Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.     

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses

Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.Recent attempts to better understand the chemistry and biology of plant cell walls have been driven by the importance of these walls as biomass sources for biofuel production systems, as sources of dietary fiber that is increasingly recognized as being highly beneficial for human health, and as key components of livestock forage and fodder. Plant cell walls consist predominantly of polysaccharides and lignin. In addition to cellulose, walls contain a wide array of complex noncellulosic polysaccharides that vary across the plant kingdom (Carpita, 1996; Popper and Fry, 2003; Niklas, 2004; Popper and Tuohy, 2010). In the dicotyledons, pectic polysaccharides and xyloglucans predominate, although smaller amounts of heteroxylans and heteromannans are also found. In evolutionary terms, a major change in noncellulosic wall composition is observed with the emergence of the Poaceae family, which contains the grasses and important cereal species. In contrast to dicots, walls of the Poaceae have relatively low levels of pectic polysaccharides and xyloglucans and correspondingly higher levels of heteroxylans, which appear to constitute the core noncellulosic wall polysaccharides in this family. In addition, walls of the Poaceae often contain (1,3;1,4)-β-glucans, which are not widely distributed in dicotyledons or other monocotyledons (Carpita, 1996; Fincher, 2009).Following the identification of the genes that encode cellulose synthases, which were designated CesA genes (Pear et al., 1996; Arioli et al., 1998), analyses of EST databases quickly revealed that the CesA group of cellulose synthase genes was in fact just one clade of a much larger superfamily that contained up to about 50 genes in most land plants (Richmond and Somerville, 2000; Hazen et al., 2002). The other members of the large gene family were designated cellulose synthase-like genes (Csl), which represent several clades in the overall phylogeny of the superfamily (Supplemental Fig. S1). The plant CesA genes were shown to have both conserved and hypervariable regions (Delmer, 1999; Doblin et al., 2002) and, together with the related Csl genes, were predicted to be integral membrane proteins and to have conserved, active-site D,D,D,QxxRW amino acid sequences. The CesA and Csl genes are members of the GT2 family of glycosyltransferases (Cantarel et al., 2009; http://www.cazy.org/).Several of the Csl genes have now been implicated in the biosynthesis of noncellulosic wall polysaccharides. Certain CslA genes mediate mannan and glucomannan synthesis (Dhugga et al., 2004; Liepman et al., 2005). Genes in the CslC clade are believed to be involved in xyloglucan biosynthesis (Cocuron et al., 2007), while genes from both the CslF and CslH clades mediate (1,3;1,4)-β-glucan synthesis in the Poaceae (Burton et al., 2006; Doblin et al., 2009). The CslJ group of enzymes is also believed to be involved in (1,3;1,4)-β-glucan synthesis (Farrokhi et al., 2006; Fincher, 2009), but the phylogeny of this group of genes remains unresolved (Yin et al., 2009). The fact that the CslF group does not form a clade with the CslH and CslJ groups on the phylogenetic tree (Supplemental Fig. S1) led to the suggestion that the genes mediating (1,3;1,4)-β-glucan synthesis have evolved independently on more than one occasion (Doblin et al., 2009; Fincher, 2009).Against this background and considering the sequence similarities between genes in the cellulose synthase gene superfamily, we have used Bayesian phylogenetic analyses of these genes from seven fully sequenced taxa to reconstruct the evolutionary origins of the CesA and Csl families in the grasses and, in particular, to investigate the evolution of the CslF, CslH, and CslJ genes. The distributions of the genes across genomes were compared, CslF gene clusters were analyzed, and the rates of synonymous and nonsynonymous nucleotide substitution were estimated to assess and compare selection histories of individual members of clades within the gene superfamily. Finally, we have constructed a refined model of the barley CslF6 enzyme to observe how selection on specific residues and regions of the enzyme has operated in a structural and functional context.     

Involvement of Intronic Sequences in Cell-Specific Expression of the Peripherin Gene

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.     

Diel Acid Fluctuations in C4 Amphibious Grasses

Orcuttieae is a small tribe of C4 grasses endemic to seasonal pools in the southwestern U.S., comprising the basal genus Neostapfia, Tuctoria, and the most derived group, Orcuttia. Growth is initiated underwater, and when pools dry, species undergo a metamorphosis replacing aquatic foliage with terrestrial foliage. O. californica and O. viscida exhibit CAM-like diel fluctuations in acidity in the aquatic foliage. Pulse-chase studies showed that although CO2 was fixed into malic acid in the dark, an overnight chase in the dark revealed that most label was not retained in organic acids, indicating a role other than CAM. Terrestrial foliage exhibited a very different diel fluctuation; acids accumulated during the day, and diminished overnight. Malic acid predominated and was secreted on the surface of the leaf in a manner similar to another arid land species. This terrestrial daytime acid accumulation may not be related to photosynthetic pathway but may play an anti-herbivore function. No acid fluctuations were observed in either N. colusana or T. greenei.     

Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell-Specific Expression in Transgenic Mice

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.     

拟南芥非特异性磷脂酶C4基因表达模式的研究

拟南芥非特异性磷脂酶C4（AtNPC4）具有降解磷脂酰胆碱（PC）,产生二酰甘油（DAG）和磷酸胆碱的活性。本研究从拟南芥基因组中分离了NPC4基因起始密码子上游1 379bp的启动子序列,与GUS报告基因融合后转化拟南芥,获得转基因植株。GUS组织化学染色表明,AtNPC4基因主要在处于衰老过程中的叶片中高水平表达,在根、茎、种荚和花中也有一定程度的表达,这种表达模式与RT-PCR结果相一致。另外,通过RT-PCR发现,AtNPC4基因在转录水平上受脱落酸的诱导,但不受水杨酸和茉莉素诱导。     

Cell-Specific Expression of the rolC Gene of the TL-DNA of Ri Plasmid in Transgenic Tobacco Plants

Transgenic tobacco plants possessing a chimeric gene of thepromoter region of the Ri plasmid ORF12 gene (rolC) and a structuralgene of bacterial ß-glucuronidase were obtained. Histochemicalanalysis showed that the chimeric gene is expressed in phloemcells throughout whole plants. Stable inheritance of such cellspecific expression of ORF12 promoter was also confirmed. ²Present address: Research Institute of Agricultural Resources,Ishikawa Agricultural College, Nonoichi-machi, Ishikawa, 921Japan. (Received September 30, 1988; Accepted March 30, 1989)     

Lateral Gene Transfer Acts As an Evolutionary Shortcut to Efficient C4 Biochemistry

The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C₄ pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C₄ trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate K_M values similar to the non-C₄ ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C₄ isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.