Functional analysis of tomato calmodulin gene family during fruit development and ripening

Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and development.However,the function of calmodulin remains elusive for fleshy fruit development.We performed expression studies of a family of six calmodulin genes(SlCaMs)in tomato fruit.All calmodulins showed a double peak expression pattern.The first flat peak appeared at 10–30 days after anthesis,but their expression rapidly declined at mature green and breaker.Then a sharp and even higher peak came at turning/pink stages.Among six calmodulins,SlCaM1 had the highest expression during fruit enlargement,whereas SlCaM2 was the major calmodulin during fruit ripening.However,SlCaMs showed different patterns in three ripening mutants rin,Nor and Nr.In particular,at the stages corresponding to mature green and breaker,the expression levels of SlCaMs in those mutants were significantly higher than wild-type.Furthermore,SlCaMs,especially SlCaM2 were upregulated by ethylene.Transiently overexpressing SlCaM2 in mature green fruit delayed ripening,while reducing SlCaM2 expression accelerated ripening.Our results suggest that SlCaMs play double roles to regulate fruit ripening.Prior to the ethylene burst,the ethylene-independent repression of SlCaMs might be critical for fruit to initiate the ripening process.After the ethylene burst,SlCaMs could participate in the ethylene coordinated rapid ripening.

Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal

Eggplant produces a variety of hydroxycinnamic acid amides(HCAAs)that have an important role in plant development and adaptation to environmental changes.In this study,we identified and characterized a spermidine hydroxycinnamoyl transferase(SHT)from eggplant(Solanum melongena)and its wild relative S.richardii,designated as SmSHT and SrSHT,respectively.SmSHT was abundant in flowers and fruits,whereas the level of SrSHT was remarkably low in all tissues.Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits,indicating its involvement in plant stress response.Both SHT polypeptides had extremely high identity with just five amino-acid substitutions.Recombinant SmSHT catalyzed the synthesis of mono-,bi-and tri-acylated polyamines.Using caffeoyl-CoA as the acyl donor,SmSHT preferred spermidine as the acyl acceptor.When spermidine was the acyl acceptor,the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA.SrSHT exhibited the same substrate specificity as SmSHT,yet exhibited significantly higher catalytic activity than SmSHT.For example,under caffeoyl-CoA and spermidine,Kcat of SrSHT was 37.3%higher than SmSHT.Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures.In particular,the conserved Lys402 adjacent to the DFGWG motif,and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT.These substitutions may contribute to the enhanced activity in SrSHT.Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.