The Plant Vascular System: Evolution, Development and FunctionsF

346 Journal of Integrative Plant Biology Vol. 55 No. 4 2013
Figure 22. Grafting experiments illustrating that transmission
of a phloem long-distance signal can induce posttranscriptional
gene silencing within developing scion tissues.
(A–C) Transgenic tobacco plants expressing a nitrate reductase
gene (Nia) segregated into silenced (S) and non-silenced (NS)
phenotypes were employed in grafting studies to test for the longdistance
propagation of the silencing condition through the phloem.
(A) Grafting of an NS scion onto an S stock resulted in silencing
of Nia within the scion leaves. (B) Placement of a wild-type (WT)
stem segment between the NS scion and S stock did not prevent
the transmission of the silencing signal. (C) Grafting of a silenced
scion (S) onto a NS stock does not activate silencing in the NS stock
tissues.
(D) Analysis of RNA samples collected from specific grafted tissues
( ∗ ) confirmed that the observed silencing phenotype reflected
sequence-specific targeting of the Nia transcripts (redrawn from
Palauqui et al. 1997).
Sequence-specificity of the silencing signal was established
by grafting studies performed with nitrite reductase (Nii) transgenic
tobacco stocks and non-silenced Nia scions (Figure 22D).
A hypothesis was advanced that phloem-mobile silencing
signals involved the translocation of antisense RNA, whose
entry into the developing scion tissues caused an enzymemediated
cleavage of the double-stranded form of the target
RNA (Jorgensen et al. 1998). Detection, in silenced tissues,
of small (20–25 nucleotide [nt]) antisense RNA complementary
to the silenced gene (sRNA) (Hamilton and Baulcombe 1999)
provided strong support for the general features of this model.
Analysis of RNA extracted from pumpkin phloem sap identified
a population of 21 nt – 24 nt sRNA. Sequencing and
bioinformatics analysis indicated that these sRNAs belong to
both the micro(mi)RNA and small interfering (si)RNA silencing
pathways (Yoo et al. 2004). Interestingly, although equal signal
strength was detected for sense and antisense sRNA probes,
they did not appear to exist in the phloem sap as duplexes.
The involvement of these phloem sRNAs in systemic silencing
was explored using silencing (stock) and non-silencing (scion)
transgenic squash (Cucurbita pepo) plants expressing a viral
coat protein gene. Phloem sap from both the stock and scion
tested positive for CP siRNA, and analysis of apical tissues
from these scions confirmed that the level of CP mRNA had
been greatly reduced. Interestingly, a low level signal for
the antisense CP transcript could also be detected in the
phloem sap of both stock and scion plants. Collectively, these
findings offered support for the hypothesis that both siRNA and
antisense RNA are likely components of the systemic silencing
machinery.
Limited information is available concerning the mechanism
by which these sRNA molecules enter and move longdistance
through the phloem. Biochemical studies performed
on pumpkin and cucumber phloem exudate identified a 20 kDa
PHLOEM SMALL RNA BINDING PROTEIN1 (PSRP1) that
bound specifically to sRNA (Yoo et al. 2004). This protein has
the capacity to traffic its sRNA cargo through PD, and in the
cucurbits, PSRP1 may be involved in shuttling sRNA from CCs
into the sieve tube system. Interestingly, PSRP1 homologues
have yet to be identified in the genomes of other plant species.
This raises the possibility that additional proteins have evolved
to carry out these same functions.
Role of phloem-mobile sRNA in directing
transcriptional gene silencing in target tissues
The phloem sap collected from pumpkin and oilseed rape
contains a significant population of 24-nt sRNA (Yoo et al.
2004; Buhtz et al. 2008), indicating a likely involvement in
transcriptional gene silencing (TGS) within sink tissues (Mosher
et al. 2008). Grafting experiments performed with various
combinations of GFP transgenic and DICER-LIKE mutant
Arabidopsis lines provided further confirmation that a significant
population of exogenous/endogenous 23 nt – 24 nt sRNA can
cross the graft union (Molnar et al. 2010). Methylation analysis
of DNA extracted from these grafted target tissues provided