Regulation Of Stomatal Development By NO And H₂S On Abaxial Cotyledon Epidermis In Arabidopsis

Stomata are valves on epidermis responsible for the exchange of gases between plant and environment.And its development is strictly regulated by endogenous signals and environmental cues.NO and H2 S,as gaseous signal molecules in animals and plants,are widely involved in plant development and adaptive responses to biotic and abiotic stresses.At present,although regulation of different hormones in stomatal development has been widely studied,little is known about whether and how NO and H2 S are involved in this development process.Pharmaceutical methods were used in this study to explore the potential function of NO and H2 S in the modulation of stomatal development on the abaxail epidermis of Arabidopsis cotyledon.Stomatal density and distribution of wild type Arabidopsis seedlings with or without chemical treatments,including donors,scavengers and endogenous synthetic blockers of NO and H2 S,were analyzed to reveal their effects on stomatal development.In addition,changes of key regulator marker genes expression were detected to unveil the effects of these chemicals on different stages during stomatal development.The main results are as follows:1.Exogenous nitric oxide donor SNP treatment resulted in decreased density of stomata and the formation of SGC on the epidermis of the wild type cotyledon of Arabidopsis.Whereas treatment of wild type cotyledon with NO scavenger c PTIO and bio-synthesis inhibitor L-NAME lead to increased stomatal density and disrupted stomatal patterning with the formation of stomatal clusters.SNP can eliminate the effect of c PTIO and L-NAME on the stomatal density and distribution pattern,indicating change of endogenous NO accumulation may affect stomatal density and distribution.2.After SNP treatment,number of mature GC maker E1728-expressing cells decreased while number of precursor marker gene TMM-expressing cells increased.These results revealed that increase of endogenous NO induced more cells entering into the early stage of stomatal development,but the final mature stomatal number decreased.Further investigation found that number of FAMA-expressing cells decreased,which marks late stages of stomatal development---before and after the GMC division specifically.It means NO might inhibit the transition of stomatal lineage precursors cells into GMC.3 Exogenous SNP treatments reduced the high stomatal density of the tmm1 and sdd1 mutants,and induced the formation of SGC.SNP treatment can restore the stomatal pattern defect of sdd1 and decrease the formation of stomatal cluster.But this rescue effect of SNP on stomatal development in sdd1 was not found in tmm1.4.Exogenous H2 S donor Na HS treatment resulted in the decrease of stomatal density and the formation of SGC on the wild-type epidermis of Arabidopsis,an effect similar to that of exogenous NO donors.Whereas H2 S scavenger HT and synthetic inhibitor AOA treatment resulted in an increase in stomatal density.HT treatment eliminates the decrease in stomatal density and SGC generated by Na HS,indicating that increased endogenous H2 S accumulation inhibited stomatal development and the GMC symmetric cell division.5.After exogenous Na HS treatment,number of all the marker gene-expressing cells including TMM,FAMA and E1728 deceased on the abaxial cotyledon epidermis of wild-type seedlings.HT can eliminate the effect of Na HS on the expression of these marker genes,indicating that the increase of endogenous H2 S can inhibit multiple stages during stomatal development from the entry of epidermis cell into the stomatal lineage to late stages of GMC division and GC differentiation.6.Exogenous Na HS treatment reduced the high stomatal density of tmm1 and sdd1 mutants and induced SGC formation on the abaxial cotyledon epidermis.Different from SNP treatment,Na HS cannot restore the stomatal pattern defect of sdd1 and tmm1.7.NO and H2 S also affect PV development.SNP treatment results more PV cells with smaller size,while Na HS induced PV enlargement and differentiation.In summary,the disturbance of endogenous NO and H2 S concentrations will lead to the change of stomata development on the abaxial cotyledon of Arabidopsis.NO acts on the early stage of stomatal development and induces more primitive epidermal cells to enter into the stomatal lineage,but it inhibits the transition of precursor cell into GMC during late stage of stomatal development.SNP treatment also induced more PV cells with smaller size on the abaxial cotyledon epidermis.Nevertheless;increasing of endogenous H2 S inhibits stomatal development from early stage of the entry of predermal cell into stomatal lineage to late stages such as the GMC division.Contrary to NO,H2 S promotes the enlargement and differentiation of PV cells.These results provide basic data for further investigation of the regulatory networks of stomatal development.