These two authors contributed equally to this work.Keywords: rice, biomass digestibility, lodging resistance, cell wall, genetic modification, GH9B and XAT. SummaryRice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with b-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinasemediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

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The major distal regulatory sequence for the ␤-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.Genes in the ␤-globin gene complex (HBBC, containing HBE1, HBG2, HBG1, HBD, and HBB), together with those in the ␣-globin gene complex (HBZ2, HBA2, and HBA1) encode the developmentally regulated, erythroid-specific family of hemoglobins in vertebrates. Transcription of the mammalian HBBC is regulated both by proximal elements, such as promoters, and by a distal regulatory element known as the locus control region (LCR). The LCR is marked by several hypersensitive sites (HSs) in erythroid chromatin (18,45) and is required for high-level expression of genes within the HBBC in erythroid cells (reviewed in references 6 and 19). Transfection and transgenic mouse studies show that the LCR confers this high-level expression at many, but perhaps not all, ectopic sites of integration (1, 21, 31). Gain-of-function experiments examining multiple integrated copies of LCR constructs revealed expression that is copy number dependent and independent of the site of integration (e.g., see references 43 and 44), suggesting that the LCR contains a dominant chromatin-opening activity. However, deletion of HS1 to HS6 from the LCR of mouse Hbbc (3, 12), as well as deletion of HS2 to HS5 from the LCR of human HBBC on chromosome 11 (37), leaves the globin genes in an open chromatin domain, albeit expressed at very low to undetectable levels. Thus, the LCR is clearly required for enhancement but it is not necessary fo...