Proteomic Analysis Of The Stress Response In Pre- And Post- Swollen Stem In Zizania Latifolia

Zizania latifolia(Griseb.) Turcz. is a perennial aquatic grass that mainly grows in eastern Asia. As the second largely cultivated aquatic vegetable in China, Z. latifolia is very popular for its unique flavor and delicacy. The swollen stem of Z. latifolia is closely rela ted to the infection of its endophytic fungus, Ustilago esculenta, which induced stress responses in the plantlets of Z. latifolia. In this research, the infection and distribution of U. esculenta between the normal Z. latifolia(―Suozi jiao‖ and ―Long jiao 2‖) and un-normal Z.latifolia with non-swollen stem were observed. Differential expression proteins of swollen stem were detected and identified by two-dimensional polyacrylamide gel electrophoresis(2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry(MALDI-TOF-TOF MS). The mechanism of stress response to the pre- and post- swollen stem in Z. latifolia was discussed.1. Observation and analysis of the differential distribution of U. esculenta among the pre- and post- swollen stems in Z. latifoliaStems of normal Z. latifolia and un-normal Z. latifolia were investigated with microscopy. T here were no U. esculenta detected in the non-swollen stem of un-normal Z. latifolia. The infection of U. esculenta had been detected in the growing point of pre-swollen stem in normal Z.latifolia at 5-leaf stage, with small clusters of hyphae growing in the parenchyma surrounding the vascular bundles. T he number of hyphae clusters increased obviously at 8-leaf stage. At the initial stage of swelling, hyphae of U.esculenta grew around the vascular bundles with a network structure, and distributed in the parenchyma of stem with lots of hyphae clusters.2. Detection and identification of differential expression proteins among the preand post- swollen stems in Z.latifoliaBy the method of 2-DE, differential proteomics on the swelling of Z.latifolia were compared among different development periods including the stages of 5-leaf, 8-leaf and the initial stage of swelling. One hundred and twenty differentially expressed proteins were detected; ninety protein spots were successfully identified by the means of MALDI-TOF-TOF MS analysis. Predicting and classification the function of the differential expressed proteins in Z.latifolia which were proceed and classed into seven categories: twenty three spots of transcription and translation-related proteins, eighteen spots of proteins involved in carbohydrate synthesis and metabolism, nine spots of fatty acid synthesis and metabolism-related proteins, fifteen spots of stress tolerance associated proteins, five spots of the cell division, nine spots related to ubiquitin proteasome pathway and five spots of energy-related. There were also six spots with unknown function. Fifty-eight percent of the differential expression proteins were related to transcription translation, carbohydrate synthesis metabolism and stress tolerance.Compared to the stem at 5-leaf stages, proteomics on stress response of pre- and post- swollen stem were compared in normal Z.latifolia. Based on the removing of differential expression proteins which also changed in unnormal Z.latifolia, swollen-related spots were selected. At 8-leaf stage, there were thirty two proteins in Longjiao 2 and thirty proteins in Suozi jiao. At the initial stage of swelling, there were sixteen spots in Longjiao 2 and seventeen in Suozi jiao. Thirteen spots with the same trends of differential expression were obtained in the normal swollen stem between Longjiao 2 and Suozi jiao. There were five up-regulated protein spots at 8-leaf stage: including triosephosphate isomerase, alcohol dehydrogenase 2-like, phenylalanine ammonia lyase, Eukaryotic translation initiation factor 3 subunit 2, glyceraldehyde-3-phosphate dehydrogenase 3; and two down-regulated protein spots: incuding glycine-trna ligase 1 and 60 S acidic ribosomal protein P0. There were four up-regulated protein spots at the initial stage of swelling: probable cytokinin riboside 5’-monophosphate phosphoribohydrolase LOG4, probable L-ascorbate peroxidase 7, phosphoglycerate kinase, ATP synthase β subunit; and two down-regulated protein spots: glutathione S-transferase F8, phospholipase A-2-activating protein-like.3. Differential expression proteins of stress response to the infection of U.escu lentaUsing the non-swollen stem in un-normal Z.latifolia as control, differential expression proteins were related to stress response in the two normal Z. latifolia which were compared and selected. There were 32 differential expression proteins at 5-leaf stage, fifteen differential expression proteins at 8-leaf stage and sixteen differential expression proteins at the initial stage of swelling. The regulation about stress response of Z.latifolia induced by the infection of U.esculenta had begun in the early development stage in Z.latifolia, corresponding with the successful infection of U.esculenta at 5-leaf stage. Many related proteins of stress tolerance distributed at three stages, with the percentage of twenty two,thirty three and twenty seven respectively, which indicated that the distribution of U.esculenta would play an important role in the stress response of Z.latifolia.Classification analysis of the function of differential expression proteins showed that there were obviously different stress responses among different development periods. For example, the differential expression proteins related with carbohydrate synthesis and metabolism were mainly detected at the stage of 5-leaf and 8-leaf. However, the differential expression proteins related with cell division were mainly detected at the stage of 8-leaf stage and the initial of the swollen. The differential expression proteins at 8-leaf stage were mostly related to the swollen stem, which indicated the importance of the 8-leaf stage during the stem swelling in Z.latifolia.4. Differential expression proteins of the interaction between U. esculenta and the callus of Z.latifoliaTo study the stress response of U. esculenta, the interaction between U. esculenta and Z. latifolia were proceed between U. escu len ta and the callus of Z.la tifo lia in vitro. Differential expression proteins were selected and identified by the methods of 2-DE and MS. Seven differential proteins were successfully identified, including six proteins of down-regulated and one spots of up-regulated. The functions of differential expression proteins were mostly associated with cell division. The changing of stress response in U. esculenta was discussed in this research.