verely suppressed line, Ri-12, in which APG mRNA accumulation was,10% of that in the wild type, HLH/bHLH Pairs for Grain Length and Weight in Rice Grain lengtha 6.960.3 7.960.1 7.760.1 Line 1000-grain weight Grain widthb 2.860.2 3.060.1 3.060.1 ns Cell lengthc 102.2620.6 140.6623.7 145.9628.1 155.1628.4 121.7626.5 Cell widthd 43.766.8 42.765.4 43.866.0ns PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22180813 47.266.9 45.265.7 44.665.3 NiWT Ri-1 Ri-2 Ri-9 Ri-12 Ni1 Ni9 Ni23 Ni37 a,b: 23.2 26.4 25.3 24.5 26.0 23.9 33.4 26.1 26.0 7.860.1 8.060.2 7.360.3 3.060.1ns 3.160.1 3.260.1 ns 8.860.3 7.960.2 3.660.1 3.260.1 ns 7.960.3 3.360.2ns 128.6629.4 data are the average of 10 samples. data are the average of 250 samples., none-significant; p,0.05; p,0.01; p,0.001. NiWT, Nipponbare wild type. Ri#, APG RNAi line. Ni#, PGL:Ox line. doi:10.1371/journal.pone.0031325.t001 c,d: ns lemma/palea, and thus grain cannot grow to a size greater than that permitted by lemma/palea. APG and PGL1 does not affect the expression of known grain length controlling genes To examine whether the increased grain sizes of the APG RNAi lines and PGL1:OX lines resulted from the alteration of known grain size-related genes, we analyzed expression levels of two known grain length controlling genes SRS3 and GS3 in our transgenics. qRT-PCR analysis indicated the expression of SRS3 and GS3 to be largely unaffected in the selected APG RNAi lines and PGL1:OX lines . PGL1 is a brassinosteroid-related gene Overexpression of the BR signaling gene BRASSINOSTEROID UPREGULATED1, an atypical bHLH gene, increased leaf inclination. In our PGL1:OX lines, we found marked leaf bending in most lines. Interestingly, levels of bending seemed to correlate to grain length and the expression level of PGL1 in lemma/palea. Analysis for coleoptile length growth under different concentrations of barassinolide revealed that the PGL1:OX lines are hypersensitive to BL. Together, these results suggest that PGL1 is a BR signaling gene like BU1. However, exogenous BL treatment had subtle or no effect on gene expression for PGL1 and APG, respectively, while it enhanced BU1 expression for, three times, being consistent with previous report on BU1 . Three APG RNAi lines showed obvious leaf bending. This is consistent with the idea that APG and PGL1 interact and have antagonistic roles. Discussion Genetic control of grain size Rice grain size is a major yield component, and controlled by at least four factors; grain width, length, thickness and the ability to fill the grain. Findings on the genetic networks underlying these factors are very limited at buy Tideglusib present. In this study, we showed the involvement of an antagonistic pair of HLH/bHLH proteins in determining grain length; the positive regulator PGL1, an atypical bHLH, and the negative regulator APG, a typical DNAbinding bHLH. To date, two grain length-controlling genes have been reported, an OSR domain-containing protein gene GS3 and a kinesin 13 protein gene SRS3. The srs3 mutant showed significantly decreased cell length in lemma but only slightly affected cell width 
compared to the wild type. Although alterations of neither cell size nor cell number were described for the GS3 mechanism, the gene negatively regulates grain length and has a small positive effect on width. The expression of GS3 and SRS3 in the PGL1:OX and APG RNAi lines was largely unaffected, suggesting that the genetic pathway through which PGL1 and APG regulate grain length is independent of these genes, although the possibility of PGL1 and APG