Function and Evolution of a MicroRNA with a Role in Reproductive Growth in Tomato

MicroRNAs (miRNAs) are a class of small RNAs that function in posttranscriptional gene regulation in eukaryotes. Plant miRNAs, which function through a combination of target cleavage and translational repression, typically show near-perfect complementarity to target sequences and thus are predicted to have a limited number of targets (reviewed in Voinnet, 2009). A number of miRNA genes are deeply conserved across plant families, indicating their ancient evolutionary origin, and these often are found to regulate major developmental processes, such as meristem functions, cell division, and organ polarity. However, there are myriad evolutionarily more recent species- or family-specific miRNAs that appear to influence a much broader range of biological processes. In most cases, the function of these more narrowly conserved miRNAs is unknown or poorly understood. Wang et al. (pages 3185–3203) explore the function and evolution of a miRNA conserved within the Solanaceae, shown to regulate a Ca²⁺-ATPase that functions during reproductive growth in tomato (Solanum lycospericum).

Wang et al. focus on the small RNA sly-miR4367, which is highly expressed in leaves and young flower buds of tomato. The authors identify the target of sly-miR4367 as a homolog of Arabidopsis thaliana ACA10, which encodes an autoinhibited Ca²⁺-ATPase. Complementation tests in yeast showed that Sl-ACA10 encodes a functional Ca²⁺-ATPase, and a 5′ rapid amplification of cDNA ends assay confirmed that sly-miR4367 is a functional miRNA that regulates the stability of Sl-ACA10 mRNA in vivo. A search of a small RNA database revealed a series of phased small RNAs derived from Sl-ACA10, having a pattern of abundance in tomato organs that mirrors the accumulation pattern of sly-miR4367. This suggests a pattern of regulation similar to miRNA cleavage-dependent generation of phased small interfering RNAs (siRNAs) in Arabidopsis (Montgomery et al., 2008).

The authors go on to show that disrupting sly-miR4367 regulation of Sl-ACA10 expression specifically alters tomato flower morphology and fruit yield (see figure). The similar phenotypes, including a dramatic decline in fruit yield, of overexpressing either a cleavage-resistant form of Sl-ACA10 or the miRNA responsible for cleavage, when they might be expected to have opposite effects, suggests that the precise regulation of ACA10 expression in tomato flowers and fruits is critical for reproductive growth. Evidence of miRNA regulation of a plant Ca²⁺-ATPase increases our understanding of molecular mechanisms contributing to calcium homeostasis in plants, which is critical for many biological processes.

sly-miR4367 regulates reproductive growth in tomato. Overexpression of sly-miR4367 (middle) or cleavage-resistant Sl-ACA10 (right) led to a drastic reduction in fruit yield compared with a control line (left). (Figure adapted and reprinted from Figure 3 of Wang et al. [2011].)

The authors explore the evolution of miR4367 and its target site within the Solanaceae, revealing a stochastic pattern of emergence that varies drastically in even closely related species. For example, they found miR4367-coding sequences in all solanaceous plants examined, but in a few species, they are only weakly expressed or not processed into a mature miRNA. The ACA10 target site was found in a number of species, but the presence of corresponding phased siRNAs was detected only in tomato and potato (Solanum tuberosum). Interestingly, attempts to create transgenic tobacco (Nicotiana tabacum) plants overexpressing miR4367 failed, suggesting possible lethality in tobacco. Although a homolog of a miRNA originally found in soybean (Glycine max), miR4376 does not appear to be deeply conserved in plants, and exact matches of the sly-miR4376 were not found in existing miRNA databases outside the Solanaceae. This work provides interesting and novel insight into possible evolutionary pathways of miRNA regulation in plants.