Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops - Crop Improvement: New Approaches and Modern Techniques - page 19

ORF Genes
Besides rol (root locus) genes, there are several ORFs (Open Reading Frames) locat-
ed on the T L -DNA (Slightom et al. 1986 ). Many of 18 open reading frames (ORFs)
nucleotide sequences identified on T L -DNA region contain 5′ and 3′ regulatory ele-
ments similar to those found in eukaryotic genes. They have at least 255 nucleotides
and start with the initiation codon ATG (Slightom et al. 1986 ; Holefors et al. 1998 ).
In many cases, CCAAT and TATA elements were situated upstream of putative tran-
scriptional initiation codons and poly(A) addition (AATAAA) elements were present
in presumed 3′-noncoding regions (Slightom et al. 1986 ). The sequence length of
coding regions of ORFs differ in ranging from 255 bp (ORF 9) up to 2280 bp (ORF8)
and encode protein products ranging in size from 9,600 to 85,000 daltons, respective-
ly. The results from analysis of insertion mutants within the T-DNA region (White
et al. 1985 ) and transformation experiments with individual or combinations of the
ORFs have showed that the open reading frames ORF10, 11 and 12, corresponding
to the genes rol A, rol B and rol C, were able to promote the formation of hairy root
syndrome (Table 1.1 ) (Jouanin et al. 1987b ; Vilaine et al. 1987 ; Spena et al. 1987 ;
Spano et al. 1988 ; Schmulling et al. 1988 ). Besides this, it has been showed that
ORF3n, ORF8 and ORF13 DNA sequences are highly conserved among all known
Ri plasmids, indicating that they alter plant morphogenesis or response of transgenic
tissues to plant hormones (Lemcke and Schmulling 1998 ; Veena and Taylor 2007 ).
The sensitivity to auxin and cytokinin in combination or auxin alone can be lowered
by expressions of both ORF3n and ORF8 (Lemcke and Schmulling 1998 ).
ORF3n
Expression of ORF3n in transgenic N. tabacum caused retarded flowering, less
dense inflorescences, altered internode elongation and leaf morphology and necrot-
ic tips of upper leaves, sepals and bracts (Lemcke and Schmulling 1998 ). Appear-
ance of localized necrosis was noticed on the tips of apical narrow leaves whereas
there was no sign of necrosis on the basal leaves. Additionally, senescence was not
altered in these leaves, and bracts became necrotic as a whole. On sepals, the ne-
crosis emerged on the tips just when the corolla was visible through the calyx (Kol-
tunow et al. 2001 ; Lemcke and Schmulling 1998 ). The ORF3n protein (48.7 kDa)
resembles phenolic-modifying enzymes and may be involved in secondary metab-
olism and/or the transport of hormones (Binns et al. 1987 ; Jacobs and Rubery 1988 ;
Lemcke and Schmulling 1998 ). A cessation was observed in the shoot formation
from ORF3n callus in response to auxin and cytokinin. Also, plantlets transferred
to the medium containing auxin and cytokinin showed decreased sensitivity lead-
ing to small and fewer calli than controls. Thus, it has been proposed that ORF3n
may act to negative regulator to the dedifferentiation of tissues as a reaction to
auxin and cytokinin, which may favor the formation of rol gene-induced roots from
such cells during pathogenesis (Britton et al. 2008 ; Dodueva 2007 ).