Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops - Crop Improvement: New Approaches and Modern Techniques - page 22

Furthermore, endoreduplication was reduced in ORF13 plants (Meyer et al. 2000 ),
indicating an interaction of ORF13 with cell cycle control. Stieger et al. ( 2004 )
claimed that a proliferative effect of ORF13 expression in the shoot apical meristem
(SAM) caused increased number of mitoses and showed no influence on meristem
structure. In consequence, the reductions of cell and meristem sizes and the retar-
dation in the formation of leaf primordia were observed. Smaller leaf sizes can be
explained by an earlier cessation of leaf growth, but not explained with a reduced
size of leaf cells, since the number of epidermal leaf cells per square millimeter was
remain unaltered. Enhanced number of cell divisions in the shoot apical meristems
and accelerated production of leaf primordia were seen in plant expressing ORF13.
ORF13 is involved in the inference of the cell cycle regulation leading to an earlier
stop in organ growth in the developing leaves. Furthermore, earlier flowering of
plants expressing ORF13 may arrest leaf initiation and leaf expansion, explaining
the fewer leaves formed in ORF13 plants (Stieger et al. 2004 ).
It has been also revealed that ORF13 protein contains a conservative retinoblas-
toma (RB)-binding motif LxCxE (Meyer et al. 2000 ). This motif was found in all
members of the ORF13 family, including agropine-, mannopine-, cucumopine-, and
mikimopine-type Ri plasmids (Stieger et al. 2004 ). When mutations are introduced
into the Rb motif, normal leaf size is restored, but plants still show stunting and
reduced apical dominance. It was also observed that ORF13 expression leads to
the formation of spur between minor veins on leaves and petals N. tabacum (Meyer
et al. 2000 ). Similar structures are formed on leaves, when KNOX (KNOTTED1-
like homeobox) genes are overexpressed (Sinha et al. 1993 ; Chuck et al. 1996 ;
Sentoku et al. 2000 ; Stieger et al. 2004 ). It was explained that cytokinin-like phe-
notype such as the formation of spikes, stunted growth, loss of apical dominance,
fusion of organs, and stem fasciations observed as consequences of ectopic expres-
sion of KNOX genes which are induced by ORF1 and cell cycle regulations (Stieger
et al. 2004 ).
Among the additional ORFs in the T L -DNA, there are two genes, which may
also contribute to the hairy root phenotype, ORF13a and ORF14. ORF13a is located
between ORF13 and ORF14 on the opposite strand. Expression of this gene is taken
place in a tissue specific manner in plants, primarily in leaf vascular tissues (Hansen
et al. 1994 b). ORF13a is necessary for root induction (Capone et al. 1989 ). ORF13a
containing motifs common to phorphorylated gene regulatory proteins codes for a
protein that may interact directly with DNA (Hansen et al. 1994 b). Despite a higher
expression rate of ORF13a was found in roots compared to leaves, its expression
did not yield a visible phenotype (Lemcke and Schmulling 1998 ; Veena and Taylor
2007 ). The putative protein encoded by ORF13a has a SPXX repeat motif and is
considered to have a regulatory function for this gene (Hansen et al. 1994 b). ORF14
is in the same gene family as rol B, rol C, ORF8 and ORF13 (Levesque et al. 1988 ).
Although overexpression of ORF14 in transgenic carrot and tobacco produced no
morphological changes (Lemcke and Schmulling 1998 ), it has been shown that the
rol genes and ORF13 act together to induce root induction (Capone et al. 1989 ;
Aoki and Syono 1999 ) (Table 1.1 ).