The Plant Vascular System: Evolution, Development and FunctionsF

function of CmGAIP and GAI from Arabidopsis was investigated
using transgenic Arabidopsis and tomato lines expressing
engineered dominant gain-of-function Cmgaip and
DELLA-gai genes. Importantly, these transgenic plants exhibit
clear morphological changes in leaf development, and this
characteristic was used to test whether phloem delivery of the
Cmgaip/DELLA-gai transcripts into sink tissues could induce
this mutant phenotype. These engineered gai transcripts were
found to move long-distance through the phloem, and could
then exit from the terminal phloem and subsequently traffic
into the apex, where they accumulated in developing leaf
primordial (Haywood et al. 2005). Parallel studies conducted
with transgenic plants expressing EGFP revealed the inability
of these transcripts to enter the phloem. This finding suggested
that phloem entry of Cmgaip/DELLA-gai transcripts must
occur by a selective process.
Analysis of WT tomato scions grafted onto Cmgaip and
DELLA-gai stocks indicated that import of these transcripts
caused highly reproducible morphological changes in leaf phenotype
(Figure 20). Unexpectedly, tomato leaflet morphology
was found to be influenced quite late in development. Of
equal importance, the presence of Cmgaip and DELLAgai
transcripts, within the various tissues of the scion, was
not correlated with overall sink strength. Strong signals were
detected in young developing flowers and the apex, but signal
could not be amplified from fruit stalks or rapidly expanding
fruit (Figure 20C). These findings indicated an unexpected
complexity in the events underlying phloem delivery
of these transcripts, suggesting a high degree of regulation
over such trafficking of macromolecules. Furthermore, these
studies revealed that phloem long-distance delivery of RNA
can afford flexibility in adjusting developmental programs to
ensure that newly emerging leaves are optimized for performance
under existing environmental conditions (Haywood et al.
2005).
A model has been proposed that selective entry of transcripts
from the CC into the sieve tube system involves specific
sequences within the RNA (Lucas et al. 2001). As both Cmgaip
and DELLA-gai transcripts were able to move within the
heterologous plant, tomato, this finding suggested that such
sequence motifs, termed “zip codes,” must be conserved and,
further, the molecular machinery required for this recognition
and trafficking must similarly be conserved between pumpkin,
tomato and Arabidopsis.
Support for this hypothesis was provided by mutational
analysis of GAI in which it was clearly established that mRNA
entry into the phloem is facilitated by a motif located within the 3 ′
region of the transcript (Huang and Yu 2009). Furthermore, this
motif was specific to GAI, as parallel experiments conducted
with the four additional members of the DELLA family failed
to detect their transcripts in heterografting assays. By testing
an extensive series of GAI mutants, it was found that two zip
Insights into Plant Vascular Biology 343
Figure 20. Phloem delivery of Cmgaip/DELLA-gai transcripts
modifies leaf development.
(A) Schematic illustrating the V-grafting method used to test the
influence of phloem-mobile transcripts, derived from the stock and
imported across the graft union (gu) into the scion, on developmental
processes occurring in the scion.
(B) Schematic showing that import of Cmgaip/DELLA-gai transcripts
into wild-type tomato scions results in the development of
scion leaves with characteristic morphological features associated
with transgenic stock plants carrying the dominant gain-of-function
Cmgaip or DELLA-gai gene (Haywood et al. 2005). Numbers
represent tomato leaflet position along the leaf axis. Leaflet L0 does
not exhibit this change in morphology as it had passed through this
developmental stage prior to formation of the graft union.
(C) Detection of Cmgaip/DELLA-gai transcripts in wild-type tomato
scions grafted onto Cmgaip/DELLA-gai transgenic tomato stocks;
presence (+), absence (-). Absence of phloem-mobile transcripts
from the rapidly growing fruit indicates the operation of a regulatory
system controlling delivery of phloem mobile transcripts to specific
tissues/organs (from Haywood et al. 2005).
codes appear to be required for phloem entry and translocation,
one being located in the coding sequence and the other in the
3 ′ untranslated region. Finally, GFP transcripts carrying these
two zip codes were detected in the scion, confirming that these
sequence motifs are necessary and sufficient for targeting GAI
transcripts to the phloem (Huang and Yu 2009).