Identification And Characterization Of The DPS1 Gene Revealed Its Role In Panicle And Leaf Development In Rice

Smarter food production is becoming imperative to feed the swelling world population.More than half of the world population consumes rice as a staple food.Rice grain yield depend on several traits particularly panicle size,number of seeds,panicle fertility and seed setting rate.Adverse environmental conditions such as cold or heat stress cause panicle degeneration(abortion)and sterility which leads to reduced grain yield in rice.Anthers are male reproductive organs which are covered by a protective layer called “cuticle” that avoid water loss from anthers.However,the molecular mechanism of anther cuticle development and panicle degeneration were still remaining to be explored.In this study,we report the functional characterization of a novel mutant called degenerated panicle and partial sterility 1(dps1)that showed panicle apical degeneration,reduced fertility and twisted flag leaves with yellow patches.The main findings of the present study are as follows.i)The dps1 mutant displayed aborted spikelets at apical portion of panicle with small whitish anthers and absence of pollen grains.ii)About 60% of the spikelets in the dps1 mutant were sterile which caused a significantly reduced seed setting rate in dps1(38 ± 5.2%)as compared to WT(95 ± 2%).iii)Scanning electron microscopy revealed altered cuticle morphology in dps1 anthers as compared to WT.Amounts of cuticular wax and cutin were also significantly reduced in dps1 anthers.iv)TUNEL and comet assays revealed increased programmed cell death(PCD)leading to enhanced DNA fragmentation in dps1 panicles and anthers.v)Panicles of dps1 plants showed an accumulation of reactive oxygen species(ROS),higher malondialdehyde(MDA)content and lower activities of antioxidant enzymes including superoxide dismutase(SOD)and catalase(CAT)as compared to WT.vi)Map-based cloning followed by sequencing revealed a point mutation from G to A at a predicted splicing site between the eleventh exon and intron of Os05g0395300(hereafter known as DPS1).vii)Genetic complementation of dps1 using genomic DNA(DPS1-gDNA-C)as well as cDNA over-expression(DPS1-CDS-C)fully restored the WT phenotype,which further confirmed that DPS1 is responsible for dps1 mutant phenotype.viii)Spatial and developmental expression patterns of DPS1 were analyzed by promoter-GUS analysis and qRT-PCR,which showed that DPS1 is constitutively expressed in all tissues with the highest expression in panicles and anthers.ix)DPS1 encodes a mitochondrial-localized cystathionine ?-synthase(CBS)domain containing protein with an additional DUF21 domain and a mitochondrial targeting peptide at N terminal.x)Protoplasts isolated from the transgenic DPS1-GFP complemented plants were observed using confocal microscopy which confirmed the mitochondrial localization of DPS1.xi)Thioredoxins h(Trx h)proteins are predicted to be localized in mitochondria and having ROS scavenging properties.Yeast two-hybrid assay revealed a potential interaction of DPS1 with Trx h proteins,OsTrx1 and OsTrx20.This direct physical interaction was further validated with in vitro MBP pull-down assays.xii)Further in vivo validation of this interaction was performed using Firefly Luciferase Complementation(LUC)Imaging Assay in tobacco leaves.These results indicate that DPS1 participates in ROS scavenging via interacting with Trx1 and Trx20,thus regulating ROS homeostasis.xiii)RNA-sequencing analysis of panicles was used to compare global gene expression changes between dps1 and WT.Global gene expression analysis in dps1 panicles revealed that biological processes related to fatty acid metabolism and ROS homeostasis were significantly affected and the expression of key genes involved in anther wax and cutin biosynthesis was significantly altered.xiv)In addition to panicle phenotype,the dps1 mutant also showed twisted yellow leaves.It was observed that chlorophyll contents were significantly reduced in dps1 with abnormal chloroplast development characterized by smaller number of starch granules and large number of osmiophilic bodies.xv)In addition,dps1 mutant leaves displayed defective cuticle development and reduced wax and cutin compounds in dps1 leaves as compared to WT.xvi)Physiological assays showed significantly higher accumulation of ROS and enhanced DNA fragmentation in dps1 leaves which could be the reason for chloroplast degeneration and defective cuticle development.In conclusion,the DPS1 gene controls panicle degeneration,anther cuticle and pollen development as well as leaf twisting and yellowing via regulating ROS homeostasis and cell death.This study revealed an important role of ROS in regulating leaf chloroplast and cuticle development in rice.The results obtained in this study also suggested that manipulating ROS level can be utilized as a strategy to develop male sterile plants for hybrid breeding.