf stage. The number below each column represents the corresponding leaf stage. Error bars represent SE (n > 3)

Fig. 5. HO-1 activity positively regulates AR formation.

Hypocotyl-derived ARs in WT (a, b) and rf (c, d) treated with 200 µM ZnPP IX for 5 days. ZnPP IX is an inhibitor of HO-1 activity. Bar = 1 cm. e HO-1 activity increases in the rf hypocotyl compared to the WT under normal growth conditions. Error bars represent SE (n = 3). f ZnPP IX decreases HO-1 activity in the rf hypocotyl. Error bars represent SE (n = 3). g The numbers of AR in WT and rf hypocotyls with (+) or without (−) ZnPP IX treatment. Error bars represent SE (n = 3). ZnPP IX zinc protoporphyrin IX

Fig. 6. Communication between leaves and hypocotyls is responsible for AR initiation.

a–d Different types of grafting combinations. rf/WT represents that scion of rf was grafted onto the WT stock, and the same designation rule applies to WT/WT, WT/rf, and rf/rf. The yellow boxes indicate the hypocotyl section that is enlarged in the corresponding insets. The yellow arrows indicate adventitious roots (c). Bar = 1 cm. e Quantification of ARs in different types of grafting. Error bars represent SE (n = 5). f Hypocotyl HO-1 activity in different types of grafting. Error bars represent SE (n = 5). g Schematic diagram of the AU–(HO-1)–heme–CO pathway in adventitious root formation. Loss of function of AU potentially leads to massive accumulation of heme in leaves and enhances HO-1 activity in the hypocotyl. The overaccumulated heme fluxes from the leaves to hypocotyls, where it is decomposed by HO-1. Through CO production, extra heme promotes the formation of adventitious roots in stems