Pereza fiscal: uno de los mega-diseños institucionales que in-

miRNA overexpression is a commonly used method for establishing the function of both a miRNA family and the target mRNA family. Some miRNAs, such as miR319 (discussed in l.F.l.A .), have even been isolated through overexpression. Many miRNAs target several members of the same protein family. Therefore, by overexpressing a single miRNA locus, multiple members of a protein family can be downregulated (Achard et al., 2004; Fujii et al., 2005; Guo et al., 2005; Kidner and Martienssen, 2004; Laufs et al., 2004; Mallory et al., 2005; Mallory et al., 2004a; Palatnik et al., 2003; Schwab et al., 2005; Wang et al., 2005b; Williams et al., 2005; Yamasaki et al., 2007). This family knock-down approach can be especially useful when multiple targets have overlapping function and may provide an alternative to the production of multiple loss-of-function mutants that are unobtainable by crossing due to gene linkage or difficult to obtain because of multiplicity (e.g. quadruple or quintuple mutants).

Whereas siRNAs can cause off-target effects (Hannon and Rossi, 2004; Jackson et al., 2003), in which not only the intended target is downregulated, but also targets with more limited complementarity, and microarray analysis of plants overexpressing various miRNAs reveals that plant miRNAs specifically regulate only their target mRNAs with little to no discernable effects on non-target mRNAs (Schwab et al., 2005); whether miRNA overexpression has effects on off-target protein accumulation has not been investigated. Others and I have overexpressed many miRNAs (Table 1-2; see also Table 3-2). Analysis of these overexpression lines has revealed that miRNA function in many aspects of plant development, from shoot development (miR170/171 is discussed further

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in Chapter 3) to flower formation (discussed further in Chapter 4) to growth tolerances (discussed further in Chapter 5).

l.F .l.A . The m iR 319/159 fam ily affects leaf m orphology

Whereas most plant microRNAs were discovered through cloning, a few have been identified through mutant screens. For example, miR319 (miR-JAW) was first identified out of an activation tagging screen (Weigel et al., 2000). Overexpression of miR-JAW results in uneven leaf shape and curvature (Palatnik et al., 2003) reminiscent of the snapdragon cin mutants (Nath et al., 2003). Microarray analysis reveals that five TCP transcription factor transcripts, related to CIN, are decreased in jaw-D (mir319) mutants (Palatnik et al., 2003). A seven amino acid stretch near the TCP domain is highly conserved in the subset of TCP genes that are decreased in jaw-D mutants (Palatnik et al., 2003). The RNA encoding this stretch displays few synonymous substitutions among the affected genes and is partially complementary to miR159 and also to a portion of the JAW locus (Palatnik et al., 2003).

RNA gel-blots probed with a 20-nt probe complementary to miR-JAW displayed accumulation in various plant tissues, whereas a 21-nt probe for miR159 did not show increased accumulation (Palatnik et al., 2003), illustrating that miR-JAW (miR319) is itself a miRNA distinct from, but related to, the miR159 family. Target mRNA

regulation was confirmed by 5'-RACE assays and exposed an overlap in miR-JAW and miR159 (targets MYB33 and MYB65) function, as miR-JAW directed cleavage sites are detected in the MYB33 and MYB65 mRNAs (Palatnik et al., 2003). However, MYB33 and MYB65 are not downregulated in the jaw -lD mutant, reconfirming that miR-JAW

Table 1-2. Consequences of miRNA overexpression

miRNA Locus T arget family C o n se q u e n c e s of overexpression miR156 M IR 156tf SPL transcription

factors

in creased leaf num ber, d e c re a se d apical dom inance, delayed flowering time miR159 MIR159ab MYB transcription

factors

sm aller rounder leaves, increased leaf num ber, m ale sterility, delayed flowering miR319 MIR31SF TCP transcription

factors

uneven leaf s h a p e and curvature, late flowering

miR160 MIR 160c? ARF transcription factors

agravitropic roots, disorganized root ca p s, in cre ased lateral roots

miR164 MIR164aeb

MIR164bem

MIR164<fb

NAC dom ain transcription factors

w eak vegetative and floral organ fusions, cu p -sh ap ed cotyledons, low levels of leaf serrations

vegetative and floral organ fusions, cu p ­ sh a p e d cotyledons, low levels of leaf serrations, few er lateral roots

floral organ fusions, low levels of leaf serrations miR165/166 M IR165d MIR 166at MIR166ct HD-ZIP transcription factors

loss of sh o o t apical m eristem , alteration of organ polarity, abnorm al formation of carpels, inhibition of vascular

developm ent, ab e rra n t differentiation of interfascicular fibers

altered vasculature, fem ale sterility, fasciated apical m eristem s

vasculature patterning defects, fasciated apical m eristem s, fem ale sterility, epinastic leav es miR167 MIR 1 6 7 ^ , MIR167bfn, M IR167dn, MIR 167cT ARF transcription factors

twisted leaves, short inflorescences and arrested flower developm ent to wild-type phenotypes depending on the locus

m iR l72 M IR 172cf° AP2-like transcription factors

early flowering, a b s e n c e of petals, transform ation of se p a ls to carpels miR398 M IR 398tf CSD, cytochrom e C

o xidase

no in cre ase in CSD1 and CSD2 protein after ex p o su re to high copper

miR399 MIR399aP, MIR399C?, MIR399cf, M IR 399fs P h o sp h ate transporter, E2 UBC

p h o sp h ate accum ulation

“(Schwab et al., 2005), b(Achard et al., 2004), c(Palatnik et al., 2003), d(Wang et al., 2005b), e(Laufs et al., 2004), f(Mallory et al., 2004a), g(Baker et al., 2005), h(Nikovics et al., 2006), '(Guo et al., 2005), j(Zhou et al., 2007), k(Kim et al., 2005), '(Williams et al., 2005), m(Wu et al., 2006), "(Aukerman and Sakai, 2003), °(Chen, 2004), p(Yamasaki et al., 2007), q(Chiou et al., 2006), r(Bari et al., 2006), s(Fujii et al., 2005)

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