Activation tagging using T-DNA vectors that contain multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene has been applied to Arabidopsis plants. New activation-tagging vectors that confer resistance to the antibiotic kanamycin or the herbicide glufosinate have been used to generate several tens of thousands of transformed plants. From these, over 30 dominant mutants with various phenotypes have been isolated. Analysis of a subset of mutants has shown that overexpressed genes are almost always found immediately adjacent to the inserted CaMV 35S enhancers, at distances ranging from 380 bp to 3.6 kb. In at least one case, the CaMV 35S enhancers led primarily to an enhancement of the endogenous expression pattern rather than to constitutive ectopic expression, suggesting that the CaMV 35S enhancers used here act differently than the complete CaMV 35Spromoter. This has important implications for the spectrum of genes that will be discovered by this method.

No abstract

The Arabidopsis genes FT and TERMINAL FLOWER1 (TFL1) encode related proteins with similarity to human Raf kinase inhibitor protein. FT, and likely also TFL1, is recruited to the promoters of floral genes through interaction with FD, a bZIP transcription factor. FT, however, induces flowering, while TFL1 represses flowering. Residues responsible for the opposite activities of FT and TFL1 were mapped by examining plants that overexpress chimeric proteins. A region important in vivo localizes to a 14-amino-acid segment that evolves very rapidly in TFL1 orthologs, but is almost invariant in FT orthologs. Crystal structures show that this segment forms an external loop of variable conformation. The only residue unambiguously distinguishing the FT and TFL1 loops makes a hydrogen bond with a residue near the entrance of a potential ligand-binding pocket in TFL1, but not in FT. This pocket is contacted by a C-terminal peptide, which also contributes to the opposite FT and TFL1 activities. In combination, these results identify a molecular surface likely to be recognized by FT-and/or TFL1-specific interactors.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHY-TOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack.Arabidopsis ͉ ARGONAUTE ͉ microRNA ͉ transacting siRNA M icroRNA (miRNA) and transacting siRNA (tasiRNA) form through distinct biogenesis pathways, but both function to guide endonucleolytic cleavage or translational modulation of target RNA transcripts (1). For miRNA, self-complementary foldback structures within primary transcripts are processed into Ϸ21-to 22-nt miRNA/miRNA* duplexes. For tasiRNA in plants, primary transcripts are first processed by miRNA-guided cleavage. One product of the cleaved transcript is stabilized, possibly by SUPPRESSOR OF GENE SILENCING3 (SGS3), and converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE6 (RDR6) (2-5). The resulting dsRNA is processed sequentially by DICER-LIKE4 (DCL4) into 21-nt siRNA duplexes in register with the miRNA-guided cleavage site (2, 6, 7). One strand of each miRNA or tasiRNA duplex is selectively sorted to one or more ARGONAUTE (AGO) proteins according to the 5Ј nucleotide or other sequence/structural elements of the small RNA (8-10). AGO proteins, which contain a 3Ј RNA binding domain (PAZ), a mid domain that confers small RNA recognition or binding function, and an RNaseH-like domain (PIWI) (11), provide the effector component for silencing complexes.Arabidopsis thaliana has eight characterized tasiRNA-generating (TAS) loci belonging to four families. TAS1 and TAS2 tasiRNA target multiple different mRNAs, including several encoding pentatricopeptide repeat (PPR) proteins (3)(4)(5)(12)(13)(14). TAS4 tasiRNA target mRNA encoding several MYB transcription factors (15). TAS3 tasiRNA target AUXIN RESPONSE FACTORs (ARF3 and ARF4) mRNA, regulation of which is important for proper patterning and developmental timing (4,(16)(17)(18)(19)(20). In addition to tasiRNA-based regulation, the RDR6/SGS3/DCL4 silencing pathway contributes to antiviral and transgene silencing (21-25).It is not clear how transcripts are routed into the RDR6/SGS3/ DCL4 s...

No abstract

Genes that are stably expressed during development or in response to environmental changes are essential for accurate normalization in qRT-PCR experiments. To prevent possible misinterpretation caused by the use of unstable housekeeping genes, such as UBQ10, ACT, TUB and EF-1α, as a reference, the use of 20 stably expressed genes identified from microarray analyses was proposed. Furthermore, it was recommended that at least four genes among them be tested to identify suitable reference genes under different experimental conditions. However, testing the 20 potential reference genes under any condition is inefficient. Furthermore, since their stability still varies, there is a need to identify a subset of genes that are more stable than others, which can be used as a starting pool for testing. Here, we validated the expression stability of the potential candidate genes together with the above-mentioned conventional reference genes under six experimental conditions commonly used in plant developmental biology. To increase fidelity, three independent validation experiments were carried out for each experimental condition. A hypothetical normalization factor, which is the geometric mean of genes that were identified as stably expressed genes in each experiment, was used to exclude unstable genes under a given condition. We identified a subset of genes showing higher expression stability under specific experimental conditions. We recommend the use of these genes as a starting pool for the identification of suitable reference genes under given experimental conditions to ensure accurate normalization in qRT-PCR analysis.

Expansin is a family of proteins that catalyze long-term expansion of cell walls and has been considered a principal protein that affects cell expansion in plants. We have identified the first root-specific expansin gene in soybean (Glycine max), GmEXP1, which may be responsible for root elongation. Expression levels of GmEXP1 were very high in the roots of 1-to 5-d-old seedlings, in which rapid root elongation takes place. Furthermore, GmEXP1 mRNA was most abundant in the root tip region, where cell elongation occurs, but scarce in the region of maturation, where cell elongation ceases, implying that its expression is closely related to root development processes. In situ hybridization showed that GmEXP1 transcripts were preferentially present in the epidermal cells and underlying cell layers in the root tip of the primary and secondary roots. Ectopic expression of GmEXP1 accelerated the root growth of transgenic tobacco (Nicotiana tabacum) seedlings, and the roots showed insensitivity to obstacle-touching stress. These results imply that the GmEXP1 gene plays an important role in root development in soybean, especially in the elongation and/or initiation of the primary and secondary roots.The root is a plant organ that has adapted to acquire water and nutrients from the environment (Schiefelbein et al., 1997). The root system has recently been the focus of interest as a useful system for understanding organ development because it is a relatively simple organ, its growth pattern is uniform, and it has a small number of differentiated cell types (Aeschbacher et al., 1994). Furthermore, the development of new roots (secondary or lateral roots) from an existing root (primary root) provides a novel opportunity to investigate cellular differentiation and development in plants.Although the primary and secondary roots share many basic structural features, they are different in their origin (Scheres et al., 1996). A basic feature of the root is its radial pattern, which is made up of concentric layers of tissues. Three fundamental types of the tissues are the epidermis, the cortex, and the vascular tissues (Esau, 1977;Dolan et al., 1993;Raven et al., 1999). In the longitudinal section, the root can be divided into three different regions: those of cell division, elongation, and maturation (specialization) (Dolan et al., 1993;Baluska et al., 1996;Howell, 1998;Raven et al., 1999). The region of cell division contains the root apical meristem, which carries out new cell divisions but does not elongate newly divided cells immediately. The cells derived from the region of cell division expand and elongate mostly in the region of elongation. After they have elongated, the cells begin to differentiate in the region of maturation, where root hairs and the secondary roots are initiated. The events of cell elongation and maturation occurring in the root have been suggested to be controlled by the extensibility of the cell wall and the turgor pressure inside the cell (Cosgrove, 1996).It has been proposed that development of the s...

Background: The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis plays an important role in regulating flowering time and in maintaining the fate of inflorescence meristem (IM). TFL1 is a homologue of CENTRORADIALIS (CEN) from Antirrhinum, which is only involved in IM maintenance. Recent mutational studies and the genome project revealed that TFL1 belongs to a small gene family in Arabidopsis, in which functional divergence may have occurred among the members.