The proportion of small RNA with same length is different in tissues.
Maize develops two morphologically distinct inflorescences that bear separate male and female flowers (Vollbrecht et al. 2005). Male flowers (anther) develop at the tip of the shoot in the tassel, and female flowers (silk) develop on the ears (Veit et al. 1993). To systematically identify uncharacterized phasiRNAs and explore its expression in maize, small RNA datasets from nine tissues including root, seedling, embryo, anther, pollen, immature ear, premature ear, immature tassel and silk were analyzed in the study. The sRNA libraries from anther (~ 3mm), embryo (12 day after pollination) and premature ear (4 ~ 5 cm) were conducted by us (see Method). The sRNA libraries from root, seedling, silk, pollen, immature ear (0.5 ~ 2cm) and immature tassel (0.5 ~ 2.5cm) were downloaded from public database (Table S1) (Zhang et al. 2009; Li et al. 2013). As reported, the 21-nt and 24-nt phasiRNAs exhibit striking temporal expression during maize male reproductive development (Zhai et al. 2015). The 21-nt phasiRNAs occur pre-meiotically while the 24-nt phasiRNAs accumulate during meiosis and persist to pollen (Zhai et al. 2015). So, the meiosis stage is a key timing. Six male and female reproductive tissues collected by us can represent the dynamic development stages of male and female florets. Immature tassel with 0.5 ~ 2.5 cm length undergoes developmental stages from paired spikelet meristems (SMs) to the premeiotic anther while 3 ~ 4 mm anther have finished meiotic progression (Kelliher and Walbot 2014; Phillips et al. 2011). Maize ear samples were also collected at two developmental stages: 0.5 ~ 2.0 cm immature ear undergoing the premeiotic stage from the development of spikelet-pair primordia (SPM) to the differentiation of pistils, and 4 ~ 5 cm premature ear bearing pistillate flowers (silk) have developed in a post-meiosis stages.
Small RNA reads in the range of 18 to 31 nucleotides were retained. In total, more than 600 million signatures from nine tissues were got, representing over 91 million unique sequences (Table S1). The length of small RNA is a significant character which usually relates to its function. For example, microRNAs are typically 20 ~ 22-nt small RNAs while great majority of endogenous siRNAs are 24-nt heterochromatic siRNAs which plays an important role in silencing transposons by methylation (Sunkar et al. 2005). In these nine tissues, the trend of small RNA size distribution is highly similarity (Fig. 1). As same as reported previously (Wang et al. 2009; Wang et al. 2011), the 24-nt and 22-nt small RNAs are the most and second abundant in all tissues. However, there was an interesting finding that the proportion of small RNA with same length was different in tissues. The difference mainly existed in the proportion of 24-nt and 22-nt small RNAs. The 24-nt small RNAs were most abundant in immature ear and premature ear with about 60% of sRNAs while the 24-nt small RNAs only accounted for 36% of sRNAs in anther. In addition, for 22-nt small RNAs, it was nearly 25% out of sRNAs in pollen corresponding to about 15% out of that in root, seedling and silk. Analysis of size distributions revealed that the proportion of small RNAs in different size classes varied among different tissues, which may be related to the tissue-specific biological progresses.
Identification of 21-nt and 24-nt phasiRNAs in maize tissues.
We performed a computational analysis to identify phasiRNAs, using an algorithm from a previous study (Song et al. 2012a). The P-score was calculated to identify phasiRNAs. Applying this algorithm, four maize TAS genes (TAS3a, TAS3b, TASb3c, TAS3d) (Nogueira et al. 2007) were identified with P-scores higher than 20 in nine tissues (Fig. 2). This result indicated that the algorithm is efficient to identify phasiRNA patterns in maize nine datasets.
We adopted a P-score of 25, a more stringent cut-off, for the identification of 21-nt and 24-nt phasiRNAs. Totally, 269 21-nt PHAS loci and 135 24-nt PHAS loci were identified in all tissues (Table 1 and Table S2). Consistent with the previous work (Zhai et al. 2015), in male reproductive tissues, we identified 68, 195 and 28 21-PHAS loci in immature tassel, anther and pollen respectively. And, 45 and 109 24-PHAS loci were also characterized in anther and pollen as predicted. Whereas to be noted, except for male reproductive tissues, 182 21-PHAS loci also were characterized in immature ear and 73 24-PHAS loci characterized in silk which is maize pistil providing the foundation and directional guidance for pollen tube navigation. Few 21-nt and 24-nt phasiRNAs were identified in root, seedling and embryo. Hence, both 21-nt and 24-nt phasiRNAs seem to be specifically generated in male and female reproductive tissues in maize.
Table 1
The 21- and 24-PHAS loci in nine datasets.
|
No.of 21-PHAS loci
|
No.of 24-PHAS loci
|
Root
|
2
|
2
|
Seedling
|
0
|
2
|
Embryo
|
2
|
0
|
Silk
|
7
|
73
|
Pollen
|
28
|
109
|
Anther
|
195
|
45
|
Immature ear
|
182
|
0
|
Premature ear
|
25
|
10
|
Immature tassel
|
68
|
0
|
Total
|
269
|
135
|
To test if the phasiRNAs identified in immature ear and silk are same with that in male reproductive tissues, we compared the 21-PHAS loci (immature ear) / 24-PHAS loci (silk) with anther (including 3mm anther in the study and 11 sequential stages of W23 fertile anthers (Zhai et al. 2015)) and immature tassel / pollen. The results are displayed on Venn diagrams. 76.4% of 21-PHAS loci identified in immature ear are overlapped with those in male reproductive tissues including immature tassel and anther (Fig. 3a). Similarly, 97.3% of 24-PHAS loci identified in silk are also overlapped with those in anther and pollen (Fig. 3b). Our results suggested that 21-nt and 24-nt phasiRNAs identified in immature ear and silk may play same roles with those in male reproductive tissues. In addition, almost all the 21-nt and 24-nt phasiRNAs identified by us in male and female reproductive tissues were contained in previous work (Zhai et al. 2015) (Fig. 3a, b), which suggested the number of 21-nt and 24-nt phasiRNAs may be saturated in maize reproductive tissues.
The 21-nt and 24-nt phasiRNAs show differential expression in male and female reproductive tissues
To analyze the dynamic expression of 21-nt and 24-nt phasiRNAs during male and female reproductive development, we compared the abundance of 21-nt and 24-nt phasiRNAs among tissues by calculating the reads per million (RPM) in each library. In male reproductive tissues, the quantity and diversity of 21-PHAS loci were detectable in immature tassel, but the peak presented at anther and declined in pollen (Fig. 3c). The comparable quantity and diversity of 24-PHAS loci were identified in anther and pollen. Therefore, the dynamic expression of 21-nt and 24-nt phasiRNAs in male reproductive tissues is consistent with the previous work (Zhai et al. 2015), which certifies the accuracy of our method and data. Additionally, in female reproductive tissues, we also observed that 21-nt phasiRNAs were activated in immature ear but silenced in premature ear (removing silk and cob) and silk. But 24-nt phasiRNAs were undetectable until in silk (Fig. 3c). Hence, the results indicated that 21-nt and 24-nt phasiRNAs may be dynamic with the development of female florets, which seems to be correlated with the development of anther. Though the clusters of 21-nt and 24-nt phasiRNAs identified in female reproductive tissues are almost as much as those in male reproductive tissues, the abundance of 24-nt phasiRNAs in silk was significantly less than that in anther. In immature ear, the abundance of 21-nt phasiRNAs was several times less than that in anther. However, the abundance of 24-nt phasiRNAs in silk was several hundred times less than that in anther, but was several times higher than that in other tissues.
The motif of 21-nt phasiRNAs and 24-nt phasiRNAs in tissues
Recent studies demonstrated that miR2118 and miR2275 act as triggers to initiate formation of 21- and 24-nt phasiRNAs in rice panicle and in maize anther, respectively (Zhai et al. 2015; Fei et al. 2016). Hence, we used the motif-identification program MEME to scan 21- and 24-PHAS loci to identify the conserved sequence (Bailey and Elkan 1994). As a result, 93.4% out of 273 21-PHAS loci contained a 22-nt motif which well matched with miR2118 and was same with that identified in rice panicle (Fig. 3d). 98.9% of 21-PHAS loci identified in immature ear also possessed the 22-nt motif, which indicated that miR2118 may mediate the biogenesis of 21-nt phasiRNAs in female reproductive tissues like that in male reproductive tissues.
Similar analysis of 24-PHAS loci, a different 22-nucleotide motif was found to be shared by 81.3% of all 24-PHAS loci, of which sequence matched well with miR2275 family (Fig. 3e). Majority of 24-PHAS loci identified in silk contained the 22-nt motif, which indicated that the biogenesis of 24-nt phasiRNAs in female inflorescence is also mediated by miR2275. Therefore, our evidences support that miR2118 and miR2275 may mediate the biogenesis of 21- and 24-nt phasiRNAs in male and female inflorescence, respectively, by initiating cleavage of their transcripts in maize.
The expression of miR2118 and miR2275 in tissues
In maize, there are seven and four members belonging to miR2118 and miR2275 families in the current version of miRBase, respectively. As we know, miR2118 and miR2275 play important roles in the biogenesis of phasiRNAs. Here, we showed the expression profiles of miR2118 and miR2275 among all tissues, which indicated that miR2118 and miR2275 exhibited significantly differential expression (Fig. 4a). Both miR2118 and miR2275 mainly expressed in anther and pollen. Besides, miR2118 also expressed in immature ear and immature tassel and miR2275 show low expression level in silk. Hence, the expression levels of miR2118 and miR2275 accord well with the abundance of 21-nt and 24-nt phasiRNAs identified in tissues.
Additionally, whether all members of miR2118 and miR2275 families contribute to the biogenesis of phasiRNAs is still exclusive. Here we have observed that each member in miR2118 and miR2275 families existed differential expression in each tissue (Fig. 4b,c). In male and female inflorescence organs, the expression levels of miR2118c, miR2118d, miR2118e and miR2118f seemed to be relative lower than that of miR2118a, miR2118b and miR2118g. miR2118g possessed significant high expression levels compared with other members of miR2118 family in anther while miR2118b expressed highest in pollen. In immature ear, miR2118a and miR2118b possessed significant higher expression levels than other members of miR2118 family. For the expression of miR2275, miR2275a, miR2275b and miR2275c showed comparable expression levels in anther. Only miR2275c expressed in pollen and silk. Whereas, miR2275d rarely expressed in all tissues. In a word, the differential expression of each member in miR2118 and miR2275 families among tissues suggested that they may provide different contributions in the biogenesis of 21-nt and 24-nt phasiRNAs.
MiR2118 and MiR2275 were less accumulated in dcl1 mutant.
MicroRNAs, processed from stem-loop transcripts by the activity of Dicer ribonucleases, are a kind of conserved small RNAs mainly with a length of 21 nucleotides (Bologna and Voinnet 2014; Rogers and Chen 2013). As reported, DCL1 is a key enzyme required for miRNA biogenesis (Bologna et al. 2009). In maize, the DCL1 gene were constructive expressed among tissues (Fig. S1). Hence, we speculated whether DCL1 is the direct Dicer ribonucleases to process the maturation of two 22-nt miR2118 and miR2275. Recently, maize fuzzy tassel (fzt) mutant has been cloned (Thompson et al. 2014), which contains a mutation in a dicer-like1 homolog. As expected, the expression levels of many miRNAs have reduced in dcl1-fzt mutants, like miR167 and miR408 (Thompson et al. 2014). To determine whether DCL1 protein generates the triggers of phasiRNA biogenesis, we detected the expression levels of miR2118 and miR2275 in dcl1-fzt seedling and tassel primordia (Fig. 4d). Though the miR2118 and miR2275 rarely expressed in seedling and tassel primordia compared with the male and female inflorescence organs, the expression levels of miR2118b, miR2118d and miR2275b were at least two-fold reduced in dcl1-fzt.