Regulation of small RNA accumulation in the maize shoot apex

Abstract

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

Figure 1. mir166 family members exhibit distinct overlapping expression profiles in the maize shoot apex.

(A) Longitudinal sections through a maize apex diagramming regions captured by laser-microdissection (left panel): 1) P2–P3 leaf primordia; 2) the incipient (P0) and P1 leaf primordia plus the base of the SAM; 3) P4–P6 leaf primordia; 4) stem tissue. The right panel shows a longitudinal section of an apex after capturing cells from the P2 and P3 leaf primordia. Note the precision with which cells are captured. (B) Transcript accumulation of tub6, rld1, rs1 and kan1 in these regions is as previously reported , –, illustrating the accuracy of laser-microdissection. (C) RT-PCR analysis of mir166a to mir166e shows that these mir166 family members exhibit distinct but overlapping expression profiles within the vegetative apex. -RT controls are also shown.

Figure 2. Accumulation of miR166 and miR390 within the SAM is regulated at the level of biogenesis and/or stability.

(A) Laser-microdissection of domains within the SAM: 5) the stem cell containing SAM tip; 6) the incipient primordium; 7) the region below the incipient leaf. (B) Expression analysis of mir166 family members by RT-PCR shows that only mir166a, -c, -f and –i precursor transcripts accumulate in or below the incipient leaf and likely contribute to the establishment of organ polarity. In addition, mir166a transcripts are expressed in the SAM tip. Whereas both mir390 precursors are expressed in the tip of the SAM and in the incipient leaf, only mir390a accumulates in the region below the incipient leaf. (C) In situ hybridization of a longitudinal section through a wild-type apex with a miR166 complementary LNA probe shows miR166 accumulates below the incipient leaf and on the abaxial side of the incipient and developing leaf primordia. Note the lack of detectable signal in the tip of the SAM (marked “M”). (D) Longitudinal section through a wild-type apex shows rld2 expression in the tip of the SAM, vasculature, and the adaxial side of leaf primordia. (E) Expression of tub6, kan1, kan2, and rld1 in the captured SAM regions is as previously reported ,, illustrating the accuracy of laser-microdissection. (F) RT-PCR analysis for the core miRNA processing genes dcl1, se1a and se1b in the SAM tip.

Figure 3. MiR390 is an upstream component in the maize leaf polarity pathway.

(A, B) In situ hybridization analyses with a miR390 complementary LNA probe shows this small RNA accumulates on the adaxial side (arrowheads) of incipient and developing leaf primorida of both wild-type (A) and lbl1-rgd1 (B) seedlings. Dashed lines mark the position of the incipient leaf. No hybridization signal is detected in the tip of the wild-type SAM. (C) In situ hybridization with an LNA-probe complementary to murine miR122a serves as a negative control.

Figure 4. mir390 precursors, unlike mature miR390, accumulate in just the epidermal (L1) layer of the maize SAM.

(A) Representative sections showing the laser-microdissection of the L1 epidermal layer of the SAM (M) including the incipient primordium (P0). The L1 layer is precisely cut (middle panel) and subsequently captured (right panel). Afterwards, the L2 layer is microdissected similarly. (B) RT-PCR analysis of the miR390 precursors shows that both mir390a and mir390b transcripts accumulate specifically in the L1 layer. Mutually exclusive expression of ocl4 and kn1 in the L1 and L2 layers, respectively, demonstrates the precision of LM ,. (C) Close-up view of the accumulation pattern of mature miR390 in the incipient leaf (dashed lines) taken from Figure 3A. Note the presence of signal in the underlying L2 layer (arrow). (D) RT-PCR analysis of mir166 and tas3 family members shows these precursor transcripts accumulate in both the L1 and L2 layers of the SAM.

Figure 5. A model for the establishment of maize leaf polarity.

The diagram on the left illustrates the spatial pattern of expression for both mir390 precursors as well as mature miR390 and miR166 in the SAM. The mir390 precursors accumulate in the L1 layer of the whole SAM (red). In contrast, mature miR390 (green) is present in both the L1 and underlying L2 layers, but just on the adaxial side of the incipient leaf. The adaxial expression of miR390 persists even in the abaxialized leaves of lbl1 mutants, demonstrating that this small RNA, which constitutes an upstream component in the maize adaxial-abaxial patterning network, maintains polar expression independent of the tas3 ta-siRNA pathway. MiR390 activity triggers ta-siRNA biogenesis on the adaxial side of the incipient primordium that indirectly restrict the expression of specific mir166 family members. Regulation of small RNAs in the ta-siRNA pathway at the level of biogenesis, stability, or possibly movement generates an inverse abaxial gradient of mature miR166 (purple) that delineates the expression of hd-zipIII genes, like rld1 and rld2, to the adaxial side of the incipient leaf establishing organ polarity.