Plant aquaporins form a large protein family including plasma membrane-type (PIPs) and tonoplast-type aquaporins (TIPs), and facilitate osmotic water transport across membranes as a key physiological function. We identified 33 genes for aquaporins in the genome sequence of rice (Oryza sativa L. cv. Nipponbare). We investigated their expression levels in leaf blades, roots and anthers of rice (cv. Akitakomachi) using semi-quantitative reverse transcription-PCR (RT-PCR). At both early tillering (21 d after germination) and panicle formation (56 d) stages, six genes, including OsPIP2;4 and OsPIP2;5, were expressed predominantly in roots, while 14 genes, including OsPIP2;7 and OsTIP1;2, were found in leaf blades. Eight genes, such as OsPIP1;1 and OsTIP4;1, were evenly expressed in leaf blades, roots and anthers. Analysis by stopped-flow spectrophotometry revealed high water channel activity when OsPIP2;4 or OsPIP2;5 were expressed in yeast but not when OsPIP1;1 or OsPIP1;2 were expressed. Furthermore, the mRNA levels of OsPIP2;4 and OsPIP2;5 showed a clear diurnal fluctuation in roots; they showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness. The mRNA levels of 10 genes including OsPIP2;4 and OsPIP2;5 markedly decreased in roots during chilling treatment and recovered after warming. The changes in mRNA levels during and after the chilling treatment were comparable with that of the bleeding sap volume. These results suggested the relationship between the root water uptake and mRNA levels of several aquaporins with high water channel activity, such as OsPIP2;4 and OsPIP2;5.

Maximum freezing tolerance of Arabidopsis fhaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2°C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 t o 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 t o 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 t o 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar t o that of leaves from which the protoplasts were isolated (-3.5OC for nonacclimated leaves; -1 0°C for cold-acclimated leaves). I n protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was ~1 0 % at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. I n protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the "fracture-jump lesion," which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.Membrane destabilization resulting from freeze-induced dehydration is the primary cause of freezing injury in plants (Steponkus, 1984;Steponkus and Webb, 1992). Although a11 cellular membranes are vulnerable to freezeinduced destabilization, the plasma membrane is of primary importance because of the central role that it plays during a freeze/thaw cycle. During cold acclimation, the cryostability of the plasma membrane is increased; in part, this is a consequence of alterations in its lipid composition ' Portions of this work were supported by grants from the U.S. 15that alter its lyotropic (dehydration-induced) phase behavior. Therefore, the mechanistic significance of changes in membrane lipid composition, ideally at the molecular species level, should be assessed from a perspective of their effect on the lyotropic rather than the thermotropic phase behavior or fluidity of the plasma membrane.We have previously demonstrated that there are severa1 alterations in the lipid composition of the plasma membrane of winter rye leaves during cold acclimation (Lynch and Steponkus, 1987;Uemura and Steponkus, 1994). The most pronounced changes are (a) an in...

To understand the mechanistic basis of cold temperature stress and the role of the auxin response, we characterized root growth and gravity response of Arabidopsis thaliana after cold stress, finding that 8 to 12 h at 48C inhibited root growth and gravity response by ;50%. The auxin-signaling mutants axr1 and tir1, which show a reduced gravity response, responded to cold treatment like the wild type, suggesting that cold stress affects auxin transport rather than auxin signaling. Consistently, expression analyses of an auxin-responsive marker, IAA2-GUS, and a direct transport assay confirmed that cold inhibits root basipetal (shootward) auxin transport. Microscopy of living cells revealed that trafficking of the auxin efflux carrier PIN2, which acts in basipetal auxin transport, was dramatically reduced by cold. The lateral relocalization of PIN3, which has been suggested to mediate the early phase of root gravity response, was also inhibited by cold stress. Additionally, cold differentially affected various protein trafficking pathways. Furthermore, the inhibition of protein trafficking by cold is independent of cellular actin organization and membrane fluidity. Taken together, these results suggest that the effect of cold stress on auxin is linked to the inhibition of intracellular trafficking of auxin efflux carriers.

Constitutive expression of the cold-regulated COR15a gene of Arabidopsis thaliana results in a significant increase in the survival of isolated protoplasts frozen over the range of ؊4.5 to ؊7°C. The increased freezing tolerance is the result of a decreased incidence of freeze-induced lamellar-tohexagonal II phase transitions that occur in regions where the plasma membrane is brought into close apposition with the chloroplast envelope as a result of freeze-induced dehydration. Moreover, the mature polypeptide encoded by this gene, COR15am, increases the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species that comprise the chloroplast envelope. We propose that COR15am, which is located in the chloroplast stroma, defers freeze-induced formation of the hexagonal II phase to lower temperatures (lower hydrations) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.The ability to endure low temperatures and freezing is a major determinant of the geographical distribution and productivity of agricultural crops. Even in areas considered suitable for the cultivation of a given species or cultivar, decreases in yield and crop failure frequently occur as a result of aberrant, freezing temperatures. In spite of attempts to minimize damage to freezing-sensitive crops-primarily by using energy-costly practices to modify the microclimate-substantial economic losses resulting from freezing are incurred annually in a diverse array of agricultural crops.Only modest increases (1-2°C) in the freezing tolerance of crop species would have a dramatic impact on agricultural productivity and profitability. The development of genotypes with increased freezing tolerance would provide a more reliable means to minimize crop losses from freezing stresses and greatly diminish the use of energy-costly practices to modify the microclimate. However, there has been little progress in improving the freezing tolerance of crop species by using classical plant breeding approaches. For example, the freezing tolerance of the best wheat varieties today is essentially the same as the most freezing-tolerant varieties developed in the early part of this century (1). Currently, there is considerable interest in the use of genetic engineering techniques for increasing the freezing tolerance of agricultural crop species.Since 1985, when Guy et al.(2) first reported that gene expression is altered during cold acclimation, remarkable progress has been made in identifying an ever-increasing number of genes that are regulated by low temperatures (3, 4). Many of these genes encode hydrophilic polypeptides with little or no homology with previously described polypeptides. These include the COR and LTI genes of Arabidopsis thaliana (5-7), the COR and pao86 genes of barley (8, 9), and the A͞ES genes of alfalfa (10). It is widely speculated that these genes might have roles in freezing tolera...