Teruaki Taji

Comparative Genomics in Salt Tolerance between Arabidopsis and Arabidopsis-Related Halophyte Salt Cress Using Arabidopsis Microarray

Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%–95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, β-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens.

Dissecting the genetic control of natural variation in salt tolerance of Arabidopsis thaliana accessions

Many accessions (ecotypes) of Arabidopsis have been collected. Although few differences exist among their nucleotide sequences, these subtle differences induce large genetic variation in phenotypic traits such as stress tolerance and flowering time. To understand the natural variability in salt tolerance, large-scale soil pot experiments were performed to evaluate salt tolerance among 350 Arabidopsis thaliana accessions. The evaluation revealed a wide variation in the salt tolerance among accessions. Several accessions, including Bu-5, Bur-0, Ll-1, Wl-0, and Zu-0, exhibited marked stress tolerance compared with a salt-sensitive experimental accession, Col-0. The salt-tolerant accessions were also evaluated by agar plate assays. The data obtained by the large-scale assay correlated well with the results of a salt acclimation (SA) assay, in which plants were transferred to high-salinity medium following placement on moderate-salinity medium for 7 d. Genetic analyses indicated that the salt tolerance without SA is a quantitative trait under polygenic control, whereas salt tolerance with SA is regulated by a single gene located on chromosome 5 that is common among the markedly salt-tolerant accessions. These results provide important information for understanding the mechanisms underlying natural variation of salt tolerance in Arabidopsis.

Regulation of the ABA-responsive Em promoter by ABI3 in the moss Physcomitrella patens: Role of the ABA response element and the RY element

The plant-specific transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) or the maize ortholog VIVIPAROUS1 (VP1) is known to regulate seed maturation and germination in concert with the phytohormone abscisic acid (ABA) but is also evolutionarily conserved among land plants including non-seed plants. An ABI3/VP1 ortholog (PpABI3A) from the moss Physcomitrella patens can activate ABA-responsive gene promoters in the moss and angiosperms; however, it failed to fully complement the phenotypes of the Arabidopsis abi3-6 mutant, suggesting that some aspects of ABI3/VP1 functions have diverged during the evolution of land plants. To gain insights into the evolution of ABI3/VP1 function, we performed a comparative analysis of the regulatory elements required for ABI3 activation in Physcomitrella using a wheat Em gene promoter, which is induced by ABA and ABI3/VP1 both in Physcomitrella and in angiosperms. Elimination of either the ACGT core motif in the ABA response element (ABRE) or the RY element, to which ABI3/VP1 binds directly, resulted in a drastic reduction of the ABA response in Physcomitrella. Arabidopsis ABI3 could effectively activate the Em promoter either in an ABRE- or RY-dependent manner, as observed in angiosperms. On the other hand, PpABI3A failed to activate an Em promoter lacking the RY element but not the ABRE. These results suggest that RY-mediated transcriptional regulation of ABI3/VP1 is evolutionarily conserved between the moss and angiosperms, whereas angiosperm ABI3/VP1 has evolved to activate ABA-inducible promoters via the ABRE sequence independently from the RY element.

Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens.

Plant hormone abscisic acid (ABA) is found in a wide range of land plants, from mosses to angiosperms. However, our knowledge concerning the function of ABA is limited to some angiosperm plant species. We have shown that the basal land plant Physcomitrella patens and the model plant Arabidopsis thaliana share a conserved abscisic acid (ABA) signaling pathway mediated through ABI1-related type 2C protein phosphatases (PP2Cs). Ectopic expression of Arabidopsis abi1-1, a dominant allele of ABI1 that functions as a negative regulator of ABA signaling, or targeted disruption of Physcomitrella ABI1-related gene (PpABI1A) resulted in altered ABA sensitivity and abiotic stress tolerance of Physcomitrella, as demonstrated by osmostress and freezing stress. Moreover, transgenic Physcomitrella overexpressing abi1-1 showed altered morphogenesis. These trangenic plants had longer stem lengths compared to the wild type, and continuous growth of archegonia (female organ) with few sporohpytes under non-stress conditions. Our results suggest that PP2C-mediated ABA signaling is involved in both the abiotic stress responses and developmental regulation of Physcomitrella.

Analyses of leaves from open field-grown transgenic poplars overexpressing xyloglucanase

The transgenic expression of Aspergillus xyloglucanase cDNA (AaXEG2) with 35S promoter in the leaves of open field-grown poplars was studied. The level of xyloglucan in the transgenic poplars was decreased to 15–16% in the non-fertile soil (forest-field soil) and to 21–22% in the fertile soil (farming-field soil) compared with that of the wild-type poplars. The leaves exhibited a smaller surface area with more rounded teeth than those of the wild-type plants, similar to the sun leaf variety that was grown in the incubation room and subsequently greenhoused. The majority of total veins with water-conducting vascular bundles were shorter in the leaves of the transgenic poplars than those of the wild type. This decrease in vein length may result from a decrease in xyloglucan during leaf development, from which large numbers of proteins were markedly downregulated in the leaves of the transgenic plants via proteomic analysis. It seems likely that the leaves of the transgenic poplars came to relax the edges of their tooth rather than extend their veins as a result of the loosening of the xyloglucan cellulose networks in the leaves.

Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones

Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.