Ear Photosynthetic Characteristics In Different Ploidy Wheat Species And Functional Characterization Of Structure Formation-related Gene

As the increasingly prominent contradictions between food supply and demand in the world balance,it is essential to increase the overall yield by increasing the yield level of each grain crop.Previous study suggested that wheat ears contribute a considerable portion of grain mass.In particular,during drought,ear may be the main photosynthetic contributor to grain filling.Studies have shown the better photosynthetic performance of wheat ear may be related to some degree of C₄metabolic pathway.C₄ photosynthetic pathway,a good complement of C₃photosynthetic pathways,has been suggested to be evolved from C₃ pathway to adapt to the external environment,and the presence of a Kranz anatomy is the most obvious feature in C₄ plants.It is generally believed that diploid and tetraploid wheat are chromosome contributors to the hexaploid wheat,carrying important genetic resources of wheat.Due to the complexity of establishing a complete C₄ pathway in C₃ crops,screening for C₄-related traits from different ploidy wheat varieties became a more viable alternative to C₃ crop transformation.Therefore,it is necessary to study photosynthetic,physiological characteristics,anatomical structure,and the molecular mechanism of the establishment and regulation of Kranz-like structure in different ploidy wheat,thus facilitating to understand the correlation between photosynthetic performance and photosynthetic structure,and to clarify the regulation mechanism of the formation photosynthetic structure,so as to provide a new idea for constructing C₄structure in C₃ crop.In the present research,the correlation between photosynthetic physiological characteristics and anatomical characteristics of flag leaf and ear organs in different ploidy wheat was studied systematically by field and pot experiment.The function of TaSCR gene in wheat seedling growth and spike formation process were verified by BSMV-VIGS and transgenic technique.The main results are as follows:1.Compared to ears,flag leaves exhibited higher photosynthetic rate（P_N）at the filling stage under well-watered（WW）and water stressed（WS）conditions,but more severe decrease under WS.The P_N of the flag leaf of the wheat during the grain filling stage increased with the increase of the ploidy,that is,P_{N hexaploid}>P_{N tetraploid}>P_{N diploid}.With the grain filling process,the flag leaf P_N of six wheat varieties decreased gradually,the diploid and hexaploid wheat decreased the most and least.While the ear P_N was increased first and then decreased with the ploidy increase,that is,P_{N tetraploid}>P_{N diploid}>P_{N hexaploid}.With the progress of grain filling,the ear PN were also decreased,and the the largest and lowest decrease were presented in the hexaploid and tetraploid wheat.Not only the P_N of flag leaf and ear,water stress also reduced the content of photosynthetic pigment,which aggravated the process of wheat senescence.Compared with the flag leaf,the wheat ear was maintained at higher RWC under WS,and the WUE improved obviously,which became the key factor to maintain the better photosynthetic performance and drought tolerance of wheat.Rubisco and PEPCase activity analysis results showed that the decrease of Rubisco activity inhibited the assimilation ability of CO₂,which was the main reason for the decrease of P_N.Along with the ¹³C isotope content,it is showed that there was no C₄ photosynthetic pathway in the wheat ear.The improved performance of the tetraploid wheat ears under WS was associated with better water relations.2.The photosynthetic rate of the ear decreased less than that of the flag leaf under water stress,and that the photosynthetic rate of tetraploid wheat decreased the least among the three wheat species,while the water use efficiency（WUE）of it increased the most among the three species under water stress.The tetraploid wheat ear organs presented lower stomatal frequency（SF）,stomatal area per organ area（SA）and higher vein density（N_V）,and bundle sheath cell number(N_BS)than hexaploid wheat species,and tetraploid wheat exhibited lower vein distance（D_V）than hexaploid wheat in the glume and lemma.Meanwhile,tetraploid wheat showed the lowest bundle sheath cell volume(V_BS)in the glume and the highest in the awn,lemma,and palea.The changes in anatomical characteristics caused by water stress occurred to a greater extent in tetraploid wheat than in the other two wheat species.3.‘Changhan 58’showed a significantly higher ear photosynthetic rate with a lower rate of variation and a significantly higher percentage variation of transpiration compared to control plants at the heading stage under WS conditions than did‘Xinong 9871’plants.Moreover,‘Changhan 58’showed lower stomatal density（SD）and higher stomatal area per unit organ area（A）under both water conditions.Water stress decreased SD,A,and stomatal width（SW）,and increased stomatal length in flag leaves（upper and lower surfaces）and ear organs（awn,glume,lemma,and palea）,with the changes more pronounced in ear organs than in flag leaves.Instantaneous WUE increased slightly,while integral WUE improved significantly in both cultivars.Integral WUE was higher in‘Changhan 58’,and increased by a greater amount,than in‘Xinong 9871’.4.Based on homologous cloning method,the TaSCR gene,which belongs to the GRAS transcription factors family,was cloned from wheat cultivar Ningchun 16.The length of the genomic DNA of TaSCR-4BS,-4AL and-4DS were 3233bp,3191bp and 3242bp,respectively.The entire deduced proteins of TaSCR–4BS,-4AL and-4DS were highly similar in amino acid sequence（more than 99%identity,679,680,679 amino acids）.All of the deduced proteins contain five distinct sequence motifs,leucine heptad repeat I（LHR I）,the VHIID motif,leucine heptad repeat II（LHR II）,the PFYRE motif,and the SAW motif,that are characteristic of GRAS family members.The expression pattern of TaSCR gene in developmental organs was significantly higher than that in mature tissue.The expression of TaSCR gene in wheat seedling and ear was silenced by BSMV-VIGS method.Roots collected from the BSMV:TaSCR-infected plants were shorter and smaller than those from BSMV:00 and BSMV:PDS infected plants.Statistical analysis showed that the tiller number,leaf number,aboveground biomass,root biomass,root surface area,and leaf surface area of BSMV:TaSCR-infected plants were significantly reduced compared to BSMV:00-infected plants.The results of TaSCR gene silencing in the ear of wheat showed that the ears from BSMV:TaSCR-infected plants were reduced in size.Moreover,the width of mature seeds from BSMV:TaSCR-infected plants were significantly reduced by approximately32.22%,compared to BSMV:00-infected plants.Analysis of the anatomical structure of the BSMV:TaSCR plants revealed that veins and BS cells are abnormal in many respects.BS cells with abnormal size or shape are common,with disappeared cell wall in some cells.Many BS cells layer merge or missing,and these merged or missing BS cell layers organize smaller veins.Some BS cells have no intact organelle,such cells may occur at any location around the circumference of the veins.Two or more abnormal cells often founded consecutively adjacent to normal cells,while they appear almost devoid of cytoplasm.The the root length for TaSCR transgenic lines was significantly longer than that of scr-1 plants,and the leaf size,both the length and width,also increased similarly.The TaSCR transgenic lines were the same size as wild type and overall the seedlings looked normal.We also observed that over-expression of TaSCR in Arabidopsis thaliana caused some degrees of overgrowth of the reproductive organ,manifested as thicker and longer compared to scr-1 plants.Examination of the cellular patterning of roots homozygous for the TaSCR over-expressing plants revealed sporadic increases in the number of ground tissue layers.The TaSCR over-expressing plants,similar to the wild type（Ws）,displayed Casparian strip marks in its designated endodermal layer.The cells surrounding the vascular tissue in the scr mutant were large and irregular in shape,while in the wild-type and TaSCR overexpression plants,BS cells may be easily recognized by their rectangular cell shape,ordered organization and intermediate cell size relative to the vascular cells and mesophyll cells,which are large and irregularly shaped.Some abnormal minor veins were recovered in TaSCR transgenic lines.In the TaSCR over-expressing plants,similar to the wild type（Ws）,the vascular cores and BS layers arranged normal with intact structure.