| Foxtail millet [Setaria italica(L.)P.Beauv.] has many excellent characters,including a small genome size with few repeated sequences,short growth period and higher propagation coefficient.In recent years,the whole genome sequencing for foxtail millet has been completed,and it is being developed into an ideal model plant especially for C4 photosynthesis,and stress biology research in Gramineae.Foxtail millet belongs to the thin sowing crop,whose yield mainly dependent on the panicle in main stem,thus panicle traits are the main factors which determining yield.Currently,there are few studies reported about genes regulating panicle development in foxtail millet.In our previous studies,we isolated two mutant lines,siaux1 and lp1 with abnormal panicle development from a normal variety ‘Yugu1’(the genome sequenced variety)using ethyl methane sulfonic acid(EMS)mutagenesis.Here,we cloned the candidate genes,and investigated the expression profile of target genes.The main results are as follows,1.Compared to wild types ‘Yugu1’,siaux1 has delayed seed germination,decreased germination percentage,decreased number of nodes on the main stem,shorter but thicker main stem,longer and wider leaves,and longer main panicle.The panicle primary branches,florets,and seed setting rate were all reduced.The most obvious characteristics were lower spikelet density,and significantly decreased yield per panicle(P<0.01).Using map cloning and genome resequencing,we found that the mutant traits in siaux1 was controlled by the nuclear recessive gene Si AUX1(Seita.5G387200),which located on Chromosome 5.A nucleotide mutation of G to A in the fourth exon of the Si AUX1 was found in siaux1,which resulted in a triplet TGG(UGG)being changed into a stop codon TGA(UGA).As a result,during translation to protein,277 amino acid residues were missed out from the C-end,due to the translation being prematurely stopped at the 214 th amino acid position.2.The transgenic mutant rice lines of knockout the gene Os AUX1(LOC_Os01g63770)resulted in significantly decrease in density of spikelet and of lateral root(P<0.05 or <0.01).This result was coincident with the fact that the transgenic mutant rice lines of heterologous overexpression of Si AUX1 resulted in significantly increase in density of spikelet and of lateral root(P<0.05 or <0.01).By resequencing the Si AUX1 of 50 representative varieties which selected from 916 sets of foxtail millet germplasm resource,we also found that 7 varieties had a 38 bp missing variation in the 3’ UTR of the Si AUX1 gene.The panicle length,number of primary branches,number of florets,and grain weight per panicle of the 7 varieties were significantly decreased compared to other varieties(P<0.05),although their spikelet density were significantly inscreased(P<0.05).3.Si AUX1 gene could response to the induction of the auxin analogue(2,4-D).The extent of upregulation of expression in Si AUX1 is directly proportional to the concentration of(2,4-D).Tissue-specific expression analysis showed that Si AUX1 had high expression level in the vigorously growing young tissues of the embryoes,central leaves,and panicles,as well as vascular tissue such as the pith of the main root and ridges of the husk,which coincided with the distribution pattern and transport model of auxin in the plant.The mutation of Si AUX1 significantly downregulated the expression level of multiple genes in the IAA signal transduction pathway in panicle(P<0.01 or <0.05).The IAA concentration in the panicle of siaux1(46.17 mg/g)was significantly higher than that of wild types(27.61 mg/g)during the grain filling stage(P<0.01).By sequence alignment analysis,five genes were found in the AUX1/LAX family in foxtail millet.Among them,only Si AUX1 was highly expressed in the panicles.The other four genes Si AUX2(Seita.3G223500),Si AUX3(Seita.9G471700),Si AUX4(Seita.9G288200),and Si AUX5(Seita.8G047400)were highly expressed in the roots at different developmental stages of foxtail millet.4.Compared to wild types ‘Yugu1’,the mutant line lp1 has shorter main stem,longer main panicle,and reduction in number of primary branches and secondary branches,and decreased number of florets and grains.The most obvious characteristics were lower spikelet density,and significantly decrease in grain yield per panicle(P<0.05),but larger grain size and extremely significantly increase in 1000-grain weight(P<0.01).lp1 mutant traits was controlled by a nuclear recessive gene LP1(Seita.2G369500)on Chromosome 2.LP1 belongs to subfamily I of the WRKY transcription factor family.A nucleotide mutation of G to A at the end of the fifth intron of the LP1 gene was found in mutant lp1,which resulted in three alternative splicing events different from that in wild types.In lp1,the three transcripts all suffered from prematurely stopped of protein translation,which caused destruction of the C2H2 zinc finger structure in the second WRKY domain of the C-terminal of the LP1 protein.5.High expression level of LP1 was found in the stem nodes at jointing stage,panicles at booting stage,and grains at grain filling stage in foxtail millet.This expression pattern was consistent with its function of regulating plant height,panicle structure,and seed size.By subcellular localization,we also found that LP1 expressed in the nucleus,which consisted with the expression profile of transcription factor gene.Conclution: Panicle development in foxtail millet is a complex process which controlled by multiple genes.This study found that the mutantions in the auxin influx transporter encoding gene Si AUX1 and the transcription factor gene LP1 of the WRKY family,all resulted in abnormal development of panicle in foxtail millet,the most obvious characteristics are reduction in spikelet density,and significantly decrease of yield per panicle(P<0.01 or <0.05). |
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