Transcriptome Sequencing And Physiological Analysis Of A Low-temperature-sensitive Purple Leaf Color Mutant Pur1 In Wheat (Triticum Aestivum L.)

Low-temperature-sensitive leaf color mutants are ideal materials for elucidating the mechanism of chloroplast development and chlorophyll synthesis under low temperature conditions.Our laboratory obtained a pair of near-isogenic lines: low-temperature-sensitive purple leaf color line(pur1)and low-temperature-insensitive green leaf color line(gre1),which was derived from a low-temperature-sensitive wheat purple striped leaf color mutant PSL1 and selected by 6 successive generations of self-crossing.In this study,the near-isogenic lines pur1 and gre1 were used for leaf color phenotypes and agronomic characterization,pigment content measurement,HPLC analysis of Carotenoid composition,chloroplast ultrastructure observation and RNA-seq,to reveal the physiological,biochemical and gene expression changes,identified key candidate genes responsible for leaf color change,and then illustrate the molecular mechanism of pur1 leaf color mutation under low temperature condition.The main findings are as follows:1.After normal autumn sowing(2017.10.01),the leaf color phenotypes of pur1 and gre1 was investigated.Compared with the gre1 plants,the pur1 plant seedlings have green tender leaves in the early autumn(2017.10.10-2017.12.8).With the progress of autumn-winter,the new leaves of pur1 turn yellow-green(2017.12.09-2018.01.23);then appears purple-red due to the temperature decreased(2018.01.24-2018.02.15);then turn to albino during late winter to early spring(2018.02.16-2018.03.16).As the temperature rises in spring,the new leaves color gradually turn to normal green(2018.03.17-2018.04.10).Only the new leaves returned to green with increasing temperature,and the whitened tissue of the original leaves would not re-green.By contrast,the tender leaves of gre1 plants remained green throughout the seedling and adult stages(2017.10.10-2018.04.10).The survey of the main agronomic traits between pur1 and gre1 showed that the panicle number per plant and Grain number per spike in pur1 were significantly lower than those of gre1 plants(P<0.05).Compared with the gre1 plants,the pur1 plants showed developmental retardation,and the heading stage and maturity stage are delayed by about 6 days.There was no significant difference in the Plant height,Flag leaf length and width,Spike length and 1000-grain weight.2.Using the nuclear genomic SSR markers(ncSSRs)and chloroplast SSR markers(cpSSRs)to investigate the genetic background between pur1 and gre1.The results showed that among the 42 pairs of ncSSR markers evenly distributed on 21 chromosomes of wheat,37 pairs of markers were monomorphic between pur1 and gre1,and only 5 pairs of markers detected differential bands.Thus,at the nuclear genome level,the genetic similarity between pur1 and gre1 was 88.1%(37/42).The results of 32 pairs of cpSSR markers showed that all cpSSR markers were amplified as monomorphic between pur1 and gre1.The above results indicated that the genetic background of pur1 and gre1 was highly consistent,and they were a pair of low-temperature sensitive/insensitive leaf color near-isogenic lines,which were an ideal material for studied the molecular mechanisms of chloroplast development and chlorophyll biosynthesis under low temperature conditions.3.RNA-seq was performed to compare the transcriptome profiles between the pur1 and gre1 leaves.Six cDNA libraries were constructed,and each sample obtained approximately 7GB clean reads.A total of 7,135 differently expressed genes(DEGs)were identified between pur1 and gre1,containing 5,857 up-regulated genes and 1,278 down-regulated genes.Functional annotation revealed that the DEGs are significantly enriched in carbon fixation,photosynthesis,photosynthesis-antenna proteins,Ribosome,porphyrin and chlorophyll metabolism,carotenoid biosynthesis,anthocyanin biosynthesis pathways.Moreover,GLK and DnaJ gene family involved in chloroplast development were down-regulated in pur1.The expression profiles of 15 key candidate genes related to leaf color mutation were verified by quantitative RT-PCR.The qRT-PCR results show similar expression pattern to the transcriptome data,indicating that the transcriptome results were highly credible.4.The results of photosynthetic pigments content determination showed that the chlorophyll and carotenoid contents were significantly decreased(P<0.05)in pur1 plants compared with gre1,which was 25.9% and 26.9% of those in gre1.The results of anthocyanin content measurement showed that the content of anthocyanin in pur1 was significantly higher than gre1(P<0.01),and the anthocyanin content in pur1 was about 20 times of gre1.5.High performance liquid chromatography(HPLC)analysis of carotenoid componds showed that the zeaxanthin content in pur1 plants was not significantly different from that in gre1,while the contents of lutein,?-carotene were highly significantly decreased(P<0.01),?-cryptoxanthin and ?-carotene contents were significantly decreased(P <0.05).In addition,violaxanthin and neoxanthin were also identified,and their contents were significantly lower in pur1 than gre1.6.The transmission electron microscopy(TEM)observation of chloroplast ultrastructure showed that the structure of chloroplasts in pur1 plants showed a series of dynamic changes with ambient temperature and leaf color.Under low temperature conditions,the chloroplasts of pur1 migrated toward the center of the cell,the shape was irregular,the internal structure was disordered,endomembrane system was disorganized,and stroma thylakoids and grana thylakoids were degraded.The chloroplasts of gre1 plants exhibit well-developed thylakoid membranes and closely packed stroma and grana lamella during the whole lifetime.