Proteomic Analysis Of Two Near Isogenic Maize Lines With Different Leaf Angles

The maize planting area and total output of China rank the second in the world,which are only after that of the United State.Maize is China's important food crops,silage and industrial raw materials.It is the main food crop.Therefore,the stability and sustained growth of maize production play a major role in maintaining and improving the national economy.However,maize production is limited by drought,low production technology and mechanization level,backward seed industry development and other constraints.Enlarging the cultivation area,which can lead to increased production directly,is an important way to relieve the above constraints.However,due to the limited arable land in China,to increase the planting density becomes the main way to increase the final yield.Increasing the density of maize can be achieved by improving the morphology of maize plants,for example,cultivating compact plants.The small leaf angle,an important trait for determining the dense properties of maize,is one of the main traits of compact plant.Therefore,it is of great significance to explore the formation mechanism of maize leaf angle at protein level.In this study,maize inbred lines Shen 137(large leaf angle)and its near isogenic line NIL(small leaf angle)were used to study the relationship between the differences of plant characteristics and the change of leaf angle at different leaf stages by mechanical strength measurement,morphological and histological observation.Three protein-extraction methods that are commonly used in plant proteomics,i.e.,phenol extraction,TCA/acetone extraction,and TCA/acetone/phenol extraction,were qualitatively and quantitatively evaluated to select the method suitable for extracting maize leaf protein rich in thick-walled tissue.Comparative proteomic analysis and glycostaining were performed to reveal protein profile differences in the midribs of the 5th,10 th and 19 th new fully expanded leaves between Shen137 and NIL.The absolute quantification(i TRAQ)were applied to the leaf blade,leaf midrib,leaf sheath and leaf ear(from the newly full-expanded leaves at the 10 th leaf stage)of both Shen137 and NIL to elucidate the possible regulation mechanism of maize leaf angle formation.The main results of this study are as follows:1.The changes of leaf angle and midrib puncture force of of maize inbred Shen 137 and NIL at different growth stages were measured.The results showed that the leaf angle difference between the new fully expanded leaves at the 9th stage to the 11 th leaf stage was large and stable.In addition,the midrib puncture force was affected by the puncture position and the leaf stage.The maximum difference in puncture force between Shen137 and NIL occurred at the 10 th leaf stage.The results showed that the differences of the leaf angle and midrib puncture force between 137 and NIL usually occurs from the 7th to 15 th leaf stage.2.The cross section of maize inbred lines Shen 137 and NIL were studied.The thick-walled cell area of ? ? the NIL is larger than that of Shen137 in the midribs near lamina joint.It suggests that the size of the thick-walled cell area plays an important role in the mechanical diversity of maize leaf midribs.In addition,the increase of leaf angle is accompanied by the increase of thick-walled cell content and vascular bundle density in plant tissue,suggesting that leaf angle might be related to the content of mechanical tissues.Combined with histological observation,it was found that the differences of leaf angle,midrib puncture force and morphological between Shen 137 and NIL were mainly occurred at the 10 th leaf stage.3.Three protein-extraction methods that are commonly used in plant proteomics were qualitatively and quantitatively evaluated based on using 2DE maps and matrix-assisted laser separation time-of-flight mass spectrometry(MALDI-TOF-MS/MS)using the leaf midrib of the new fully expanded leaf at the 10 th leaf stage.The number of points identified on the 2-DE profiles was as follows: phenol extraction(655)> TCA/ acetone extraction(589)> TCA/ acetone / phenol extraction(545).MS/MS analysis identified a total of 17 spots that exhibited 2-fold changes in abundance among the three methods(using phenol extraction as a control).Sixteen of the proteins identified were hydrophilic.For all three methods,we were able to obtain high-quality protein samples and good 2DE maps for the maize leaf midrib.However,phenol extraction produced a better 2DE map with greater resolution between spots,and TCA/aceton eextraction produced higher protein yields.The results provide useful information that can be used to select suitable protein extraction methods for proteome analysis of recalcitrant plant tissue that are rich in sclerenchyma cells.4.Comparative proteomic analysis was performed to revealprotein profile differences in the midribs of the 5th,10 th and 19 th newly expanded leaves between Shen137 and NIL.Quantitative analysis of 24 identified midrib proteins indicated that the maximum changes in abundance of 22 proteins between Shen137 and NIL appeared at the 10 th leaf stage,of which phosphoglycerate kinase,adenosine kinase,fructose-bisphosphate aldolase and adenylate kinase were implicated in glycometabolism.Thus,glycometabolism might be associated with leaf angle formation and the mechanical properties of the midribs.These results proved the importance of10 th leaf stage in the morphorlogical and microstructural differences between Shen137 and NIL.5.Peroxidase 42 and peroxidase were identified by glycoprotein staining.The content of peroxidase 42 was relatively abundant in NIL.Increased levels of auxin lead to more erect leaf morphorlogy.And peroxidase regulates auxin levels.The results suggested that glycoprotein peroxidase 42 is not only associated with cell wall synthesis of thick-walled cells,but also have the capacity to affect leaf angle by regulating auxin levels.6.The leaf blade,midrib,leaf sheath and lamina joint of maize inbred line Shen 137 and its near isogenic lines NIL were compared using absolute quantitative technique(i TRAQ).The proteins abundance change greater than 1.5 times was identified.The results showed that PER family members are not only associated with the synthesis of cell wall in thick-walled cells in maize tissues,but also closely related to the formation of leaf angle.Moreover,the abundant CAD and LOX family proteins contents suggested a strong lignin metabolism.The study also validated the important role of PER family proteins in the synthesis of lignin and catabolism that associated with cell wall synthesis and finally affect the formation of leaf angle differences between Shen137 and NIL.The results indicated that the PER protein family members contribute to the mechanical strength of the leaf midrib,and may also influence the leaf angle formation by interacting with LOX and CAD through lignin synthesis and degradation.