Small RNAs, microRNAs (miRNAs) and short interfering RNAs (siRNAs) are important gene-regulatory molecules at both the transcriptional and post-transcriptional levels in eukaryotic cells . Plant miRNAs are derived from single RNA molecules. Primary RNA precursors (pri-miRNA) can form imperfect stem-loop structures where a miRNA/miRNA* duplex is processed from the stem by Dicer-like 1 (DCL1) or DCL4 [2–4]. Plant miRNAs negatively regulate their cognate mRNAs by fully or partly binding to complementary sites. After being methylated at the 3′ end by Hua Enhancer 1 (HEN1) , the mature miRNA with a length of 20–24 nucleotides (nt) is loaded onto the RNA-induced silencing complex (RISC) to direct the cleavage of its mRNA targets based on extensive complementarity. Plant miRNAs predominantly modulate their targets by mRNA cleavage, and some classes of 24 nt miRNAs direct cytosine DNA methylation at target genes to regulate their expression [6–8]. More recently, miRNA regulation of gene expression via DNA methylation and chromatin modification has been suggested [9, 10]. The nearly perfect complementarity between miRNAs and their target sites makes it possible to predict their targets by computational approaches. miRNAs were shown to regulate genes involved in basic developmental processes, such as leaf development, flowering time, organ polarity and auxin signaling [11, 12], as well as stress responses and disease resistance [13, 14].
High-throughput sequencing technologies allow the discovery of a large set of diverse plant miRNAs. Thousands of miRNAs have been identified in different plant species, rapidly enlarging the identified plant miRNA pool, including miRNAs from different tissues or developmental stages. Based on the recent version of miRBase (http://www.mirbase.org/), over 400 miRNAs have been identified in rice. Among them, 21 miRNA families are evolutionarily conserved between Arabidopsis and rice [15–18]. Some of the miRNAs are conserved only among closely related monocots, suggesting the emergence of novel miRNAs after divergence of monocots and dicots [19, 20].
As one of the most important food sources for the world’s population, rice is also an ideal model plant representing cereal crops. The grain-filling phase is a major stage of plant development that largely determines yield and quality . During this process, all resources of the plant contribute toward a steady rate of starch accumulation in the storage units of rice grains [22, 23]. In general, the grain development process can be divided into early development and filling phases. The former is characterized by high biosynthetic activity in grain formation when the total dry matter starts to increase and endosperm starch begins to accumulate rapidly in the seed (6–17 days after flowering, or DAF), whereas during the latter phase (from 18 DAF) the grain usually exhibits a slower increase in dry weight until maximum values are reached and grain weight becomes constant. Global gene expression profiling studies of mRNAs have shown that many genes in multiple pathways participate in grain filling processes, such as those involved in nutrient synthesis, starch synthesis and transport [24, 25].
On the other hand, miRNAs were identified as preferentially expressed in various rice organs, including leaf, root, panicle and stem, as well as in seedlings under various stress treatments [17, 26, 27]. A number of studies were also carried out on small RNAs in the grains of japonica varieties [26, 27]. Some miRNAs were preferentially expressed in early developing rice grains, such as 1–10 DAF and 3–12 DAF [28, 29], suggesting regulatory roles of miRNAs during grain development. These studies, mainly in subspecies of japonica, also identified significant numbers of both conserved and non-conserved miRNAs. We report here the generation and sequencing of a small RNA library from grain tissues sampled during the entire grain filling stage of an indica cultivar. In addition to numerous conserved miRNAs, we identified 11 novel miRNAs. Subsequently, a customized miRNA chip was generated and miRNA expression profiling was studied using RNA samples from grains of each of the three filling stages: viz. milk-ripe (6–12 DAF), soft-dough (13–17 DAF), and hard-dough (18–20 DAF). Our results showed that most of the widely conserved miRNAs were down-regulated during grain develop-ment whereas rice or grass-specific miRNAs were up-regulated. The targets of differentially expressed miRNAs appeared to be involved in multiple biological processes, such as carbohydrate metabolism, hormone signaling and pathways associated with seed maturity, suggesting that rice miRNAs may play important roles during grain development.