The Function Of SpMPKs On Tomato Tolerance To Abiotic Stresses

Tomato as the second most consumed vegetable is widely grown around the world andconstitutes a major agricultural industry. However, abiotic stresses like drought and salinehave been threatening the growth and development of tomato plants and the quality of tomatofruits. Recently, there are studies have shown that MAPK (mitogen activated proteinkinase)cascade plays an important role in plant defense signaling, but little about MAPKs intomato. It is meaningful to study the gene expression profiles of tomato MAPKs, clarifytheir functions in stress tolerances of tomato at growth and development stage. Herein, theexpression patterns of SpMPK1, SpMPK2and SpMPK3in Solanum pimpinellifolium andSlMPK1，SlMPK2and SlMPK3in Solanum lycopersicum tomato plants exposed to drought,salt and cold stresses were studied. A novel method to evaluate virus-induced gene silencingefficiency was established. The functions of SpMPK1,-2and-3in plant tolerance to abioticstresses like drought, salt, were clarified by VIGS（virus-induced gene silencing） andAgrobacterium-mediated genetic transformation techniques. The main results are as follows:1. The expression patterns of SpMPK1/SlMPK1, SpMPK2/SlMPK2andSpMPK3/SlMPK3genes induced by abiotic stresses were analyzed. It was found that theexpression patterns of SpMPK1/SlMPK1, SpMPK2/SlMPK2and SpMPK3/SlMPK3inducedby drought, salt and low temperature stresses were similar in L03708and M82-graduallyincreased to a peak and declined to background with longer treatment, but the abundance atthe same time were markedly different in the two materials. When plants were subjected to10%PEG solution, the transcription levels of SpMPK1/SlMPK1, SpMPK2/SlMPK2andSpMPK3/SlMPK3were increased by2-5folds compared to control (0h), and they were higherin L03708than that of M82with a1.5-2folds. Similarly, when the plants were subjected to300mM NaCl stress, the transcription levels of MPK1and MPK3were increased by2-5foldscompared to control, and the expression of SpMPK1and SpMPK3were higher than that ofSlMPK1and SpMPK3with a2and-2.5folds, respectively. In addition, the transcriptionlevels of MPK1and MPK2were increased by2-9folds under4°C stress, and they werehigher in L03708than that of M82with a1.5-5folds. The highest transcription level ofMPK3was detected at12h after4°C stress，when the transcription level of SpMPK3and SlMPK3were induced to80-fold in L03708and20-fold in M82compared to control (0h).This result suggested that MPK1,-2and-3were induced by drought, salt and low temperaturestresses, and the transcription levels of SpMPK1,-2and-3in L03708were higher thanSlMPK1,-2and-3in M82under each stress.2An evaluation system was established for the evaluation of VIGS silencing efficiency.Because of the limitation of the application of VIGS technology—unstable silencingefficiency and localized tissue-silencing, four indexes—silencing frequency of plants,silencing efficiency of a plant, silencing frequency of each leaf and silencing efficiency ofgene were defined and utilized to evaluate VIGS silencing efficiency by silencing theendogenous PDS (phytoene desaturase) gene with a TRV (Tobacco rattle virus)—based VIGSvector. The silencing results of SpPDS showed that an optical density at600nm of2.0wasmore suitable than the others for VIGS in S. pimpinellifolium, at which the silencingfrequency of plant reached to97%at25dpi and SpPDS gene was highly silenced in the4th9th leaves during25-40dpi.3. The function of SpMPK1,-2and-3genes were identified using VIGS technology.Individual or combined gene silencing of SpMPK1,-2and-3attenuated the drought toleranceof tomato plants by varying degrees. In the presence of100μM ABA, H2O2production ingene silenced plants increased by3-6folds compared with plants infiltrated with pTRV2:00.In the dehydration trial, survival rates of gene-silenced plants were reduced by4%-65%compared to the control plants. Co-silencing SpMPK1and SpMPK2impaired ABA (abscisicacid)-induced and H2O2(hydrogen peroxide)-induced stomatal closure. Similar results wereobserved when silencing SpMPK3alone, but not when SpMPK1and SpMPK2wereindividually silenced. SpMPK1,-2and-3may play crucial roles in enhancing tomato plants’drought tolerance by influencing stomatal activity and H2O2production via the ABA-H2O2pathway. The functions of SpMPK1and SpMPK2may be redundant, and they overlap withthat of SpMPK3in this process.4. The function of SpMPK1,-2and-3genes were identified using an Agrobacteriumtumefaciens-mediated floral dip method. The overexpression of SpMPK1,-2and-3improvedArabidopsis plants’ tolerance to stresses at germination and development stage. At thegermination stage, when exposed to10%PEG and100,200mM NaCl stress, transgenicArabidopsis plants overexpressing SpMPK1,-2and-3showed higher germination rate(>90%), while in WT（wild-type）plants it was10%、31%and15%, respectively. Buttransgenic seeds of SpMPK1,-2and-3showed lower germination rate-30%,10%and2%,respectively, than wild-type (86%) when exposed to0.5μM ABA. At development stage, theleaves of transgenic Arabidopsis plants showed decreased productions of O2-and H2O2 compared with WT plants under150mM NaCl and100μM ABA stresses, and the leaves oftransgenic plants lost less water than WT plants at6h post-detached. In addition, theiroverexpression enhanced the transcription levels of ABA inducible genes—RD29A, RAB18and RD22, and transcription factor genes—ZAT6, ZAT10and MYB44, in transgenicArabidopsis compared with WT plants in response to ABA or salt stress. These results suggestthat SpMPK1,-2and-3are positive regulators in response to osmotic stress in Arabidopsis atgermination and development stage.