Evolution Of Stomatal Immunity Against Fungi Invasion In Different Vascular Plants

Fungi are the most common plant pathogens,accounting for 70%-80%of plant diseases,posing a serious threat to global food security.Fungi infect plants by infection hyphae,meanwhile stomata is an important pathway for infection.At the same time,plants can actively close stomata to resist the invasion of pathogenic fungi,and stomatal closure induced by fungi and ABA share the same downstream elements of the pathway.Studies have shown that there are ABA-regulated stomatal closure behaviors and pathways in seed plants,while ferns and lycophytes have not evolved these pathways,and there stomata are insensitive to ABA.Therefore,we hypothesized that the responses to pathogenic fungi of different evolutionary groups of vascular plants were different.In this study the effect of Puccinia triticina and exogenous chitin on stomatal conductance,stomatal aperture,signaling components(H₂O₂,NO,and Ca²⁺),guard cell ion channels(Ca²⁺and K⁺)and vacuolar dynamics were analyzed in species of vascular land plants including 1 lycophyte,5 ferns,4 gymnosperms and 12 angiosperms,to reveal the differences and basis of stomatal immunity against pathogenic fungal invasion in different evolutionary groups of plants.Through a large number of analytical studies,we mainly achieved the following results:1.Puccinia triticina could induce the stomatal conductance of 4 seed plants to decrease by 42%to 56%,and chitin could induce the stomatal conductance of 16 seed plants to decrease by 38%to 95%,but ferns did not respond.Puccinia triticina and chitin induced a decrease in stomatal aperture of 9%-60%in all tested seed plants,while the stomatal aperture of lycophyte and ferns remained unchanged.Since the selected plants came from different families and genera,the study suggested that the response of stomata to pathogen fungal infection varied along the evolutionary type.Seed plants closed stomata to resist pathogen infection,while ferns or lycophytes did not evolve this innate immune function.2.The pathway of stomatal closure signaling element studies showed that after exogenous chitin induction treatment,all the plants H₂O₂ content in the guard cells were increased,but the expression of NO and the downstream signaling components including the guard cell cytoplasmic calcium ion concentration rise,Ca²⁺and K⁺ion channels activation,etc.,only expressed in seed plants,but not in guard cells of ferns or lycophytes.The results suggested that the stomatal closure pathway induced by chitin has only evolved in seed plants,but not in ferns or lycophytes.3.The biophysical characteristics of guard cell closure process showed that the vacuoles of guard cell in Vicia faba were highly fragmented under exogenous chitin induction,but not in Matteuccia struthiopteris.It was preliminarily found that the biophysical basis of the process of chitin-induced stomatal closure from ferns to seed plants also has a gradual evolution.4.The leaves of all lycophytes,ferns and seed plants showed oxidative bursts and callose deposition induced by exogenous chitin,indicating that although the lycophytes or ferns not equipped with stomatal immunity,there were other defense mechanisms.In brief,stomatal immunity evolved from nothing in the evolution of vascular plants,and the early lycophytes or ferns did not evolve the function of stomatal immunity against pathogen fungi,which evolved in seed plants.These results provided an important basis for understanding the evolution and formation of innate immunity in lycophytes,ferns,gymnosperms and angiosperms.