In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

The NAD(P)H dehydrogenase complex is encoded by 11 ndh genes in plant chloroplast (cp) genomes. However, ndh genes are truncated or deleted in some autotrophic Epidendroideae orchid cp genomes. To determine the evolutionary timing of the gene deletions and the genomic locations of the various ndh genes in orchids, the cp genomes of Vanilla planifolia, Paphiopedilum armeniacum, Paphiopedilum niveum, Cypripedium formosanum, Habenaria longidenticulata, Goodyera fumata and Masdevallia picturata were sequenced; these genomes represent Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae subfamilies. Four orchid cp genome sequences were found to contain a complete set of ndh genes. In other genomes, ndh deletions did not correlate to known taxonomic or evolutionary relationships and deletions occurred independently after the orchid family split into different subfamilies. In orchids lacking cp encoded ndh genes, non cp localized ndh sequences were identified. In Erycina pusilla, at least 10 truncated ndh gene fragments were found transferred to the mitochondrial (mt) genome. The phenomenon of orchid ndh transfer to the mt genome existed in ndh-deleted orchids and also in ndh containing species.

SummaryPlant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

(M.-C.S.) Transcription factors (TFs), which are central to the regulation of gene expression, are usually members of multigene families. In plants, they are involved in diverse processes such as developmental control and elicitation of defense and stress responses. To investigate if differences exist in the expansion patterns of TF gene families between plants and other eukaryotes, we first used Arabidopsis (Arabidopsis thaliana) TFs to identify TF DNA-binding domains. These DNA-binding domains were then used to identify related sequences in 25 other eukaryotic genomes. Interestingly, among 19 families that are shared between animals and plants, more than 14 are larger in plants than in animals. After examining the lineage-specific expansion of TF families in two plants, eight animals, and two fungi, we found that TF families shared among these organisms have undergone much more dramatic expansion in plants than in other eukaryotes. Moreover, this elevated expansion rate of plant TF is not simply due to higher duplication rates of plant genomes but also to a higher degree of expansion compared to other plant genes. Further, in many Arabidopsis-rice (Oryza sativa) TF orthologous groups, the degree of lineage-specific expansion in Arabidopsis is correlated with that in rice. This pattern of parallel expansion is much more pronounced than the wholegenome trend in rice and Arabidopsis. The high rate of expansion among plant TF genes and their propensity for parallel expansion suggest frequent adaptive responses to selection pressure common among higher plants.Regulation of gene expression is central to a myriad of biological processes at the molecular level and is to a significant extent controlled by transcription factors (TFs). Most TFs are modular proteins consisting of a DNA-binding domain that interacts with cis-regulatory elements of its target genes and a protein-protein interaction domain that facilitates oligomerization between TFs or other regulators (Wray et al., 2003). Sequence divergence in the DNA-binding domains of related TFs may lead to differences in affinities to a set of cis-regulatory elements. Together with the propensity for TFs to homodimerize and/or heterodimerize, the large TF repertoire in a eukaryote genome provides a wide range of combinatorial relationships for transcriptional regulation. TFs usually form gene families that vary considerably in size among organisms (Riechmann et al., 2000;Wray et al., 2003). The reasons behind such differences are not known, although it is suggested that organismal complexity correlates with an increase in the absolute number and the proportion of TFs in a proteome (Levine and Tjian, 2003).In Arabidopsis (Arabidopsis thaliana), at least 1,500 genes are TFs, and 45% of these TFs belong to families common to Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae (Riechmann et al., 2000). Some of these TF families are much larger in Arabidopsis, suggesting differential expansion. For example, the Myb family has 190 members in Ar...