MicroRNA-mediated regulatory functions under abiotic stresses in legumes - Legumes Under Environmental Stress: Yield, Improvement and Adaptations - page 238

14.5 MicrorNas play important
roles in nodulation and symbiosis
in legumes

(Boualem et al., 2008; Subramanian et al., 2008;
Nogueira et al., 2009; Devers et al., 2011; Wang et al.,
2011). Overexpression of miR166 in M. truncatula led
to a reduction in nodules and lateral root number in
addition to ectopic development of vascular bundles in
transgenic roots. Several lines of evidence suggest that
miR166 activity during nodule development most
probably relies on the conserved functions of its target
in root cell differentiation. In M. truncatula, miR169
regulates the expression of MtHAP2-1 (Combier et al.,
2006), whose expression is strongly upregulated during
nodule development (El Yahyaoui et al., 2004).
MtHAP2-1 is a member of the NF-Y transcription factor
family; it comprises three subunits - HAP2 (CBF-B,
NF-YA), HAP3 (CBF-A, NF-YB) and HAP5 (CBF-C,
NF-YC) - and binds the CCAAT motif present in many
eukaryotic promoters (Maity & de Crombrugghe,
1998).
Overexpression of miR169 resulted in non-functional
nodules and delays of 8-10 days in nodule development.
Furthermore, the downregulation of MtHAP2-1 by
RNAi showed a similar arrest in nodule development.
On the other hand, overexpression of miR169-resistant
MtHAP2-1 under its native promoter restored tissue
differentiation. However, a downregulation of miR169
in early development of soybean nodules was noticed
(Subramanian et al., 2008), suggesting that miR169-
dependent regulation of MtHAP2-1 may also play a role
in the early events of symbiosis.
In addition to miR166 and miR169, a number of
conserved miRNAs, including miR156, miR160,
miR167, miR172, miR398 and miR399, are also
expressed in other nodule tissues (Lelandais-Briere
et al., 2009; Yan et al., 2013). However, the dynamics of
miRNA expression at the late stages of nodule
development and symbiosis have so far not been inves-
tigated. Recently, Dong et al. (2013) performed deep
sequencing of miRNAs from both normal and salt-
stressed mature soybean nodules. They identified 110
known miRNAs belonging to 61 miRNA families, and
128 novel miRNAs belonging to 64 miRNA families.
Both salt-stressed and control libraries shared nearly
all the known miRNAs, and for 128 novel miRNAs
only 82 were detected in both libraries. Of the remain-
ing 34, 12 novel miRNAs were expressed only in one
of the libraries, although the average abundance of
each novel miRNA was much higher in the salt-
stressed library.

Nodules are specialized root lateral organs that result
from symbiotic interaction between legumes and
nitrogen-fixing bacteria (collectively called rhizobia);
they have both agronomic and ecological importance
for plants (Murray, 2011). Symbiosis begins with the
exchange of certain chemical signals between the part-
ners, which determines the host specificity. Rhizobial
infection of legumes occurs primarily through root
hairs. Legume roots release specific flavonoid com-
pounds that are recognized by compatible bacterial
species, which in turn release lipo-chito-oligosaccharides,
called the 'Nod' signal. The bacteria colonize the root
hair and gain access to the nodule cells through a spe-
cialized structure called an infection thread. This is
followed by cell division in the cortex and pericycle cell
layer to form a nodule primordial, thereby ultimately
leading to nodule formation. A specialized structure in
the nodule called a symbiosome is the functional unit
of biological nitrogen fixation (Kereszt et al., 2011). A
number of different plant hormones are shown to play
significant roles in the initiation, development and ulti-
mately establishment of symbiosis in legumes (Suzaki
et al., 2013). Recent studies have shown that some
legume miRNAs are functionally associated with
nodulation and the establishment of symbiosis
(Lelandais-Briere et al., 2009; Simon et al., 2009;
Subramanian et al., 2008; Yan et al., 2013). Several
miRNAs (miR156, miR160, miR166, miR167, miR169,
miR319 and miR393) that are involved in auxin signal-
ling were shown to be regulated in the early events of
nodulation and symbiosis (Subramanian et al., 2008;
Li et al., 2010).
Several researchers have identified and functionally
characterized miRNAs in legume nodules using
sequencing and bioinformatics approaches (Zhang
et al., 2008; Jagadeeswaran et al., 2009; Wang et al., 2009;
Joshi et al., 2010; Li et al., 2010). Early investigations
revealed the involvement of only two miRNAs,
miR166 and miR169, in Bradyrhizobium infection
and nodule development in soybean and M. truncatula
(Combier et al., 2006; Boualem et al., 2008;
Subramanian et al., 2008). It is well known that
miR166 regulates a family of HD-ZIP III transcription
factors in a number of plant species including legumes