| Maize(Zea mays)is not only one of the most productive crops in the world,but also as food source and industrial raw materials.Because of the intensified food crisis and energy crisis,the corn production demand will continue to increase.How to improve the maize yield with insufficient land is a wide question attended to be further researched.The maize yield is greatly depended on the ear numbers in the unit area,the kernel number of each ear and the seed weight.The final seed size and weight are controlled by multiple factors,such as genetics,hormonal regulation and environmental impact.Thus,people can improve maize yield from various perspectives.Identifying the genes that regulate the crop yield,exploring these genes'function,and obtaining high production maize germplasm resources by transgenic breeding have become the inevitable trend of modern agricultural breeding.In this study,the maize ubiquitin receptors ZmDA1 and ZmDAR1 were studied.The transgenic and MU insertion mutant materials were used to explore their mechanism in regulating maize kernel development.Maize ZmDAl and ZmDARl are the homologies of the AtDAl and AtDARl in ArabidopsisDA1 was firstly reported as an ubiquitin receptor which regulated the organ size in Arabidopsis by Li Yuhai et al.AtDAl belongs to a small gene family in Arabidopsis.And the members play important roles during the plant development with the function that they cleave substrates by its peptidase activity.AtDA1 is a negative regulator in organ and seed size control.The dal-1 phenotype could be observed when there is an arginine to lysine change at amino acid position 358(DA1R358K).When compare with the wild-type,the dal-1 mutant's organ size has greatly increased with larger seeds.AtDAR1 is another important member of DA1 gene family which has a closest evolutionary relationship with AtDAl.AtDAR1 also has a conservative arginine position in its amino acid sequence,and the mutant has a similar phenotype with dal-1.In Arabidopsis,the AtDA1 and AtDAR1 have functional redundancy with each other.Bioinformatics analysis showed that,there are six members in maize DA1 gene family;all of them have close relationship with AtDA1,AtDAR1 and AtDAR2.The members of maize DA1 gene family contain two UIM domains,a single LIM domain and a DUF3363 domain.The phylogenetic tree shows that GRMZM2G017845 and GRMZM2G099328 are the homologies of the AtDA1 and AtDAR1,and there is a conservative arginine site at 333 or 322 positive in their amino acid sequence,respectively.Based on these results,the two proteins were named as ZmDA1 and ZmDAR1.In addition,the ZmDA1 and ZmDAR1 have similar expression patterns with AtDA1 and AtDAR1 that they ubiquitously express in mostly organs and during mostly development stages.Over-expressing the Zmda1 or Zmdar1 improves transgenic grain yieldThe cDNA from maize inbred line B73 was used as the template to clone ZmDA1 and ZmDAR1.The G?A single base mutant at 998 bp was introduced into the CDS region of ZmDA1 which would lead a DA1R333K mutant in the amino acid sequence.A similar system was to produce a G?A single base mutant at 965 bp in the CDS region of ZmDAR1 which would lead a DA1R322K mutant.The ZmDA1,ZmDAR1,Zmda1 and Zmdar1 were cloned into the plant expression vector,separately.The genes were transformed into maize inbred line DH4866 by the Agrobacterium-induced maize shoot-tip system.The transgenic plants were detected by herbicide screening and molecular analysis,and the results showed that the genes have been integrated into the maize genome.During the vegetative growth stage,it seems that there was no significant difference in the phenotype such as plant type,plant height or leaf area between the tr-ansgenic plants and the wild-type.When the plants got into the loose powder period,the Zmda1 and Zmdar1 had more developed tassel and increased filament number.But there were no differences in plant type,plant height and ear location between the transgenic plants and the wild-type.The three consecutive year of field yield test showed that,over-expression the Zmda1 or Zmdar1 could improve the maize yield.The ear length significantly increased to 15.3-15.8 cm in Zmda1 transgenic lines,and 14.6-15.8 cm in Zmdarl transgenic lines when compared with 14.3 cm in wild-type,the kernel number was also improved as a result of the increased ear length.The Zmdal and Zmdarl transgenic plants also had larger seed size and heavier seed weight.Due to the increased seed number and heavier seed weight,the plot yield was 15-22%higher in Zmdal transgenic lines and 18-22%higher in Zmdarl transgenic lines than the wild-type.But it was opposite in ZmDAl and ZmDAR1 over-expression lines,the shorter ear length and decreased 100-grain weight lead to a poor plot yield.These results suggest,over-expression Zmdal or Zmdarl in maize could improve the yield by affect the seed weight and seed number.However,in the study of Arabidopsis showed that the seed size was significant increased in the dal-1 mutant.ZmDAl and ZmDARl have effects on carbohydrate transport and starch synthesisIn monocot crops,the endosperm weight is the major factor in yield determination.Starch is the main component of endosperm in maize kernel.Therefore,the starch content in the mature endosperm of Zmda1 and Zmdar1 transgenic materials were determined.As the results shown,the starch content was significant improved in Zmdal and Zmdarl transgenic seed mature endosperm,the total starch was increased by approximately 11%in the Zmdal transgenic endosperm and 14%in Zmdarl transgenic endosperm.The Schiff's reaction treated the paraffin sections which could stain demonstrates starch granule displayed that the Zmdal and Zmdarl transgenic seed at DAP 12 had accumulated more starch granules.This demonstrated that the starch synthesis rate was enhanced in mutant gene transgenic seed.Used the semi-quantitative PCR and real time RT-PCR detected the relate genes in the starch synthesis pathway,ZmGBSSI and ZmWxl were up-regulated in Zmdal and Zmdarl transgenic seeds,the two genes were down-regulated in ZmDAl and ZmDAR1 over-expression lines.All these results suggest that ZmDA1 and ZmDARl could affect the starch synthesis processes.The starch granule morphology of the maize silty endosperm was observed used the scanning electron microscope.The starch granules were more rounded with an uneven size in the Zmdal and Zmdarl transgenic endosperm.To quantization the difference in granule size,the granules' diameters were measured and divided into three groups.The big granules were significant decreased;meanwhile the granules in the other two groups with the smaller diameter were increased.The average starch diameter was decreased in Zmdal and Zmdarl transgenic lines.We speculated that the smaller starch granule provided a larger superficial area that could bind more protein for starch synthesis,the morphological change of the starch granules was corresponded to the increased starch synthesis rate.The sugar plays an important role during the seed development;it is a signal molecular which can regulate the gene expression at the early stage during the maize seed development;and also serves as a nutrient substance for the seed development.In addition,at the later stage of grain development,soluble sugar plays as a precursor of starch synthesis which has a great effect on the starch synthesis rate and accumulation.At DAP 10,in the Zmdal and Zmdarl transgenic lines it has shown a higher soluble sugar level,that might be able to stimulate the cell proliferation,meanwhile,the ZMDA1 and ZmDAR1 transgenic lines had a lowest content when compared with the wild-type.But,the sugar content was increased in ZmDA1 and ZmDAR1 transgenic lines after DAP 10.We speculate that it may be due to the slower starch synthesis rate.ZmDAl and ZmDARl regulate the development of maize transfer cellsUsed the ZmDA1 as a bait,the maize kernel(DAP 10)cDNA library was screened.The maize glucose transporter ZmSWEET4c was got,and the proteins interaction was confirmed by Co-IP.During the kernel development,the ZmSWEET4c imports glucose from the panicle into the endosperm,and also regulates the formation of the basal endosperm transfer lays(BETLs).The glucose is an important growth regulator;the high level of glucose concentration stimulates cell proliferation in seeds.As the higher soluble sugar content in the Zmdal and Zmdarl transgenic lines at the early stage of the seed development,we speculate,the establishment and the morphology of the transfer cells may be having some difference between the transgenic plants and the wild-type.The seeds at DAP3,DAP8 and DAP15 were collected and sectioned,the longitudinal sections were observed.The early embryo in the Zmdal over-expression was growing faster than other samples;the transfer cells of the Zmdal and Zmdarl transgenic lines were well-developed when compared with the wild-type and the ZMDA1 and ZMDAR1 transgenic lines.The similar phenotypes were observed in the ZmDA1 and ZMDAR1 MU mutants'kernels.Consistent with the microscopic observation,ZmMRPl which is the key regulator during the BETLs development had significant up-regulation in the Zmda1 and Zmdarl transgenic seeds.The results suggest ZmDAl and ZmDAR1 have effects on the development of maize transfer cells.The more developed transfer cells could provide more nutrients into the kernels,on the one hand,the nutrients encourage the seed growth;on the other hand,there are more kernels would have opportunity to develop,especially those located at the top of the ears,which are aborted under normal conditions.ZmDA1 and ZmDARl regulate the cell proliferationIn the observation of the maize leaves,the area was rarely difference between transgenic plant and the wild-type.But,the leaf epidermis cell wide was decreased in Zmdal and Zmdarl transgenic lines.The leaf epidermis cell length was decreased in Zmdal transgenic lines but was increased Zmdarl transgenic lines.The average cell area was decreased with no significant change in leaf area;these suggested that the total cell number was increased in Zmda1 and 7mdar1 transgenic leaves.In addition,the cell number in the endosperm of the Zmdal and Zmdarl transgenic lines were also increased.As a result,in the semi-quantitative PCR analysis,in the mutant gene over-expression lines,the positive cell number regulator ZmCycD2;3 has been up-regulated.These demonstrated that the Zmdal and Zmdar1 have promoted the cell proliferation in the transgenic plants.ZmDAl and ZmDAR1 are both functional redundancy and mutual independenceIn this study,we found that both ZmDAl and ZmDAR1 have similar protein structures,and they can regulate the maize seed size by influence the development of transfer cells and starch synthesis,which suggest they may be have functional redundancy with each other.However,the two genes have shown some difference in their expression patterns and regulatory modes.From the gene expression level,ZMDA1 is primarily expressed in leaves and immature tassels,ears and embryos,while ZmDARl is primarily expressed in the endosperm at DAP 10 and DAP 15.These suggest that ZmDAR1 may function primarily in the endosperm,whereas ZmDAl plays a major role in embryonic development.In the three years' field trials,the Zmdarl transgenic lines have a greater kernel number and higher starch content,also with a higher crop yield when compared with the Zmdal transgenic plants.In the real-time PCR analysis,the ZmGSBBI and ZmBt2 were greatly up-regulated in Zmdarl transgenic lines.The results demonstrate ZmDAR1 may be an important regulator in the endosperm development,but also support the hypothesis that ZmDARl plays a more important role in the maize endosperm development stage.In the Y2H assay,the ZmDA1 and ZmDAR1 were used as baits,separately.The results shown that these two genes interacted with different proteins.There are two proteins can interact with both with ZmDA1 and ZmDAR1,on is the protein elongation factor EF1-? like,the other one is one of the L-asparaginases ZmASPGB1 in maize.ZmDA1 and ZmDARl are the ubiquitin receptors in maize with variety substrates and they have the same substrates suggest that they may be regulators of common pathways.But,their mostly substrates are different suggest that ZmDA1 and ZmDAR1 may be involved in different regulate pathways during the maize development.In this study,the maize gene DA1,DAR1,dal and dar1 were cloned and introduced into maize elite inbred line.The field trials shown that,over-expression Zmdal or Zmdarl gene could improve the kernel number in each row and increase the ear kernel number.The 100-grain weight was also increased in the Zmdal or Zmdarl transgenic lines.The follow-up experiment showed that the increased grain weight was due to the increased starch content.The hexose transporter ZmSWEET4c was identified to interact with ZmDA1 by Y2H analysis and Co-IP analysis.It was speculated that ZmDAl could regulate the degradation of ZmSWEET4c,as a result,the development of the transfer cells,the nutrient inputted into the endosperm and the starch content were affected.The study provides important clues for exploring the role of ubiquitin proteasome system in carbohydrate transport and maize kernel development. |
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