Summary In plants, the chilling response involves decreased photosynthetic capacity and increased starch accumulation in chloroplasts. However, the mechanisms that modulate these processes remain unclear. We found that the SlWHY1 gene is significantly induced by chilling stress (4°C) in tomato. Three SlWHY1 overexpression (OE) lines grew better than the wild type (WT) under chilling stress; the OE plants retained intact photosynthetic grana lamellae and showed enhanced hydrolysis of starch. By contrast, RNAi lines that inhibited SlWHY1 were more affected than the corresponding WT cultivar. Their grana lamellae were damaged and starch content increased. The psbA gene encodes the key photosystem II (PSII) protein D1. We show that SlWHY1 binds to the upstream region (A/GTTACCCT/A) of SlpsbA and enhances the de novo synthesis of D1 in chloroplasts. Additionally, SlWHY1 regulates the expression of the starch‐degrading enzyme α‐amylase (SlAMY3‐L) and the starch synthesis‐related enzyme isoamylase gene (SlISA2) in the nucleus, thus modulating the starch content in chloroplasts. We demonstrate that SlWHY1 enhances the resistance of tomato to chilling stress by maintaining the function of PSII and degrading starch. Thus, overexpression of WHY1 may be an effective strategy for enhancing resistance to chilling stress of chilling‐sensitive crops in agricultural production.

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Rubisco, which consists of eight large subunits (RBCLs) and eight small subunits (RBCSs), is a major photosynthetic enzyme that is sensitive to chilling stress. However, it is largely unclear how plants maintain high Rubisco content under low temperature conditions. Here, we report that tomato WHIRLY1 (SlWHY1) positively regulates the Rubisco level under chilling stress by directly binding to the promoter region of SlRbcS1, resulting in the activation of SlRbcS1 expression. SlRbcS1-overexpressing lines had higher Rubisco contents and were more resistant to chilling stress compared with the wild type. Quantitative real-time PCR analyses showed that, among the five RbcS genes, only SlRbcS1 expression is up-regulated by chilling treatment. These results indicate that SlWHIRLY1 specifically enhances the levels of SlRbcS1 and confers tolerance to chilling stress. The amino acid sequence of SlRBCS1 shows 92.67% identity with those of another two RBCS proteins and three residues are specifically found in SlRBCS1. However, mutation of these residues to alanine in SlRBCS1 does not influence its function during cold adaptation. Thus, we conclude that high levels of Rubisco, but not the specific residues in SlRBCS1, play important roles in tolerance to chilling stress in tomato.

SUMOylation is an important post-translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS-type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was upregulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H 2 O 2 ) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1-2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.

Heat stress poses a major threat to plant productivity and crop yields. The induction of heat shock proteins (HSPs) by heat shock factors is a principal defense response of plants exposed to heat stress. In this study, we identified and analyzed the heat stress-induced Whirly1 (SlWHY1) gene in tomato (Solanum lycopersicum). We generated various SlWHY1-overexpressing (OE) and SlWHY1-RNA interference (RNAi) lines to investigate the role of WHIRLY1 in thermotolerance. Compared with the wild type (WT), the OE lines showed less wilting, as reflected by their increased membrane stability and soluble sugar content and reduced reactive oxygen species (ROS) accumulation under heat stress. By contrast, RNAi lines with inhibited SlWHY1 expression showed the opposite phenotype and corresponding physiological indices under heat stress. The heat-induced gene SlHSP21.5A, encoding an endoplasmic reticulum-localized HSP, was upregulated in the OE lines and downregulated in the RNAi lines compared with the WT. RNAi-mediated inhibition of SlHSP21.5A expression also resulted in reduced membrane stability and soluble sugar content and increased ROS accumulation under heat stress compared with the WT. SlWHY1 binds to the elicitor response element-like element in the promoter of SlHSP21.5A to activate its transcription. These findings suggest that SlWHY1 promotes thermotolerance in tomato by regulating SlHSP21.5A expression.

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Introduction: bHLH family proteins play an important role in plant stress response. However, the molecular mechanism regulating salt response of bHLH is largely unknown.Materials and Methods: Here, we investigated the function and regulating mechanism of the sweet sorghum SbbHLH85 during salt stress.Results: Results show that SbbHLH85, different from its homologs in other species, is a new atypical bHLH transcription factor and is a key gene for root development in sweet sorghum. Knockout of SbbHLH85 in sorghum by CRISPR-Cas9 results in the inhibition of root development. Overexpression of SbbHLH85 resulted in signi cantly increased number and length of root hairs by participating in ABA and auxin signaling pathways, which can increase the absorption of Na + . While SbbHLH85 plays a negative regulatory role in salt tolerance of sorghum. Through screening yeast two-hybrid library, we identi ed a potential interaction partner of SbbHLH85 that is phosphate transporter chaperone PHF1, which modulates the distribution of phosphate. Both yeast two-hybrid and BiFC experiments con rmed interaction between SbbHLH85 and PHF1.Discussion: Based on these results, we suggest that the increase of Na + content and the decrease of Pi content result in the salt sensitivity of transgenic sorghum.

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