Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5′ coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.     

Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA₁ levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.     

One Divinyl Reductase Reduces the 8-Vinyl Groups in Various Intermediates of Chlorophyll Biosynthesis in a Given Higher Plant Species,But the Isozyme Differs between Species

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. As the main component of the photosynthetic pigments, Chl molecules perform essential processes of absorbing light and transferring the light energy in the reaction center of the photosystems (Fromme et al., 2003). Based on the number of vinyl side chains, Chls are classified into two groups, 3,8-divinyl (DV)-Chl and 3-monovinyl (MV)-Chl. The DV-Chl molecule contains two vinyl groups at positions 3 and 8 of the tetrapyrrole macrocycle, whereas the MV-Chl molecule contains a vinyl group at position 3 and an ethyl group at position 8 of the macrocycle. Almost all of the oxygenic photosynthetic organisms contain MV-Chls, with the exceptions of some marine picophytoplankton species that contain only DV-Chls as their primary photosynthetic pigments (Chisholm et al., 1992; Goericke and Repeta, 1992; Porra, 1997).The classical single-branched Chl biosynthetic pathway proposed by Granick (1950) and modified by Jones (1963) assumed the rapid reduction of the 8-vinyl group of DV-protochlorophyllide (Pchlide) catalyzed by a putative 8-vinyl reductase. Ellsworth and Aronoff (1969) found evidence for both MV and DV forms of several Chl biosynthetic intermediates between magnesium-protoporphyrin IX monomethyl ester (MPE) and Pchlide in Chlorella spp. mutants. Belanger and Rebeiz (1979, 1980) reported that the Pchlide pool of etiolated higher plants contains both MV- and DV-Pchlide. Afterward, following the further detection of MV- and DV-tetrapyrrole intermediates and their biosynthetic interconversion in tissues and extracts of different plants (Belanger and Rebeiz, 1982; Duggan and Rebeiz, 1982; Tripathy and Rebeiz, 1986, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996), a multibranched Chl biosynthetic heterogeneity was proposed (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010).Biosynthetic heterogeneity refers to the biosynthesis of a particular metabolite by an organelle, tissue, or organism via multiple biosynthetic routes. Varieties of reports lead to the assumption that Chl biosynthetic heterogeneity originates mainly in parallel DV- and MV-Chl biosynthetic routes. These routes are interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). DV-MPE could be converted to MV-MPE in crude homogenates from etiolated wheat (Triticum aestivum) seedlings (Ellsworth and Hsing, 1974). Exogenous DV-Pchlide could be partially converted to MV-Pchlide in barley (Hordeum vulgare) plastids (Tripathy and Rebeiz, 1988). 8-Vinyl chlorophyllide (Chlide) a reductases in etioplast membranes isolated from etiolated cucumber (Cucumis sativus) cotyledons and barley and maize (Zea mays) leaves were found to be very active in the conversion of exogenous DV-Chlide a to MV-Chlide a (Parham and Rebeiz, 1992, 1995). Kim and Rebeiz (1996) suggested that Chl biosynthetic heterogeneity in higher plants may originate at the level of DV magnesium-protoporphyrin IX (Mg-Proto) and would be mediated by the activity of a putative 8-vinyl Mg-Proto reductase in barley etiochloroplasts and plastid membranes. However, since these reports did not use purified or recombinant enzyme, it is not clear whether the reductions of the 8-vinyl groups of various Chl intermediates are catalyzed by one enzyme of broad specificity or by multiple enzymes of narrow specificity, which actually has become one of the focus issues in Chl biosynthesis.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis (Arabidopsis thaliana) as an 8-vinyl reductase, namely, divinyl reductase (DVR). Chew and Bryant (2007) identified the DVR BciA (CT1063) gene of the green sulfur bacterium Chlorobium tepidum, which is homologous to AT5G18660. An enzymatic assay using a recombinant Arabidopsis DVR (AtDVR) on five DV substrates revealed that the major substrate of AtDVR is DV-Chlide a, while the other four DV substrates could not be converted to corresponding MV compounds (Nagata et al., 2007). Nevertheless, a recombinant BciA is able to reduce the 8-vinyl group of DV-Pchlide to generate MV-Pchlide (Chew and Bryant, 2007). Recently, we identified the rice (Oryza sativa) DVR encoded by Os03g22780 that has sequence similarity with the Arabidopsis DVR gene AT5G18660. We also confirmed that the recombinant rice DVR (OsDVR) is able to not only convert DV-Chlide a to MV-Chlide a but also to convert DV-Chl a to MV-Chl a (Wang et al., 2010). Thus, it is possible that the reductions of the 8-vinyl groups of various Chl biosynthetic intermediates are catalyzed by one enzyme of broad specificity.In this report, we extended our studies to four DVR proteins and five DV substrates. First, ZmDVR and CsDVR genes were isolated from maize and cucumber genomes, respectively, using a homology-based cloning approach. Second, enzymatic assays were systematically carried out using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates. Third, we examined the in vivo accumulations of various Chl intermediates in rice, maize, and cucumber. Finally, we systematically investigated the in vivo accumulations of Chl and its various intermediates in the OsDVR (Os03g22780)-inactivated 824ys mutant of rice (Wang et al., 2010). The results strongly suggested that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but DVR proteins from different species could have diverse and differing substrate preferences even though they are homologous.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.