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)
Hideyuki Hirano (Nagoya University)
Masamitsu Kuriyama (Nagoya University)
Yukiko Iwata (Nagoya University)


Our research attention has been focused in the area of developmental control of gene expression and cell function related to the storage tissues and organs in plants, in particular mechanisms involved in the regulation of gene expression in response to the level of sugars and mechanisms involved in the targeting of storage proteins to the vacuole are studied.


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

Availability of sugars and inter-organ transport of sugars are essential in the growth and development of plants. Expression of a variety of genes is now known to be 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 macromolecules but also as signalling molecules controlling the growth and development by changing the pattern of gene expression in plants.

Although genes coding for sporamin and b-amylase, two major proteins of the tuberous root, are expressed specifically in tuberous roots of sweet potato under the normal growth conditions, their expression is inducible by high levels of sugars in other vegetative tissues. Expression of GUS reporter genes under the control of promoters of the sporamin gene or the b-amylase gene is inducible by sugars in leaves of transgenic tobacco plants, and these fusion genes are expressed in tubers of transgenic potato plants. It is suggested that massive transport of sugars to developing tuberous roots plays an important role in maintaining the high-level expression of sporamin and b-amylase genes. The sugar-inducible expression of sporamin and b-amylase genes requires synergistic action of two separate cis regulatory elements in each case, and one of the element is shared between the two genes. The activity to bind this conserved sequence motif in the nuclear extract increased upon treatment of the tissue with sucrose. A bZIP-type DNA binding protein and a novel DNA binding protein, which contains unique Zn-finger motif conserved among several plant DNA binding proteins, bind to other regulatory elements.

Experiments with various inhibitors suggested that the sugar-inducible expression of sporamin and b-amylase genes requires continuous dephosphorylation of proteins, Ca2+-signalling and the activity of protein kinase. It was also found that in leaves of tobacco, the level of 54 kDa-protein with autophosphorylation acitivity in the plasma membrane increases upon treatment of the tissue with sucrose. The 54 kDa autophosphorylation protein was solubilized from the plasma membrane by 1% sodium deoxycholate (DOC), and it was purified to about 1,000-fold compared to the crude extract. The 54 kDa-protein phosphorylated histone IIIS in a Ca2+-dependent manner and it cross-reacted with antibody raised against calcium-dependent protein kinase (CDPK) of Arabidopsis thaliana. By RT-PCR reaction with primers designed from sequence motifs highly conserved among plant CDPKs, we identified 8 cDNA fragments from leaves of tobacco which codes for different isoforms of CDPKs. Antibodies raised against kinase domains of two of these isoforms did not cross-reacted with the 54 kDa-protein. These results suggest that the 54 kDa-protein is a novel isoform of tobacco CDPK which is tightly associated with the plasma membrane.


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

Similar to the b-amylase gene of sweet potato, expression of the b-amylase gene of Arabidopsis thaliana (ATb-Amy) occurs in response to high levels of sugars. We identified several mutants of Arabidopsis which showed altered response of the expression of ATb-Amy to sugars in leaves. A recessive mutation, lba1, significantly reduced the level of expression of ATb-Amy under high levels of sugars. By contrast, a recessive mutation, hba1, caused increased levels of expression of ATb-Amy in response to lower levels of sugars, the results suggesting that HBA1 might function to maintain low level expression of ATb-Amy until the level of sugars reaches some high levels. It is suggested that the expression of ATb-Amy is regulated by a combination of both positive and negative regulation depending on the level of sugars.


III. Derepression from negative regulation in the sugar-inducible expression of b-amylase gene of Arabidopsis thaliana

In addition to the ATb-Amy:GUS fusion gene, in which the sequence from -1,587 to +136 of ATb-Amy was fused with gus, the ATb-Amy[D-172]:GUS fusion gene where the 5'-upstream of the promoter was truncated to -172, showed sugar-inducible expression in leaves of transgenic Arabidopsis plants in a manner similar to the endogenous ATb-Amy. A sequence between -172 and -61 of ATb-Amy was essential for the sugar-inducible expression and it conferred sugar-inducibility on the -89 core promoter of CaMV 35S. Linker scanning of the sequence between -172 and -62 in the ATb-Amy[D-172]:GUS fusion gene identified two cis-regulatory elements. When sequence substitutions were made in the negative regulatory region A, levels of GUS activity in non-treated leaves increased to those in sugar-treated leaves. On the other hand, substitutions in the positive regulatory region B abolished the expression. The region 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.

In contrast to ATb-Amy:GUS, ATb-Amy[D-172]: GUS did not show increased sensitivity to low levels of sugars in hba1 mutant plants. In the wild type plants, the 5'-truncation of the promoter to -172, but not to -310, caused increased sensitivity to low levels of sugars similar to the pattern of expression of ATb-Amy in hba1 plants. These results suggest that the negative regulation by HBA1 might require cis-element(s) located between -310 and -173, and further support that the sugar-inducible expression of ATb-Amy might involve derepression from negative regulations.


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

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. In particular, several growth and developmental properties of Arabidopsis plants, which in wild type plants occurs in response to changing levels of sugars, are affected in these mutant plants. To obtain further insights into the role of the sugar-regulated gene expression in the growth and development of plants, research attention will be focused on screening of mutants of Arabidopsis thaliana with defects or anomalies in the growth and organ development that are affected by the availability of sugars in the wild-type plants. To aid this purpose, we are constructing increasing number of mutagenized plants after transformation with multiple copies of enhancer fragments in the T-DNA.


nibb-adm@nibb.ac.jp
Last Modified: 12:00, June 27, 1997