MicroRNA-mediated regulatory functions under abiotic stresses in legumes - Legumes Under Environmental Stress: Yield, Improvement and Adaptations - page 239

14.6 MicrorNa-mediated approaches
for functional genomics in legumes

Rhizophagus irregularis genes showing reduced mycor-
rhizal colonization. Furthermore, knock-down of MtErf1
by amiRNA-mediated gene silencing indicated a poten-
tial function of this gene during arbuscule development,
since only truncated, non-fully branched arbuscules
were observed in roots. It is further emphasized that
application of this potentially efficient and effective
method will likely revolutionize functional genomics in
a wide variety of legumes as many reports of successful
use of this approach have recently been documented for
both M. truncatula (Haney & Long, 2010) and soybean
(Melito et al., 2010).

Current molecular approaches to investigate plant
biology/physiology using transcriptional profiling or
large-scale DNA/protein analysis often lead to the
identification of large groups of novel genes with
unknown functions. One of the methods to determine
the function of a new gene is to knock-down this gene
using RNA interference (RNAi) technology, which can
be delivered to plants by virus-induced gene silencing
(VIGS) vectors. In soybean VIGS has successfully been
employed to develop mutant resources (Subramanian
et al., 2005). However, the core of RNAi is in fact two
types of small RNAs, small interfering RNAs (siRNA)
and miRNAs. RNAi through siRNA-mediated gene
silencing has identified several genes involved in specific
developmental processes in soybean (Libault et al., 2010;
Takagi et al., 2011). Although RNAi and VIGS have been
extensively applied to develop knock-down plant
mutants in the past, RNAi constructs often did not pro-
duce consistent results due to off-target effects of RNAi
approaches. Therefore, a new approach based on
artificial miRNAs (amiRNAs) was developed for silencing
genes (Khraiwesh et al., 2008; Ossowski et al., 2008;
Schwab et al., 2010). AmiRNAs are obtained by replacing
native miRNA duplexes from a natural miRNA pre-
cursor and have been shown to function as native
miRNAs. Recent studies have successfully used amiRNA-
based silencing constructs in M. truncatula (Haney &
Long, 2010) and soybean (Melito et al., 2010). Very
recently, Verdouk and Sullivan (2013) employed an
amiRNA sequence to silence the GUS Plus gene in trans-
genic alfalfa plants. Analysis of gene expression in the
transgenic plants indicated that the amiRNA construct
was effective at reducing both GUS Plus transcript levels
and GUS activity. Based on these results, it is further
demonstrated that amiRNA silencing constructs based
on the Arabidopsis miR319c can be successfully utilized
to specifically silence gene expression in alfalfa.
Similarly, Devers et al. (2013) presented compelling evi-
dence that an amiRNA-mediated gene silencing system
is a useful tool to investigate functions of genes involved
in root endosymbiosis in M. truncatula . With the amiR-
NAs the authors unravelled the function of a putative
transcription factor gene, MtErf1 , which showed strong
induction during mycorrhizal symbiosis. Knock-down
of MtErf1 expression reduced expression levels of

14.7 Conclusions and future
prospects

Current research is directed towards exploring the
essential roles in gene regulation of miRNAs, which are
the largest group of gene regulatory molecules in
legumes. However, these efforts provide a foundation
for the evaluation of the individual roles of miRNAs
in post-transcriptional regulation of developmental
processes and stress responses in economically impor-
tant legumes. Interestingly, miRNAs were long believed
to act only in a cis- or trans- way on their targets.
However, the finding that miRNAs can also transmit
information over a long distance or in a signalling way
regarding the nutrient status of plant organs is sur-
prising. For example, grafting experiments provided
convincing evidence that miR395 and miR399 are
phloem mobile under specific nutrient deficiency (Pant
et al., 2009; Buhtz et al., 2010). Considering the
numerous regulatory functions of miRNAs, detailed fol-
low-up studies will be invaluable to provide clear
evidence of in vivo mobility and for exploring the pos-
sible roles of additional miRNAs in systemic regulation.
Therefore, future efforts should be directed towards
supplementary experimental approaches for answers to
specific questions and in turn to understand the com-
plex gene regulatory networks of miRNAs.

references

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