In Arabidopsis, miR395 expression is highly induced in response to S deprivation. It targets three ATP sulfurylase genes (APS1, APS3 and APS4) and a low-affinity sulfate transporter, AST68, which facilitates S transport between plant organs. miR395 and target genes are conserved in angiosperms and in more primitive plants, such as the moss Physcomitrella patens (Jones-Rhoades and Bartel, 2004; Allen et al., 2005; Fattash et al., 2007; Sunkar and Jagadeeswaran, 2008). Of the four targets, miR395-guided cleavage on APS1, APS4 and AST68 mRNAs was confirmed both in leaves and roots (Kawashima et al., 2009). However, miR395-directed cleavage of APS3 was detected in leaves but not in roots, which led the authors to conclude that miR395 does not regulate APS3 in roots (Kawashima et al., 2009). In this study, we confirmed miR395-guided cleavage on four miR395 targets (data not shown), including APS3 in leaves and roots of Arabidopsis during S deprivation (Figure 1(a)). In addition to the four conserved targets, At2g28780 was predicted as a target for miR395 because of mere 1.5 mismatches between miR395 and At2g28780 (Adai et al., 2005); however, previous attempts to confirm miR395-guided cleavage on At2g28780 were unsuccessful, which led authors to suggest that miR395 does not regulate the At2g28780 transcript despite high complementarity (Kawashima et al., 2009). In this study, we detected canonical cleavage in the complementary region (between 10th and 11th nucleotides) of At2g28780 transcripts in roots and shoots of Arabidopsis exposed to S deprivation (Figure 1(b)), which confirms that miR395 regulates At2g28780 expression in Arabidopsis. Because conserved miRNA target sites are usually conserved, we searched for complementary sites on homologous transcripts available at NCBI. This analysis revealed that the miR395 target site is conserved in Arabidopsis lyrata, Capsella rubella, and Populus trichocarpa (1.5 mismatches). However in other dicots, the number of mismatches increased (Figure 1(c)). For example, 2.5 mismatches in tomato (Solanum lycopersicum) and Ricinus communis; 3.5 in Brassica napus and Prunus persica; 4.0 in Fragaria vesca; 5.0 in grape (Vitis vinifera); and 6.0–6.5 in legumes (Glycine max, and Medicago truncatula). Interestingly, in sacred lotus (Nelumbo nucifera), a basal eudicot, there are 6.5 mismatches. In Cucumis sativus, there are 10 mismatches in the complementary region (Figure 1(c)). It is noteworthy that the CCS1 transcript (encoding a chaperone for Cu/ZnSOD) contains 5.5 mismatches with miR398 and has been confirmed as a target in Arabidopsis and rice (Beauclair et al., 2010; Li et al., 2010). More recently, several transcripts were confirmed as genuine miRNA targets despite weak complementarity (5 or more mismatches) (Li et al., 2010; Chorostecki et al., 2012; Zheng et al., 2012). Although we only validated At2g28780 as a miR395 target in Arabidopsis, homologous transcripts are likely to be regulated by miR395 in A. lyrata, B. napus, and possibly in distantly-related plants such as P. trichocarpa, S. lycopersicum, and R. communis.