A Genetic Study Of A Rolling Leaf Mutant Shallot-like 2 Reveals Complex Nature In Regulation Of Bulliform Cell Development In Rice(Oryza Sativa L.)

Bulliform cells are large,thin-walled and highly vacuolated cells,and play an important role in controlling leaf rolling in response to drought and high temperature.Meanwhile,leaf rolling is considered an important agronomic trait in rice(Oryza sativa)breeding.To understand the molecular mechanism controlling leaf rolling,here,we report the isolation and characterisation of a rice leaf rolling mutant,named shallot-like 2(sll2).The sll2 plants exhibit adaxially rolled leaves,starting from the sixth leaf stage,accompanied by increased photosynthesis and reduced plant height and tiller number.Histological analyses showed shrinkage of bulliform cells,resulting in inward-curved leaves.The mutant is recessive and revertible at a rate of 9%.The leaf rolling is caused by a T-DNA insertion.Cloning of the insertion using TAIL-PCR revealed that the T-DNA was inserted in the promoter region of LOC_Os07g38664.Unexpectedly,the enhanced expression of LOC_Os07g38664 by the 35S enhancer in the T-DNA is not responsible for the leaf rolling phenotype.Further,the enhancer also exerted a long-distance effect,including up-regulation of several bulliform cell-related genes.sll2 suppressed the outward leaf rolling of oull in the sll2oull double mutant.We conclude that leaf rolling in sll2 could be a result of the combined effect of multi-genes,implying a complex network in regulation of bulliform cell development.Results from our research are summarized as follows:1.The sll2 plants were fully fertile and had normal seed size,but reduced plant height and tiller number compared to WT in field condition.In addition,the sll2 leaf was slightly narrowed and darker.The physiological measurements indicated that sll2 leaves contained higher chlorophyll and carotenoid than WT.Accordingly,higher photosynthesis was observed in sll2.Moreover,sll2 had slightly reduced transpiration rate,but not significantly different from WT.To understand what structural changes in sll2 cause such an extreme rolling leaf phenotype,we performed transverse section of leaves.We concluded thatreduction of bulliform cell area along the leaf width,due to cell size change at large,causesstepwise folding,and the bulliform cell area in sll2 was reduced to nearly 60%of WT,thusresulting in the shallot-like leaf in sll2.2.Genetic analysis showed the leaf rolling phenotype is recessive and controlled by asingle locus and the rolling leaf was associated with the presence of T-DNA.We employedthe Thermal Asymmetric Interlaced(TAIL)-PCR to clone genomic sequences flanking theinserted T-DNA.The T-DNA was inserted 126-bp upstream of the 5'UTR ofLOC_Os07g38664,and the LB,together with part of enhancer in 5' region of the 35Spromoter,was deleted during integration with rice genome.The downstream geneLOC_Os07g38664 was up-regulated nearly 13-fold in sll2.LOC_Os07g38664 encodes anunknown protein with 3 exons and 2 introns.Therefore,we performed vector constructionand transformation(LOC_Os07g38664 was overexpressed in WT and interference in sll2),surprisingly,no visible phenotype was observed in over-expression lines and the RNAitransgenic plants.We also constructed an F2 population to map SLL2 by crossing sll2 withan indica variety 93-11 and the gene was localized to a 28.6-kb region on chromosome 7spanning the T-DNA site using 459 individual plants with rolled leaves.Ten genes werepredicted within this region,including LOC_Os07g38664.However,we sequenced thewhole mapping region of sll2 and WT and found no nucleotide changes.It has beendocumented that CaMV35S promoter harbors two tandem enhancer elements,which notonly affect promoter activity of its own but also can influence expression of nearby as wellas the distant genes.Therefore,we propose an intact enhancer remain in T-DNA could leadto the change of gene expression out of the mapping region.We performed RNA-seqanalysis of sll2 and WT plants to identify genes whose expression level were altered in sll2.Globally,5 genes,including LOC_Os07g38664,were up-regulated 10-fold at least in sll2,however,no gene contribute leaf rolling was found.3.We observed about 9%revertants after two years9 observation among the progeny ofthe sll2.The sll2-Rev plants restored rolled to fully expanded leaves with LRI similar toWT.Transverse section of the sll2-Rev leaves showed that the bulliform cells had normalsize,resulting in restoration of bulliform cell area.In addition,other traits such as plantheight,tiller number,seed weight and leaf width were also restored in s112-Rqv.Wedetected presence of the inserted T-DNA in sll2-Rev plants without changes in its insertionsite.The possible mechanism is that:the enhancer in 35S promoter could change theexpression of the SLL2 in proper chromatin structure and cause leaf rolling.When the chromatin structure changed,the enhancer failed to regulate SLL2 and brought about flatleaf in sll2-Rev.4.To understand the relationship between sll2 and the outcurved leaf mutant oull(anoutcurved leaf mutant due to the increased bulliform cell number and size),we produceddouble mutant sll2oul1.The double mutant sll2oul1 exhibited the typical adaxial leafrolling phenotype as sll2,and the abaxial rolling leaf phenotype in oull which was causedby loss-of-function of Roc5,a homeodomain leucine zipper class IV gene which negativelyregulates bulliform cell development,was completely suppressed.Microscopic observationshowed shrinkage of bulliform cells in sll2oul1,similar to sll2 but opposite to oull.Thus,SLL2 is epistatic to Roc5.oull and sll2 complement each other:in double mutant oullsll2,and the reduced seed set and tiller number are recovered.The double mutant is also darkgreen in field condition.This observation suggests that an ideal plant type,which ismorphologically normal and has moderate rolling leaf,may be achieved by combiningdifferent leaf rolling mutants or by manipulating different leaf rolling-related genes.It hasbeen reported that Protodermal Factor-Like(PFL)gene is downstream of Roc5.Both Roc5and PFL were up-regulated in sll2 in our qRT-PCR analysis.Interestingly,PFL expressionwas down to the level of WT in the sll2oul1 double mutant plants.Those results suggestthat SLL2 may negatively regulate Roc5 and in turn PFL.5.SLL1 encodes a KANADI transcription factor and the extremely incurved leafphenotype in sll1-1 was due to the defective development of sclerenchymatous cells on theabaxial side.The double mutant sll1-1sll2 still showed the extremely incurved leafphenotype with a LRI between sll1-1 and sll2.The reduced bulliform cell number and sizeas well as defective sclerenchymatous cells were present in sll1-1sll2 plants.In addition,there was slightly higher expression of SLL1 in sll2 but no significant change in sll2-Rev.Those observations suggest that SLL1 and SLL2 may be independent in regulating bulliformcell development.