Abiotic Stress Responses in Plants: Unraveling the Complexity of Genes and Networks to Survive - Abiotic Stress: Plant Responses and Applications in Agriculture - page 59

The miRNA395 gene-family targets genes involved in sulphate translocation (the low-affinity
transporter SULTR2;1 ) and assimilation (the ATP sulphurylases, APS ) [134, 140,141]. Impor‐
tantly, miR395 itself is regulated by a transcription factor, the SULFUR LIMITATION 1 (SLIM1)
[141]. The miR395/APS-SULTR2;1/SLIM1 regulatory module is involved in root-to-shoot
sulphate translocation as a strategy to improve sulphate assimilation in the leaves during
sulphate starvation [142].
The miR399 gene-family is strongly and specifically induced by inorganic phosphate limi‐
tation in the shoot and targets PHO2, an E2 ubiquitin-conjugating enzyme that represses
Pi uptake [109, 140,143-144]. As for miR395, also the expression of miR399 is regulated
by a transcription factor, the MYB TF PHOSPHATE STARVATION RESPONSIVE1
(PHR1; [109]). The miR399/PHO2/PHR1 regulatory module operates under Pi depriva‐
tion: miR399 is induced by PHR1 in the leaves, travels along the phloem to repress
PHO2 expression in the roots thereby releasing several protein targets from ubiquitinyla‐
tion-dependent degradation, including transporters involved in Pi allocation inside the
plants and increasing Pi content in the shoot. A worth mentioning aspect of the miR399
regulatory module is the extra layer of miR399 activity regulation exerted by IPS1 (in‐
duced by phosphate starvation1) [145]. IPS1 is a non-protein coding transcript with se‐
quence complementarity to miR399 that sequesters miR399 thus inhibiting its repressing
activity over its target. This mechanism designated as target mimicry was first described
in plants [145] and more recently discovered in animals [146] and expands the regulatory
post-transcriptional gene expression network in which miRNAs are involved.
The miR398 (and miR408) are induced by copper limitation and target genes encondig copper
proteins like Copper/Zinc superoxide dismutases, cytochrome c oxidase and plantacyanin
[147, 148]. Similar to miR395 and miR399, also miR398 and miR408 are regulated by a tran‐
scription factor, the SQUAMOSA promoter binding protein-like7 (SPL7) that regulates the
expression of several copper-responsive genes [149]. Copper in contrast to sulphate and
phosphate is a micronutrient but still the regulation of this nutrient homeostasis is basically
similar, as it involves sistemic signalling, a well established regulatory module involving a
transcription factor, the miRNA and its target.
The miR395, miR399 miR398 and miR408 were identified in M. truncatula by sequencing
libraries of small RNAs from the aerial part [135]. Homologs of known miRNA target genes
were identified, such as low affinity sulphur transporter for miR395, COX5b (subunit 5b of
mitochondrial cytochrome c oxidase) for miR398, PHO2 for miR399 or plantacyanin for
miR408. However, our computational prediction identified many hypothetical genes for
miRNA targeting ([135] - Additional File 1), rendering experimental confirmation a laborious
and unsuccessful task (Trindade, unpublished data).
Some miR398 and miR408 predicted targets were validated by 5'RACE and miR398 and
miR408 expression was further investigated in different plant parts and in specific water deficit
conditions, showing up-regulation in water deprivation and concomitant down-regulation of
their validated targets [129]. These targets were further confirmed by deep sequencing of
cleaved miRNA targets (Parallel Analysis of RNA Ends - PARE) [150-151] in M. truncatula in