NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF DEVELOPMENTAL BIOLOGY

(Adjunct)

Professor:
Kenzo Nakamura (Nagoya University)
Associate Professor:
Tsukaho Hattori (Mie University)
Research Associate:
Masa-aki Ohto
Graduate Students:
Yasuhiro Koide (Nagoya University)
Yukiko Iwata (Nagoya University)


Availability of sugars and the inter-organ transport and distribution of sugars are essential in the growth and development of the plant body. Expression of a variety of plant genes is regulated, either positively or negatively depending on the gene, by the level of sugars at the sugar-importing sink sites as well as at the sugar-exporting source sites of the plant body. Thus sugars are not only important as sources for cellular energy and the synthesis of macromolecules but also as a signal controlling the growth and development of plants by changing the pattern of gene expression. Our research attention is focused to eludicate the mechanisms involved in the regulation of gene expression in response to sugars, especially the activation of gene expression by increased-levels of sugars, and the role of such regulation in the organ development in plants. In addition, mechanisms involved in the accumulation of storage proteins in plant vacuoles are also studied.


I. Regulatory factors involved in the sugar-inducible expression of plant genes

EExpression of genes coding for sporamin and b-amylase, two major proteins of the storage roots of sweet potato, is inducible by high levels of sugars in various vegetative tissues. The GUS reporter genes under the control of the promoters of these genes are also inducible by sugars in leaves of transgenic tobacco plants, and these fusion genes are expressed in tubers of transgenic potato plants. Although the induction of expression of these fusion genes requires the activity of hexokinase, phosphorylation of hexose by hexokinase is not sufficient to cause the induction. The induction requires Ca2+-signalling and the activity of protein kinase. Eight different cDNAs for the isoforms of calcium-dependent protein kinase (CDPK) were isolated from leaves of tobacco, and transcripts of two of them were found to be increased upon treatment of leaves with various metabolizable sugars. Antibodies against a fragment of one of these isoforms cross-reacted strongly with the 57 kDa-protein in the soluble fraction from the young leaves. The level of this 57 kDa-protein decreased significantly as leaf matures, while the level of this 57-kDa protein in mature leaves increased significantly after the treatment of leaves with sugars. The sugar-induction of the 57-kDa protein occured preceding the induction of expression of the b-amylase:GUS reporter gene. In addition, a 54 kDa-protein with autophosphorylation acitivity in the plasma membrane of mature leaves also increased significantly upon treatment of leaves with sugars. This protein was purified to about 1,000-fold compared to the crude extract. It phosphorylated histone IIIS in a Ca2+-dependent manner and cross-reacted with an antibody against CDPK of Arabidopsis thaliana. These results suggest the possible involvement of CDPKs in the sugar-inducible gene expression and the development of leaves.


II. Mutants of Arabidopsis thaliana with altered patterns of the sugar-inducible gene expression

Expression of the b-amylase gene of Arabidopsis thaliana (Atb-Amy) in vegetative tissues occurs in response to high levels of sugars. A recessive mutation, lba1, caused significantly reduced-level of expression of Atb-Amy under high levels of sugars, while a recessive mutation, hba1, caused increased levels of expression in response to lower levels of sugars. It is suggested that HBA1 might function to maintain low-level expression of Atb-Amy until the level of sugars reaches some high levels and that the expression of Atb-Amy is regulated by a combination of both positive and negative regulation depending on the level of sugars.

A sequence between -172 and -61 of Atb-Amy was not only essential to drive the sugar-inducible expression of the GUS reporter gene but also able to confer sugar-inducibility to the core promoter of the constitutive gene. Linker scanning analysis identified two cis-regulatory elements between -172 and -62 of Atb-Amy, namely the negative element A and the positive element B. The element A contained a sequence which is highly homologous to the sequence motif that is conserved between sporamin and b-amylase genes of sweet potato and required for their sugar-inducible expression. The gel-mobility shift assay of the nuclear extracts identified activities binding to these regions.

Neither lba1 nor hba1 mutation affected the sugar-regulated gene expression in general suggesting that sugar-regulated expression of a variety of plant genes is mediated by multiple mechanisms. Nevertheless, lba1 and hba1 mutant plants showed pleiotropic effects on the sugar-inducible accumulation of anthocyanin. Furthermore, these mutant plants showed several characteristic growth properties. For example, lba1 mutant plants showed defective leaf development under the carbohydrate-limiting growth conditions and altered patterns of root development. The flowering time of the Arabidopsis plants under different carbohydrate availability was altered in the hba1 mutant plants. These results suggest that products of LBA1 and HBA1 genes might affect the organ development either directly or indirectly.


III. Screening of mutants of Arabidopsis thaliana with defective growth and development.

During the growth of plants, new organs develop as carbohydrate sink, and many vegetative organs shows sink to source transition after their maturation. Many aspects of the organ development in higher plants are thought to be affected, to some degree, by the levels of sugars. Sugars seem to have influence on the meristematic transition in long day plants from vegetative to reproductive growth. To obtain insights into the role of sugar-regulated gene expression in the growth and organ development in plants, we are screening for mutants of Arabidopsis thaliana with defects or anomalies both in the sugar-regulated gene expression and in the developmental processes such as leaf development and the determination of the flowering time. To aid this purpose, we have established more than 4,000 independent lines of Arabidopsis plants transformed with T-DNA containing multiple copies of the enhancer sequence. We have identified several plant lines with defects in the development of leaves or anormalies in the flowering time which also show the altered patterns of the sugar-regulated gene expression (Fig. 1).

Fig. 1
Phenotypes of the wild type plants (WT) and homozygous mutant line (#154) that were grown for 3 weeks. The chlorophyll content of the mutant plants is much lower than that of the wild type plants (Fig.1A). Mutant plants also showed the reduced levels of the sugar-inducible increase of b-amylase (left) and of anthocyanin (right) when leaf explants were treated with high levels (5%) of sucrose (Fig.1B).


Selected Publications:
Mita, S., Murano, N.,Akaike, M. and Nakamura, K. (1997) Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for b-amylase and of the accumulation of anthocyanin that are inducible by sugars. Plant J. 11: 841-851.
Matsuoka, K., Higuchi, T., Maeshima, M. and Nakamura, K. (1997) A vacuolar-type H+-ATPase in non-vacuolar organelle is required for the sorting of soluble vacuolar protein precursors in tobacco cells. Plant Cell 9: 533-546.
Mita, S., Hirano, H. and Nakamura, K. (1997) Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana; a recessive mutation causing enhanced expression of a gene for b-amylase. Plant Physiol. 114: 575-582.
Koide, Y., Hirano, H., Matsuoka, K. and Nakamura, K. (1997) The N-terminal propeptide of the precursor to sporamin acts as a vacuolar targeting signal even at the C-terminus of the mature part in tobacco cells. Plant Physiol. 114: 863-870.


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Last Modified: 12:00, May 28, 1998