Preliminary Study On Pod Valve Anatomy,physiological Characteristics And Pod Dehiscence Mechanism Of Medicago Ruthenica

Medicago ruthenica is an important native grass species in northern China.It has high application value of ecological restoration,improvement and breeding value of legume crops,and feeding value in arid and cold areas,which is a very valuable strategic resource for China.This superior forage has a long florescence,and the pod dehiscence and seed dropping off obviously after pod maturation,which not only brings inconvenience to seed harvesting and affects seed yield,but also restricts its promotion and application in grassland agricultural production and degraded grassland ecological restoration.To clarify the pod cracking mechanism of M.ruthenica has an important theoretical guiding significance for effectively controlling pod cracking,new varieties of pod cracking resistance breeding,and practical problems solving in seed production,greatly shortened the process of field anti-cracking pod material breeding.In the previous study,we found that the pod of M.ruthenica cracked only along the ventral suture,and the peel did not undergo spiral distortion.There also has no abnormal lignified cell structure at the area of the ventral suture,which is different from other reported pod dehiscence mechanisms of legumes.On this basis,this study selected materials of pod shattering-susceptible(SPD,pod cracking rate≥76%)and pod shattering-resistant(RPD pod cracking rate<10%).By comparing the differences in microscopic anatomical structure,physiological and biochemical characteristics and transcriptome of pod walls,we comprehensively analyzed the factors leading to pod dehiscence,and screened the differentially expressed genes related to pod dehiscence.Meanwhile,we had to screene the reference genes for better understanding those genes expression and function regulating pod dehiscence in the future.The main results are as follows:(1)EF-1a is the best candidate reference gene in M.ruthenica via comprehensive evaluation of expression stability.In order to make better use of quantitative real-time PCR(q RT-PCR)analysis in genes expression and function regulating pod dehiscence in the future.It is necessary to comprehensively analyzed and evaluated gene expression stability of the 9 candidate reference genes(EF-1a,a-TUB,b-TUB,ACT,U6 sn RNA,5.8S r RNA,5S r RNA,18 S r RNA,GAPDH)by useing five evaluation softwares,which was treated under different genotypes,different developmental stages,different environmental stresses and different tissues.EF-1a is the best reference gene in our comprehensive evaluation by Ref Finder software.(2)Water loss of pod walls and highly lignified of endocarp promoted pod active dehiscence.The pod development period was 36 days.Through the observation of pods dynamic development,we found that the lignification of endocarp was higher and the cell wall of sclereids was thicker in SPD material during the stage from 12 to 16 days after flowering.During the stage of 20 to 28 days after flowering,parenchyma cells in mesocarp of SPD material lost water and shrank together or even disappeared,the endocarp was also broken.Nevertheless,the mesocarp was still visible and the endocarp is almost intact in RPD material.It indicated that pod walls water loss and highly lignified endocarp closely related to the occurrence of pod dehiscence.(3)The main components of pod walls and their related physiological processes play an auxiliary role in pod dehiscence.Through the determination and comparison of pod walls physiological characteristics,we found that the water content of pod walls was lower,the cellulose content,hemicellulose content and lignin content were higher in SPD material.The activity of Ces A,PAL,4CL,CAD and POD which synthetize principal component of pod wall were higher in SPD material,and the activities of PG and CE which hydrolyze cell wall were also higher in SPD material.It indicated that the principal components of the pod walls and related synthetase indirectly assisted in pod active dehiscence,and the component-related hydrolase directly assisted in pod passive dehiscence.(4)Phenylpropanoid biosynthesis is an important metabolic pathway affecting pod dehiscence of M.ruthenica.RNA-seq technology was used to analyze the differential genes during the four stages of pod development in SPD material and RPD material.The results of GO annotation showed that a total of 17,072 unigenes and 2,706 differentially expressed genes(DEGs)matched to metabolic process and ranked first in the biological process category.The results of KEGG annotation showed 498 unigenes and 91 differentially expressed genes matched to phenylpropanoid biosynthesis(ko00940),respectively,ranking higher in the metabolic category,which is important to pod dehiscence.According to phenylpropanoid biosynthesis,totally of 58 DEGs related to lignin synthesis were screened,and most of these DEGs were significantly up regulated in SPD materials.When the lignin-related DEGs strongly expressed,the expression of flavonoid-related DEGs were inhibited(the branch of phenylpropanoid biosynthesis).The results constructed important gene expression information for the regulation network of pod dehiscence.The experiment further selected 19 DEGs of 58 DEGs for performing q RT-PCR verification on the transcriptome.The results showed that the linear fitting degree of transcriptome results and q RT-PCR results was good,and the transcriptome data were accurate and reliable(5)Transcription factors played a positive role and auxin played a negative role in pod dehiscence.A total of 23 NAC,74 MYB,39 WRKY and 46 b HLH DEGs were screened.The four transcription factor families all showed that the number of up-regulated DEGs was higher than the number of down-regulated DEGs.The DEGs expression pattern was consistent with the expression pattern of lignin biosynthesis genes in SPD,which is a positive regulator for pod dehiscence of M.ruthenica.Five DEGs of auxin biosynthesis were screened that showed down-regulated in SPD,which was opposite to the expression pattern of lignin biosynthesis genes and the change pattern of cell wall hydrolase activity.Auxin is a negative regulator of pod dehiscence in of M.ruthenica.(6)Lignin content of petal could be used as early predictor for M.ruthenica pod dehiscence.Lignin synthesis genes and flavonoid synthesis genes showed down-regulated in low pod-cracking-rate with yellow-flowered material(YFM).It is similar with RPD material.The lignin synthesis of YFM was on the contrary with SPD material,while it is similar with the flavonoid synthesis during 16 days after flowering of SPD material.The results showed that the color of flowers and pods could not use as an index to judge whether pod dehiscence of M.ruthenica,but the lignin content could.The field pre-screening period of anti-cracking pod materials of M.ruthenica can be advanced to the full flowering stage.In summary,this study is the first systematic analysis of the role of M.ruthenica pod walls in pod dehiscence.We found that the water content reduction of pericarp and the high lignification of endocarp played an important role in pod active dehiscence.The highly activity of cell wall synthase indirectly affected the pod dehiscence.The highly activity cell wall hydrolase played an important role in the pod passive dehiscence.Phenylpropanoid biosynthesis is an important metabolic pathway affecting M.ruthenica pod dehiscence.Transcription factors related to lignin synthesis were positive regulators of pod dehiscence,while auxin was a negative regulator of pod dehiscence.This study clarified the mechanism of M.ruthenica pod dehiscence.Our results showed great significance to reduce the loss of seed production caused by pod dehiscence.