Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress–responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis . Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of ∼30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

Pentatricopeptide repeat (PPR) proteins form a huge family in plants (450 members in Arabidopsis and 477 in rice) defined by tandem repetitions of characteristic sequence motifs. Some of these proteins have been shown to play a role in posttranscriptional processes within organelles, and they are thought to be sequence-specific RNA-binding proteins. The origins of this family are obscure as they are lacking from almost all prokaryotes, and the spectacular expansion of the family in land plants is equally enigmatic. In this study, we investigate the growth of the family in plants by undertaking a genome-wide identification and comparison of the PPR genes of 3 organisms: the flowering plants Arabidopsis thaliana and Oryza sativa and the moss Physcomitrella patens. A large majority of the PPR genes in each of the flowering plants are intron less. In contrast, most of the 103 PPR genes in Physcomitrella are intron rich. A phylogenetic comparison of the PPR genes in all 3 species shows similarities between the intron-rich PPR genes in Physcomitrella and the few intron-rich PPR genes in higher plants. Intron-poor PPR genes in all 3 species also display a bias toward a position of their introns at their 5' ends. These results provide compelling evidence that one or more waves of retrotransposition were responsible for the expansion of the PPR gene family in flowering plants. The differing numbers of PPR proteins are highly correlated with differences in organellar RNA editing between the 3 species.

It is likely that many small ORFs (sORFs; 30–100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.

We mapped RIKEN Arabidopsis full-length (RAFL) cDNAs to the Arabidopsis thaliana genome to search for alternative splicing events. We used 278,734 full-length and 3'/5' terminal reads of the sequences of 220,214 RAFL cDNA clones for the analysis. Eighty-nine percent of the cDNA sequences could be mapped to the genome and were clustered in 17,130 transcription units (TUs). Alternative splicing events were found in 1764 out of 15,214 TUs (11.6%) with multiple sequences. We collected full-length cDNA clones from plants grown under various environmental conditions or from various organs. We then analyzed the correlation between alternative splicing events and environmental stress conditions. Alternative splicing profiles changed according to environmental stress conditions and the various developmental stages of plant organs. In particular, cold-stress conditions affected alternative splicing profiles. The change in alternative splicing profiles under cold stress may be mediated by alternative splicing and transcriptional regulation of splicing factors.

Summary: Plants respond and adapt to drought stress in order to survive under stress conditions. Several genes that respond to drought at the transcriptional level have been described, but there are few reports on genes involved in the recovery from dehydration. Analysis of rehydration‐inducible genes should help not only to understand the molecular mechanisms of stress responses in higher plants, but also to improve the stress tolerance of crops by gene manipulation. We used a full‐length cDNA microarray containing ca. 7000 Arabidopsis full‐length cDNAs and identified 152 rehydration‐inducible genes. Venn diagram analysis showed relationship of the rehydration‐inducible genes to proline‐inducible and water‐treatment‐inducible genes. Among the 152 rehydration‐inducible genes, 58 genes contained the ACTCAT sequence involved in proline‐ and hypoosmolarity‐inducible gene expression in their promoter regions, suggesting that ACTCAT sequence is a major cis‐acting element involved in rehydration‐inducible gene expression, and that some novel cis‐acting elements are involved in rehydration‐inducible gene expression. Functional analysis of rehydration‐inducible and rehydration‐repressed genes revealed their functions not only in the release from a stressed status but also in the recovery of growth in plants.