Microarray analysis was performed on RNA isolated from guard cells that were manually dissected from leaves of Arabidopsis. By pooling our data with those of two earlier studies on Arabidopsis guard cell protoplasts, we provide a robust view of the guard-cell transcriptome, which is rich in transcripts for transcription factors, signaling proteins, transporters, and carbohydrate-modifying enzymes. To test the hypothesis that photosynthesis-derived sugar signals guard cells to adjust stomatal opening, we determined the profile of genes expressed in guard cells from leaves that had been treated with sucrose. The results revealed that expression of 440 genes changed in guard cells in response to sucrose. Consistent with this hypothesis, these genes encoded cellular functions for photosynthesis and transport of sugars, water, amino acids, and ions. Plants of T-DNA insertion lines for 50 genes highly responsive to sucrose were examined for defects in guard cell function. Twelve genes not previously known to function in guard cells were shown to be important in leaf conductance, water-use efficiency, and/or stomate development. Of these, three are of particular interest, having shown effects in nearly every test of stomatal function without a change in stomatal density: TPS5 (At4g17770), a TRAF domain-containing protein (At1g65370), and a WD repeat–containing protein (At1g15440).

Circularization of cellular mRNAs is a key event prior to translation initiation. We report that efficient translation of Saguaro cactus virus (SCV) requires a 3' translational enhancer (PTE) located partially in coding sequences. Unlike a similar PTE reported in the 3' UTR of Pea enation mosaic virus that does not engage in an RNA:RNA interaction (Wang Z. et al., J. Biol. Chem. 284, 14189–14202, 2009), the SCV PTE participates in long distance RNA:RNA interactions with hairpins located in the p26 ORF and in the 5' UTR of one subgenomic RNA. At least two additional RNA:RNA interactions are also present, one of which involves the p26 initiation codon. Similar PTE can be found in six additional carmoviruses that can putatively form long-distance interactions with 5' hairpins located in comparable positions.

Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3= end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes. IMPORTANCERibosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin and that this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3= end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough. P ositive-sense RNA viruses employ a variety of gene expression strategies for the diversification of their proteomes (1, 2). Two noncanonical mechanisms, Ϫ1 programmed ribosomal frameshifting (Ϫ1PRF) and programmed ribosomal readthrough (PRT), circumvent stop codons for the expression of carboxyterminal extension products, effectively economizing on the limited genome size of most RNA viruses (3, 4). In Ϫ1PRF, the elongating ribosome shifts back 1 residue at a 7-residue...

The Panicum mosaic virus-like translation enhancer (PTE) functions as a cap-independent translation enhancer (3’CITE) in members of several Tombusviridae genera including 7/19 carmoviruses. For nearly all PTE, a kissing-loop connects the element with a hairpin found in several conserved locations in the genomic RNA (5’ terminal hairpin or ~100 nt from the 5’end) and small subgenomic RNA (~63 nt from the 5’end). Moving the interaction closer to the 5’end in reporter mRNAs using Saguaro cactus virus (SCV) sequences had either a minimal or substantial negative effect on translation. Movement of the kissing loop from position 104 to the SCV 5’ terminal hairpin also reduced translation by 4-fold. These results suggest that relocating the PTE kissing loop closer to the 5’end reduces PTE efficiency, in contrast to results for the Barley yellow dwarf BTE and Tomato bushy stunt virus Y-shaped 3’CITEs , suggesting that different 3’CITEs have different bridging requirements.