Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extension tolerates little variation in protein structure.

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl-and amino-terminal fusions of the R gene to the reporter gene encoding 8-glucuronidase (CUS) were sufficient to direct CUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the CUS protein to the nucleus. Aminoterminal NLS-A (amino acids 100-109, CDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few vira1 proteins. The media1 NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRK-AICKR) is a mating type 012 type. NLSs M and C are independently sufficient to direct the CUS protein to the nucleus when it is fused at the amino terminus of CUS, whereas NLS-A fused to CUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of CUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and CUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed.

Three nuclear localization signals (NLS), including an unusual Mat a2-like N L S from maize (Zea mays) R, were found to compete for binding to plant nuclei. In addition, the authentic yeast Mat a 2 NLS, which does not function in mammals, was shown to function in plants in vivo. Our results indicate that plants possess a site at the nuclear pore complex that recognizes the three known classes of NLSs.

Biochemical dissection of the "acid-growth" process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit meristems and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.