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Loblolly pine (Pinus taeda) is an economically important gymnosperm and is used extensively for pulp, paper and lumber. Recent work has explored its utility as a source of biomass for sustainable energy (Ragauskas et al., 2006). Gymnosperms and angiosperms are thought to have diverged from a common ancestor c. 300 million years ago (Bowe et al., 2000; Kuzoff & Gasser, 2000). Most gymnosperms and angiosperms show distinct differences during seed development, such as single vs double fertilizations, haploid female gametophyte (FG) vs triploid endosperm, and advanced development of FG before fertilization vs establishment of endosperm after fertilization (Singh, 1978; Attree & Fowke, 1993; Nagmani et al., 1995; von Arnold et al., 2002; Nowack et al., 2006).
One of the most distinctive features of conifer embryogenesis is the development of a diploid conifer embryo that is surrounded by the haploid FG (Fig. 1a). The FG functionally corresponds to the endosperm in angiosperms. The relatively large size of P. taeda embryos permits the study of stage-specific development in embryo and FG tissues. Figure 1(b) illustrates embryo development for both zygotic embryos (ZEs) and somatic embryos (SEs) (for description, see Pullman & Webb, 1994). Somatic embryogenesis is a useful method for studying conifer embryogenesis and has great potential to generate superior growing stock for plantation forestry (McKeand et al., 2007). During somatic embryogenesis, the culture consists of suspensor-like cells and SEs at various stages of development, but the extra-embryonal FG tissues are not present (Zoglauer et al., 2003). If the haploid FG produces compounds necessary for normal embryo development, these potentially important compounds would be missing in culture and must be added as medium supplements. The percentage of SEs that complete all stages of development is small (Pullman et al., 2003). Transcript analysis of P. taeda embryos has revealed over 3000 novel expressed sequence tags (ESTs) unreported in any other conifer tissue (Cairney et al., 2006; Cairney & Pullman, 2007). Molecular mechanisms regulating the development of P. taeda embryos are poorly understood.
Figure 1. Loblolly pine (Pinus taeda) embryos (EMs) and female gametophytes (FGs). (a) A stage 8 zygotic embryo (ZE) is dissected from a FG and the FG is being secured with forceps. (b) P. taeda zygotic embryos (ZE, upper panels) and somatic embryos (SE, lower panels) from sequential developmental stages. Note that the ZEs, but not the SEs, continue growth through stage 9.2 to stage 9.10 (10 wk).
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Evidence supporting a major role for microRNAs (miRNAs) in regulating plant development has been obtained from a number of studies. MicroRNAs are small, noncoding, endogenous RNA molecules, 21 nucleotides in length, that regulate gene expression in animals and plants (Jones-Rhoades et al., 2006). Plant miRNAs function in post-transcriptional gene regulation by targeting mRNAs for degradation or translational repression by hybridizing to complementary sequences within target mRNA molecules (Bartel, 2004). To date, many miRNAs isolated from Arabidopsis and other plants have been shown to influence development by silencing their target genes (Llave et al., 2002; Reinhart et al., 2002; Rhoades et al., 2002; Baulcombe, 2004; Juarez et al., 2004; Kidner & Martienssen, 2004; Mallory et al., 2005; Williams et al., 2005; Jones-Rhoades et al., 2006; Zhou et al., 2007). Mutations in the ‘Dicer-like’ and argonaute proteins, which process miRNAs from their precursors and bind miRNAs as a component of the RNA-induced silencing complex (RISC), respectively, cause developmental defects (Park et al., 2002; Han et al., 2004; Kidner & Martienssen, 2004). MicroRNA expression may exhibit both tissue and developmental specificity. miR157 is expressed strongly in seedlings, but weakly, if at all, in leaves, flowers and other tissues of mature plants (Axtell & Bartel, 2005). Recent work has shown that the absence of a specific miRNA in Arabidopsis can cause embryo abortion (Williams et al., 2005).
One of the best characterized miRNA families in plants is miR165/166. Eight copies of DNA sequences encoding this family of miRNAs are present in the Arabidopsis genome. miR165/166 molecules are localized in shoot apical meristem (SAM), leaf primordia and vascular tissues in Arabidopsis. Five class III homeodomain leucine-zipper (HD-ZIP III) genes, namely Revoluta (REV), Phavoluta (PHV), Phabulosa (PHB), ATHB-8 and ATHB-15, are predicted target genes, and these mRNAs have been shown, in biochemical assays, to be cleaved at the miR165/166 target sequence (Tang et al., 2003; Kim et al., 2005; Jung & Park, 2007; Zhou et al., 2007). Phenotype analyses of plants with target genes mutated at the cleavage site reveal alterations in meristem functions, organ polarity, and vascular differentiation and patterning.
Another well-studied pair of miRNAs, namely miR160/miR167, regulate the mRNAs of auxin response factors (ARFs) that are associated with auxin homeostasis (Tian et al., 2004; Mallory et al., 2005; Sorin et al., 2005; Wang et al., 2005; Yang et al., 2006). In Arabidopsis, a transcription factor ARF17 mRNA is cleaved by miR160. The ARF17 represses downstream expression of the GH3 family that encodes auxin-conjugating proteins, resulting in an increase of the active indole-3-acetic acid (IAA) level in the cell. Cleavage of ARF17 mRNA, mediated by miR160, may reduce the amount of ARF protein, thus releasing the repression and decreasing the levels of free IAA (Mallory et al., 2005; Sorin et al., 2005; Wang et al., 2005). A recent report showed that regulation of ARF8L by miR167 is positively linked to the expression of GH3 (Yang et al., 2006). Thus, miRNAs that target positive regulators, such as ARF6 and ARF8, and negative regulators, such as ARF17, appear to fine-tune the expression of GH3 family genes, thereby regulating the availability of free IAA.
While a growing number of plant miRNAs have been found and predicted both by direct cloning approaches and by bioinformatics analyses, only two published reports include information on miRNAs in a gymnosperm (Axtell & Bartel, 2005; Lu et al., 2007). In the first study the presence of 11 well-conserved miRNAs was detected in needle tissues of the gymnosperm, Pinus resinosa (red pine) (Axtell & Bartel, 2005). In the second study, 26 miRNAs in xylem tissues were reported and their expression was investigated in tissue infected with a fungus that caused fusiform rust galls (Lu et al., 2007).
In the current work, we explored the presence of miRNAs in embryos and female gametophytes of loblolly pine (P. taeda). We chose five well-studied miRNAs: miR159, Pta-miR166, Pta-miR167, miR171 and miR172. These miRNAs were identified in ZE and FG tissues and were examined for stage-specific modulation. We also examined several of the known mRNA targets for miRNA-guided cleavage in seed tissues as a function of stage of development and the expression of different argonaute genes possibly involved in miRNA metabolism. A polymerase chain reaction (PCR) validation method was used to reveal partial sequences of Pta-miR166 and Pta-miR167 (Lim et al., 2003).
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In this study, five different P. taeda miRNAs, corresponding in sequence to Arabidopsis miR159, miR166, miR167, miR171 and miR172, were detected in zygotic embryos and female gametophytes of seed tissues. This is the first report of miRNAs in a seed gametophytic tissue. Pta-miR166 was detected in SEs, and Pta-miR167 was found in FGs, needles and seedlings, of loblolly pine using northern blots. All five miRNAs were also detected in ZEs and FGs using PAP-mediated RT-PCR. Northern blots for Pta-miR167 showed that this miRNA was highly expressed in needle tissues and in germinating seedlings. The authors noted that the amounts of total RNA loaded were different – 130 µg and 70 µg for SEs and needles, respectively – and both blots were run with the same amounts and sources of total RNAs. Pta-miR167 signal was not observed in SE tissues when almost twice as much total RNA was employed (Fig. 2b). Pta-miR166 was not detected in needles but gave a signal in SE tissues using the same RNA sources probed for Pta-miR167. These observations suggest that these two miRNAs are differentially expressed in these P. taeda tissues. We cannot exclude the possibility that Pta-miR166 is also expressed in needles, but at levels below the detection limits of this study. It may also be worth noting that the total RNA isolated from FGs was obtained from a mixture of developmental stages 1 to 9.12.
Poly(A) polymerase-mediated qRT-PCR was used to investigate the expression of five different miRNAs in zygotic embryo and female gametophyte tissues. Female gametophyte tissue from two different trees showed relatively similar expression patterns for the miRNAs examined. Zygotic embryos and FGs showed very different miRNA expression patterns during seed development. The statistically significant increase in the level of Pta-miR166 in stage 9.1 FG (Fig. 3d) strongly suggests that this miRNA is triggering an event required for embryo and/or FG development. However, a corresponding stage-specific change was not observed in the degradation pattern of its potential mRNA target HB15L. The ratio of RA-PCR products for the target region of HB15L mRNA did not show significant modulation from stages 4 to 9.2 for either ZE or FG tissues. HB15L may be controlled by multiple factors. These factors may be supervening miRNA-mediated cleavage of this gene in pine embryos (Ohashi-Ito & Fukuda, 2003). Although an extensive embryo-expressed P. taeda EST database is available, HB15L mRNA was the only potential target we identified for Pta-miR166. A more comprehensive analysis of Pta-miR166 target mRNAs is needed to gain an understanding of the consequences of the rise in levels of Pta-miR166.
The potential mRNA target of Pta-miR167, ARF8L, was found in the P. taeda EST database based on the sequence of a 5′-RACE product from red pine (Axtell & Bartel, 2005). The sequences of ARF8L mRNA in loblolly pine and red pine are highly conserved (96% nucleotide identity). It was annotated as ARF8L after analysis using clustalw with other ARF genes in Arabidopsis. Our RA-PCR approach showed different degradation patterns for ARF8L mRNA in ZE vs FG. Significant reductions in the target region compared with the 3′ region were observed in ZE tissues, whereas in FG tissues this did not occur. Multiple restriction digestions of the three DNA fragments confirmed the homogeneity of the cDNA sequence in both tissues. These results clearly show that ARF8L mRNA stability is regulated in a different manner in ZE and FG tissues. The observations are consistent with Pta-miR167-mediated cleavage of ARF8L mRNA in ZEs but not in FGs; however, additional studies are needed to confirm this interpretation.
One aspect of the regional amplification results of ARF8L mRNA in ZEs does not appear to be consistent with a simplified picture of miRNA-mediated cleavage and mRNA stability. If miRNA-induced cleavage occurs to a messenger RNA, mRNA fragments upstream of the cleavage site will no longer have the poly(A) tail and would not be subject to reverse transcription using the oligoT-anchored primer. Thus, the amount of 5′ region amplified by qRT-PCR should be less than or equal to the amount amplified from the target region. Unlike the results shown in Figs 4(b) and 5(a,b), Fig. 5(c) shows that the amount of 5′ region product is higher than the amount of target region product. Unknown rates of 5′ and 3′ degradation, or aberrant secondary structures introduced by miRNA-guided cleavage of this specific mRNA, may cause the RA-PCR data to vary from expectations. This observation suggests caution in interpreting the RA-PCR results for the ARF8L mRNA in terms of the miRNA-mediated degradation. We are currently exploring further for evidence of self-priming caused by RNA secondary structures or unusual degradation patterns of this mRNA (Haddad et al., 2007).
Two homologs of argonaute family genes were profiled as a function of developmental stage in embryo and female gametophyte tissues. Other studies of argonaute genes have shown that they are involved in many different functions of cell metabolism such as regulation of gene expression with small RNAs including miRNAs, schematic silencing of genes on the chromosomes and defense mechanisms against invasion of foreign agents. The mutation studies of Baumberger & Baulcombe (2005) indicate that the catalytic Asp-Asp-His (DDH) triad and additional histidine of the PIWI domain are necessary to function as an RNA slicer. In addition, they illustrated AtAGO4, AtAGO6, AtAGO8 and AtAGO9 as producing proteins having a different amino acid residue when aligned with AGO1 position 798. They therefore suggest that AtAGO4, AtAGO6, AtAGO8 and AtAGO9 do not produce active slicer proteins. However, Qi et al. (2006) tested this speculation and they have shown that AtAGO4 with serine instead of histidine at position 798 along with the triad, still has slicer activity. This suggests that ptAGO9L with proline at position 798 along with the conserved triad may still function as a slicer, and possibly this gene is a tissue-specific functional homolog of a miRNA/siRNA carrier (with a PAZ domain) and the slicer for reproductive tissue, as suggested by Scutt et al (2003). Our observation of inverted or complementary expression of ptAGO9L in ZE and FG tissues may indicate cross-talk between tissues through uses of small RNAs.
While a number of questions remain to be answered regarding targets and function of miRNAs in P. taeda embryos, the results from this study demonstrate stage-specific and tissue-specific modulation for some miRNAs and miRNA processing components in seed tissues. Given the importance of miRNAs to general developmental processes in plants, it is likely that the miRNAs identified in the FGs play a role in embryogenesis. Our observation of differential Pta-miR167-mediated cleavage in FG vs ZE tissues suggest that miRNAs expressed in FG may be involved in regulating embryo development in loblolly pine. An understanding of the role of miRNAs in FGs on the regulation of embryo development may lead to advancements in clonal technology for improved sustainable forestry.