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Increasing evidences highlight the importance of DEAD‐box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt‐stress conditions. Map‐based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast‐targeted DEAD‐box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break‐B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast‐targeted DEAD‐box proteins are induced by salt‐stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD‐box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops., The role in plant development and fruit yield of a chloroplast‐targeted tomato (Solanum lycopersicum) DEAD‐box RNA helicase, SlDEAD39, is described. We propose that other DEAD‐box RHs might be partially redundant to SlDEAD39, mediating their specific functions in salt stress response.

Agricultural systems currently face important issues mainly related to global climate change as well as to world population growing rates. Global warming has set up a scenario were increasing temperatures promote plant heat stress thus affecting crop productivity and yield. Furthermore, heat is often linked to a limited water availability in Mediterranean agricultural areas where the combined action of heat and drought stresses increases plant damage causing reduced plant growth and productivity loss. Therefore, developing heat-drought tolerant crop varieties is crucial for enhancing yield under these challenging conditions. Reports show that tomato reproduction is highly sensitive to extreme temperatures, which can cause male gametophyte abortion and drastically reduce fruit set. Given this crop importance, we have undertaken a classical genetic approach in order to identify tomato variants showing a resilience response to combined heat and drought stress. With this aim, a phenotype screening of a natural variant collection and an EMS mutagenised population was performed. As a result, a total number of 142 tolerant variants were selected and re-screened to confirm the tolerant phenotype and to isolate the genes participating in plant response. Additionally, a Tilling analysis involving several genes previously reported as key regulators of abiotic stress responses in different species has been initiated. Hopefully, results will contribute to a better knowledge of the genetic factors promoting abiotic stress tolerance as well to coming tomato breeding initiatives aimed to the development of new varieties better adapted to climate emergency conditions.

Figure S2. Identification in vitro of mutants altered in trichome development. a Most type I trichomes of the mutant 744-P73 have abnormal terminal cells. b The leaves of 1491-MM have fewer type I trichomes. c The leaves of 912-P73 have fewer trichomes of types I and VI. d The leaves (right, up) and stem (right, down) of the mutant 4728-SP have a greater number of trichomes with respect to Solanum pennellii wild-type plants (left, up and down). Bar = 1 mm. (PPTX 2580 kb)

Figure S6. Flowering time in plants of the mutant Altered in all reproductive traits (Art). a. Number of phytomers up to the first inflorescence (0–1) and between inflorescences (1–2, 2–3, …). b Length from base up to the first inflorescence (0–1) and between inflorescences (1–2, 2–3, …). (PPTX 63 kb)

Table S1. Phenotype inheritance in tomato mutants altered in leaf development. (DOCX 20 kb)

Table S3. Co-segregation analysis between phenotype and a T-DNA insert with a functional nptII gene in mutants altered in leaf development. (DOCX 14 kb)

Figure S5. Vegetative and reproductive development of the tomato mutant necrotic and small leaflets (nsl). a. Mutant nsl seedlings (right) develop smaller leaves than WT (left). b. In the mutant nsl the leaves of shoot apex-derived plants have small necrotic spots (right), which does not occur in the WT (left). c. In nsl greenhouse-grown plants adult leaves have a great number of small leaflets that exhibit necrotic spots in the leaf blade. d. In nsl mutant plants a decrease in quantitative parameters related to the number of fruits per inflorescence, fruit set rate and fruit size is observed. Bar = 1 cm. (PPTX 627 kb)

Figure S1. Morphology of the leaves of greenhouse-grown plants of tomato cv ‘P73’ and some dominant mutants. a Fifth leaf of a tomato plant cv. ‘P73’. b-c The mutants 816-P73 (b) and 740-P73 (c) have darker, less lobed leaves. d-f The mutants 136-P73 (d), 605-P73 (e) and 14-P73 (f) develop leaflets of different morphology. g The mutant 630-P73 develops small leaves with irregular arrangement of leaflets. h The mutant 860-P73 has more complex leaves with smaller leaflets. Bar = 1 cm. (PPTX 922 kb)

Table S2. Number of T-DNA inserts with a functional nptII marker gene in tomato mutants altered in leaf development. (DOCX 19 kb)

Figure S3. Vegetative and reproductive development of the tomato mutant wiry-like-150 (wl-150). a Basal leaves of wl-150 have a certain degree of leaf blade expansion, while the following exhibit shoestring shape. b The inflorescence of wl-150 (right) is more branched than that of wild-type ‘P73’ plants (left). Note that the inflorescences of wl-150 also alternate vegetative and reproductive traits. c Flowers of wl-150 have thread-like sepals and petals as well as an open anther cone. d The mutant wl-150 is partially fertile since it develops fruits ranging from small seedless (right, up) to others of normal size with some seeds (right, down) similar to that of wild-type ‘P73’ (fruit on the left). Bar = 1 cm. (PPTX 492 kb)

Figure S3. Mapman stress diagrams. Differentially-expressed genes (DEGs) between res and WT in control and salt-stressed roots and leaves (200 mM NaCl for 5 days) involved in stress responses. Positive fold change values (red) indicate up-regulation (minimum fold-chang of 2.0) in res mutant compared to WT in each condition, whereas negative fold change values (blue) indicate down-regulation (minimum fold-change of − 2.0). Each coloured square represents an individual DEG. (PPTX 1566 kb)

Tomato CALCINEURIN B-LIKE PROTEIN 10 (SlCBL10) ensures plant growth by regulating proper distribution of Na+ and Ca2+ in the shoot apical meristem and developing organs under salt stress.

C3′H and HQT gene structures and primer pairs (triangle arrows) designed for HRM analysis. (*) SNPs position; (#) a.a. alteration. The introns are indicated in grey. (PDF 57 kb)