The miR164a‐NAM3 module confers cold tolerance by inducing ethylene production in tomato

Abstract

SUMMARY Because of a high sensitivity to cold, both the yield and quality of tomato (Solanum lycopersicum L.) are severely restricted by cold stress. The NAC transcription factor (TF) family has been characterized as an important player in plant growth, development, and the stress response, but the role of NAC TFs in cold stress and their interaction with other post‐transcriptional regulators such as microRNAs in cold tolerance remains elusive. Here, we demonstrated that SlNAM3, the predicted target of Sl‐miR164a/b‐5p, improved cold tolerance as indicated by a higher maximum quantum efficiency of photosystem II (Fv/Fm), lower relative electrolyte leakage, and less wilting in SlNAM3‐overexpression plants compared to wild‐type. Further genetic and molecular confirmation revealed that Sl‐miR164a/b‐5p functioned upstream of SlNAM3 by inhibiting the expression of the latter, thus playing a negative role in cold tolerance. Interestingly, this role is partially mediated by an ethylene‐dependent pathway because either Sl‐miR164a/b‐5p silencing or SlNAM3 overexpression improved cold tolerance in the transgenic lines by promoting ethylene production. Moreover, silencing of the ethylene synthesis genes, SlACS1A, SlACS1B, SlACO1, and SlACO4, resulted in a significant decrease in cold tolerance. Further experiments demonstrated that NAM3 activates SlACS1A, SlACS1B, SlACO1, and SlACO4 transcription by directly binding to their promoters. Taken together, the present study identified the miR164a‐NAM3 module conferring cold tolerance in tomato plants via the direct regulation of SlACS1A, SlACS1B, SlACO1, and SlACO4 expression to induce ethylene synthesis.

Transcriptomic Responses to Chilling Reveal Potential Chilling Tolerance Mechanisms in Cucumber

“…It is well known that hormones play critical roles in chilling tolerance in plants [ 3 , 5 , 6 , 7 , 8 , 12 , 29 ]. In our data, 7 of the 59 BP terms were related to hormones and their signaling pathways, including “response to salicylic acid”, “response to jasmonic acid”, “response to ethylene”, “induced systemic resistance”, “jasmonic acid mediated signaling pathway”, “ethylene-activated signaling pathway”, and “auxin-activated signaling pathway” ( Figure 2 C,D), suggesting a large change in hormone responses upon chilling in cucumber.…”

“…As ethylene signaling is involved in the regulation of plant responses to several stresses, including chilling stress [ 12 , 30 , 31 , 32 , 33 ]. Variation in gene expression in response to chilling in nature may confer chilling tolerance in plants [ 34 ].…”

“…Previously studies have indicated that plant hormones and their respective signaling pathways, including auxin (indole-3-acetic acid, IAA) [ 3 ], abscisic acid (ABA) [ 4 , 5 , 6 ], salicylic acid (SA) [ 7 , 8 ], jasmonic acid (JA) [ 9 ], and brassinosteroids (BR) [ 10 ], play important roles in improving cucumber chilling tolerance. Ethylene is a gaseous plant hormone that also regulates plant chilling stress [ 11 , 12 , 13 ]. However, this hormone’s role in cucumber chilling tolerance regulation has not been well characterized.…”

“…In Arabidopsis thaliana , ethylene production decreased when plants were exposed to chilling temperature, exogenous ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or loss of function of ethylene signaling genes ETR1 , EIN2 , EIN3 , and EIN4 enhance Arabidopsis freezing tolerance, suggesting that ethylene has a negative effect on freezing tolerance in Arabidopsis [ 13 ]. In tomato, ethylene production increased upon chilling treatment [ 12 ]; exogenous ACC significantly enhanced tomato chilling tolerance; and the SI-miR164a functioned upstream of SINAM3 , which directly activated the expression of ACS1A , ACS1B , ACO1 , and ACO4 , thereby inducing ethylene synthesis upon chilling and thus enhancing tomato chilling tolerance [ 12 ]. These results suggested that ethylene plays distinct roles in the regulation of chilling tolerance in different plant species.…”

Tomato MicroRNAs and Their Functions

“…It is concluded that sly-miR164a is required for normal fruit development but that additional evidence is needed to support its involvement in ripening per se. Recently, it was found that sly-miR164a/b silencing via STTM and SlNAM3 ectopic expression improved cold tolerance in corresponding transgenic plants by promoting ethylene production, suggesting the involvement of the sly-miR164/ SlNAM3 module in tomato cold tolerance [ 86 ].…”