regulatory regions. One of the factors, ABF-1, was 
found to be present in photosynthetically active tis- 
sues but not in etiolated or root tissue. 
The second factor, ABF-2, is present in all tissues 
analyzed and binds to a DNA sequence that contains 
a direct heptamer repeat, TCTCAAA. It was found 
that both repeats are essential for binding. ABF-2 is 
evolutionarily conserved in several plant species. 
Computer analysis showed that the TCTCAAA motif 
is present in the 5'-flanking region of several highly 
inducible plant genes and that the distance between 
the two motifs required for binding seems to be im- 
portant for their function. The next important step 
is to determine which combination of these DNA 
motifs determines the light-inducible properties of 
photosynthetic genes. In addition, protocols for the 
transformation of tomatillo {Physalis ixocarpd) 
are being developed to enable the study of the 
regulation of photosynthetic genes during fruit 
development. 
Molecular Approaches to Understanding 
the Function of Chaperonin 60 Genes 
Chaperonin 60 a and |8 polypeptides have been 
implicated in the assembly of rubisco, acting as 
molecular chaperones. However, apart from the 
strong indirect evidence supporting this role for 
chaperonin 60 (cpn60) polypeptides, no direct evi- 
dence is available to confirm the hypothesis. 
To study the function of these polypeptides. 
Dr. Herrera-Estrella's group has isolated the Ara- 
bidopsis thaliana genes encoding the cpn60 ^ 
polypeptides. 
In this plant the cpn60 /3 gene family is composed 
of three functional genes. In the past year. Dr. 
Herrera-Estrella's laboratory has focused on deter- 
mining the tissue-specific expression directed by 
the 5'-flanking region of the cpn60 ^ genes and on 
studying their function by generating transgenic 
plants harboring cpn60 ^ antisense gene constructs. 
Using chimeric genes in which the coding sequence 
of the bacterial (8-glucuronidase gene is under the 
control of cpn60 /3 promoter, the group found that 
these genes have high levels of expression in the 
mesophyll cells of photosynthetic tissues and in the 
male and female reproductive organs. A develop- 
mentally regulated expression was also observed in 
stems, where expression is initially detected in the 
peripheral-photosynthetic area but is later observed 
only in vascular tissue. This is an important step in 
the understanding of the function of cpn60 (8 genes, 
because their expression in nonphotosynthetic tis- 
sues suggests that the encoded polypeptide must 
have functions other than the assembly of rubisco. 
Transgenic plants expressing antisense cpn60 ^ 
RNA show different degrees of stunting and have 
chlorotic mature leaves. In contrast to what could 
be expected from the proposed function of cpn60, 
the antisense transgenic plants have higher levels of 
rubisco activity than nontransformed control plants. 
The most conspicuous biochemical alteration in 
these plants is that the level of sucrose phosphate 
synthase is drastically reduced. Starch content analy- 
sis and electron microscopy showed that the mature 
leaves of these plants contain large quantities of 
starch accumulated in the plastids of the cells. This 
finding suggests that cpn60 ^ polypeptides may not 
be involved in rubisco assembly but that they affect 
either the systems involved in the transport of triose 
phosphate molecules from the chloroplast to the cy- 
toplasm or in the cytoplasmic conversion of triose 
phosphate molecules into sucrose. 
Molecular Analysis of Sucrose Phosphate 
Synthase Genes 
Sucrose phosphate synthase (SPS) could be a key 
enzyme in the assimilation of photosynthates. Dr. 
Herrera-Estrella and his colleagues, in collaboration 
with the group of Dr. Horacio Pontis in Argentina, 
have sought to isolate and characterize the genes 
encoding SPS. Using antibodies and polymerase 
chain reaction technology, cDNAs encoding SPS 
have been isolated. SPS is present as a single-copy 
gene in maize, rice, and Arabidopsis. Molecular 
characterization of these genes and studies aimed to 
investigate the importance of this enzyme in carbon 
assimilation, using transgenic plants containing 
sense and antisense gene constructs, are in progress. 
Dr. Herrera-Estrella is Professor and Chairman 
of the Department of Plant Genetic Engineering at 
the Center for Research and Advanced Studies, 
National Polytechnic Institute, Irapuato. 
Articles 
Arguello, G., Garcia-Hernandez, E., Sanchez, M., 
Gariglio, P., Herrera-Estrella, L., and Simpson, 
J. 1992. Characterization of DNA sequences that 
mediate nuclear protein binding to the regulatory 
region of the Pisum sativum (pea) chlorophyll 
a/b binding protein gene AB80: identification of a 
repeated heptamer motif. Plant / 2:301-309. 
Zabaleta, E., Oropeza, A., Jimenez, B., Salerno, G., 
Crespi, M., and Herrera-Estrella, L. 1992. Isola- 
tion and characterization of genes encoding cha- 
peronin 60 (8 from Arabidopsis thaliana. Gene 
111:175-181. 
516 
